NZ712344B2 - Non-replicative transduction particles and transduction particle-based reporter systems - Google Patents
Non-replicative transduction particles and transduction particle-based reporter systems Download PDFInfo
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- NZ712344B2 NZ712344B2 NZ712344A NZ71234414A NZ712344B2 NZ 712344 B2 NZ712344 B2 NZ 712344B2 NZ 712344 A NZ712344 A NZ 712344A NZ 71234414 A NZ71234414 A NZ 71234414A NZ 712344 B2 NZ712344 B2 NZ 712344B2
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- C12N2795/10341—Use of virus, viral particle or viral elements as a vector
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Abstract
Methods and systems are provided for packaging reporter nucleic acid molecules into non-replicative transduction particles for use as reporter molecules. The non-replicative transduction particles can be constructed from viruses and use viral transduction and replication systems. The reporter nucleic acid molecules include a reporter gene, such as a reporter molecule or selectable marker, for detecting target genes or cells. Methods and systems are provided for detection of cells and target nucleic acid molecules using the non- replicative transduction particles as reporter molecules. c acid molecules include a reporter gene, such as a reporter molecule or selectable marker, for detecting target genes or cells. Methods and systems are provided for detection of cells and target nucleic acid molecules using the non- replicative transduction particles as reporter molecules.
Description
TITLE
Non-Replicative Transduction Particles and Transduction Particle-Based Reporter
Systems
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 61/779,177,
filed on March 13, 2013, U.S. Provisional Application No. 61/897,040, filed on Oct. 29,
2013, and U.S. Provisional Application No. 61/939,126, filed on Feb. 12, 2014, each of
which is hereby incorporated in its entirety by reference.
SEQUENCE LISTING
[0002.1] The instant application contains a Sequence Listing which has been submitted
electronically in ASCII format and is hereby incorporated by reference in its entirety. Said
ASCII copy, created on April 10, 2014, is named 25938PCT_CRF_SequenceListing.txt and
is 65,305 bytes in size.
BACKGROUND OF THE INVENTION
Field of the invention
The invention relates to methods and compositions for packaging and delivery of non-
replicative transduction reporter molecules into cells for detecting target genes in cells.
Description of the Related Art
A transduction particle refers to a virus capable of delivering a non-viral nucleic acid
into a cell. Viral-based reporter systems have been used to detect the presence of cells and
rely on the lysogenic phase of the virus to allow expression of a reporter molecule from the
cell. These viral-based reporter systems use replication-competent transduction particles that
express reporter molecules and cause a target cell to emit a detectable signal.
However, the lytic cycle of the virus has been shown to be deleterious to viral-based
reporter assays. Carriere, C. et al., Conditionally replicating luciferase reporter phages:
Improved sensitivity for rapid detection and assessment of drug susceptibility of
Mycobacterium tuberculosis. Journal of Clinical Microbiology, 1997. 35(12): p. 3232-3239.
Carrière et al. developed M. tuberculosis/bacillus Calmette-Guérin (BCG) luciferase reporter
phages that have their lytic cycles suppressed at 30°C, but active at 37°C. Using this system,
Carrière et al. have demonstrated the detection of BCG using phage reporters with a
suppressed lytic cycle.
There are disadvantages, however, associated with suppressing but not eliminating the
replication functions of the bacteriophage in bacteriophage-based reporter assays. First,
controlling replication functions of the bacteriophage imposes limiting assay conditions. For
example, the lytic cycle of the reporter phage phAE40 used by Carrière et al. was repressed
when the phage was used to infect cells at the non-permissive temperature of 30°C. This
temperature requirement imposed limiting conditions on the reporter assay in that the
optimum temperature for the target bacteria was 37°C. These limiting conditions hinder
optimum assay performance.
Moreover, the replication functions of the virus are difficult to control. The
replication of the virus should be suppressed during the use of the transduction particles as a
reporter system. For example, the lytic activity of the reporter phage phAE40 reported by
Carriere et al. was reduced but was not eliminated, resulting in a drop in luciferase signal in
the assay. Carriere et al. highlighted possible causes for the resulting drop in reporter signal,
such as intact phage-expressed genes and temperature limitations of the assay, all stemming
from the fact that the lytic cycle of the phage reporter was not eliminated.
Reporter assays relying on the natural lysogenic cycle of phages can be expected to
exhibit lytic activity sporadically. In addition, assays that rely on the lysogenic cycle of the
phage can be prone to superinfection immunity from target cells already lysogenized with a
similar phage, as well as naturally occurring host restriction systems that target incoming
virus nucleic acid, thus limiting the host range of these reporter phages.
In other examples, transduction particle production systems are designed to package
exogenous nucleic acid molecules, but the transduction particle often contains a combination
of exogenous nucleic acid molecules and native progeny virus nucleic acid molecules. The
native virus can exhibit lytic activity that is a hindrance to assay performance, and the lytic
activity of the virus must be eliminated in order to purify transduction particles. However,
this purification is generally not possible. In U.S. 2009/0155768 A, entitled Reporter
Plasmid Packaging System for Detection of Bacteria, Scholl et al. describes the development
of such a transduction particle system. The product of the system is a combination of
reporter transduction particles and native bacteriophage (Figure 8 in the reference). Although
the authors indicate that the transduction particle and native bacteriophage can be separated
by ultracentrifugation, this separation is only possible in a system where the transduction
particle and the native virus exhibit different densities that would allow separation by
ultracentrifugation. While this characteristic is exhibited by the bacteriophage T7-based
packaging system described in the reference, this is not a characteristic that is generally
applicable for other virus systems. It is common for viral packaging machinery to exhibit
headful packaging that would result in native virus and transduction particles to exhibit
indistinguishable densities that cannot be separated by ultracentrifugation. Virus packaging
systems also rely on a minimum amount of packaging as a requirement for proper virus
structural assembly that results in native virus and transduction particles with
indistinguishable densities.
Thus, there is a need for non-replicative transduction particles that do not suffer from
the deleterious effects from lytic functions of the virus and the possibility of being limited by
superinfection immunity and host restriction mechanisms that target virus nucleic acid
molecules and viral functions, all of which can limit the performance of the reporter assay by
increasing limits of detection and resulting in false negative results.
Even where transduction particles have been engineered, methods for using the
transduction particles to detect and report the presence of target nucleic acid molecules in
cells have limitations. Some methods require disruption of the cell and cumbersome
techniques to isolate and detect transcripts in the lysate. Detection methods include using
labeled probes such as antibodies, aptamers, or nucleic acid probes. Labeled probes directed
to a target gene can result in non-specific binding to unintended targets or generate signals
that have a high signal-to-noise ratio. Therefore, there is a need for specific, effective and
accurate methods for detection and reporting of endogenous nucleic acid molecules in cells.
Accordingly, methods and systems are needed for generating non-replicative
transduction particles that allow packaging and expression of reporter molecules in cells,
while eliminating replication-competent progeny virus. Effective and accurate methods for
detecting molecules in cells using the expressed reporter molecules are also needed.
SUMMARY OF THE INVENTION
Disclosed herein is a bacterial cell packaging system for packaging a reporter nucleic
acid molecule into a non-replicative transduction particle, said bacterial cell comprising a
lysogenized bacteriophage genome lacking a bacteriophage gene encoding a packaging
initiation site sequence, wherein deletion of said bacteriophage gene prevents packaging of a
bacteriophage nucleic acid molecule into said non-replicative transduction particle; and a
reporter nucleic acid molecule comprising a second bacteriophage gene, wherein said second
bacteriophage gene encodes a packaging initiation site sequence and facilitates the packaging
a replica of said reporter nucleic acid molecule into said non-replicative transduction particle,
wherein said second bacteriophage gene is capable of expressing a protein that is encoded by
said gene, wherein said replica of said reporter nucleic acid molecule forms a replicon
amenable to packaging into said non-replicative transduction particle.
In some embodiments, the reporter nucleic acid molecule is operatively linked to a
promoter. In another embodiment, the promoter is selected for contributing to reactivity of a
reporter molecule expressed from said reporter nucleic acid molecule in said bacterial cell. In
one embodiment, the reporter nucleic acid molecule comprises an origin of replication. In yet
another embodiment, the replicon comprises a concatamer amenable to packaging into said
non-replicative transduction particle.
In an embodiment, the first and said second bacteriophage genes each comprises a
pacA gene of the Enterobacteriaceae bacteriophage Pl and comprises said packaging
initiation site sequence. In one embodiment, the second bacteriophage gene comprises the
sequence of SEQ ID N0:9. In another embodiment, the replicon is the Enterobacteriaceae
bacteriophage P 1 lytic replicon. In certain embodiments, the replicon comprises a C 1
repressor-controlled P53 promoter, a promoter P53 antisense, a repL gene, and an in-frame
deletion of a kilA gene. In one embodiment, the replicon comprises of the sequence of SEQ
IDN0:3.
In yet another embodiment, the first and said second bacteriophage genes each
comprises a small terminase (terS) gene comprising said packaging initiation site sequence.
In one embodiment, the terS gene is a S. aureus bacteriophage cp 11 or cp80a terS gene.
In another embodiment, the replicon is derived from a S. aureus pTI 81 plasmid origin
of replication. In yet another embodiment, the replicon comprises the sequence of SEQ ID
N0:5. In some embodiments, the packaging initiation site sequence of said second
bacteriophage gene comprises a pac-site. In other embodiments, the pac-site of said second
bacteriophage gene comprises the sequence of SEQ ID N0:7. In one aspect, the packaging
initiation site sequence of said second bacteriophage gene comprises a cos-site. In another
aspect, the packaging initiation site sequence of said second bacteriophage gene comprises a
concatamer junction.
In another aspect, a plasmid comprises said reporter nucleic acid molecule. In one
aspect, the second bacteriophage gene is operatively linked to a promoter. In another
embodiment, the promoter is an inducible promoter or a constitutive promoter. In one
embodiment, the bacteriophage comprises the Enterobacteriaceae bacteriophage Pl. In yet
another embodiment, the bacteriophage comprises a S. aureus bacteriophage cp80a or a
bacteriophage cp 11. In one aspect, the bacterial cell comprises an E. coli cell. In another
aspect, the bacterial cell comprises an S. aureus cell. In yet another embodiment, the
bacterial cell comprises a Gram-negative cell. In other embodiments, the bacterial cell
comprises a Gram-positive cell.
In another aspect, the reporter nucleic acid molecule comprises a reporter gene. In
one aspect, the reporter gene encodes a detectable and/or a selectable marker. In certain
aspects, the reporter gene is selected from the group consisting of enzymes mediating
luminescence reactions (luxA, luxB, luxAB, luc, rue, nluc ), enzymes mediating colorimetric
reactions (lacZ, HRP), fluorescent proteins (GFP, eGFP, YFP, RFP, CFP, BFP, mCherry,
near-infrared fluorescent proteins), affinity peptides (His-tag, 3X-FLAG), and selectable
markers (ampC, tet(M), CAT, erm). In another aspect, the reporter nucleic acid molecule
comprises an aptamer. In yet another aspect, the reporter nucleic acid molecule comprises a
nucleic acid transcript sequence that is complementary to a second sequence in said reporter
nucleic acid molecule.
In one embodiment, the nucleic acid transcript sequence is complementary to a
cellular transcript. In another embodiment, the nucleic acid transcript sequence comprises a
cis-repressing sequence. In yet another embodiment, the replica of said reporter nucleic acid
molecule comprises a nucleic acid transcript sequence that is complementary to a second
sequence in said replica of said reporter nucleic acid molecule, wherein the nucleic acid
transcript sequence is complementary to a cellular transcript and wherein said nucleic acid
transcript sequence comprises a cis-repressing sequence.
In some embodiments, the method for packaging a reporter nucleic acid molecule into
a non-replicative transduction particle, comprising providing conditions to said bacterial cell
described herein that induce a lytic phase of said bacteriophage to produce non-replicative
transduction particles packaged with said reporter nucleic acid molecule; and isolating said
non-replicative transduction particle comprising said reporter nucleic acid molecule. In one
embodiment, the non-replicative transduction particle does not contain a replicated
bacteriophage genome. In another embodiment, induction of said lytic phase triggers excision
of said genomic island nucleic acid molecule from said genome of said bacterial cell.
In another embodiment, the composition comprising said non-replicative transduction
particle comprising a replica of said reporter nucleic acid molecule produced from the
method described herein.
The invention comprises a bacterial cell packaging system for packaging a reporter
nucleic acid molecule into a non-replicative transduction particle, said bacterial cell
comprising a lysogenized bacteriophage genome comprising a first bacteriophage packaging
initiation site sequence, wherein said first bacteriophage packaging initiation site sequence
comprises a mutation that prevents packaging of a bacteriophage nucleic acid molecule into
said non-replicative transduction particle; and a reporter nucleic acid molecule comprising a
second bacteriophage packaging initiation site sequence, wherein said second bacteriophage
packaging initiation site sequence lacks said mutation and facilitates the packaging of a
replica of said reporter nucleic acid molecule into said non-replicative transduction particle,
wherein said replica of said reporter nucleic acid molecule forms a replicon for packaging
into said non-replicative transduction particle.
In one embodiment, the reporter nucleic acid molecule is operatively linked to a
promoter. In another embodiment, the promoter is selected for contributing to reactivity of a
reporter molecule expressed from said reporter nucleic acid molecule in said bacterial cell. In
yet another embodiment, the reporter nucleic acid molecule comprises an origin of
replication. In one embodiment, the replicon comprises a concatamer amenable to packaging
into said non-replicative transduction particle. In another aspect, the first and said second
bacteriophage packaging initiation site sequences each comprise a packaging initiation site
sequence from a small terminase gene. In one aspect, the first and said second bacteriophage
packaging initiation site sequences each comprise a pac-site sequence from a pacA gene of
the Enterobacteriaceae bacteriophage Pl. In another aspect, the first bacteriophage packaging
initiation site sequence comprises SEQ ID N0:2. In yet another aspect, the second
bacteriophage packaging initiation site sequence comprises SEQ ID NO: 1. In one
embodiment, the replicon comprises an Enterobacteriaceae bacteriophage Pl lytic replicon.
In another embodiment, the replicon comprises a C 1 repressor-controlled P53 promoter, a
promoter P53 antisense, a repL gene, and an in-frame deletion of a kilA gene. In another
aspect, the replicon comprises the sequence of SEQ ID N0:3. In certain aspects, the first and
said second bacteriophage packaging initiation site sequences each comprise a pac-site
sequence from a small terminase (terS) gene of an S. aureus bacteriophage cpl l or cp80a. In
another aspect, the replicon is derived from a S. aureus pT181 plasmid origin ofreplication.
In yet another aspect, the replicon comprises the sequence of SEQ ID N0:5. In one aspect,
the first bacteriophage packaging initiation site sequence comprises the sequence of SEQ ID
N0:2. In some embodiments, the second bacteriophage packaging initiation site sequence
comprises the sequence of SEQ ID NO: 1. In other embodiments, the packaging initiation site
sequence comprises a pac-site. In another embodiment, the packaging initiation site sequence
comprises a cos-site. In yet another embodiment, the packaging initiation site sequence
comprises a concatamer junction. In some embodiments, the mutation in said first
bacteriophage packaging initiation site sequence comprises a silent mutation. In another
embodiment, the mutation in said first bacteriophage packaging initiation site sequence
prevents cleavage of said packaging initiation sequence. In another embodiment, a plasmid
comprises said reporter nucleic acid molecule. In one embodiment, the bacteriophage
comprises Enterobacteriaceae bacteriophage Pl.
In another embodiment, the bacteriophage comprises the S. aureus bacteriophage cpl l
or cp80a. In one embodiment, the bacterial cell comprises an E. coli cell. In another
embodiment, the bacterial cell comprises an S. aureus cell. In some embodiments, the
bacterial cell comprises a Gram-negative bacterial cell. In one aspect, the bacterial cell
comprises a Gram-positive bacterial cell. In another aspect, the reporter nucleic acid molecule
comprises a reporter gene. In yet another aspect, the reporter gene encodes a detectable
marker and/or a selectable marker.
In other aspects, the reporter gene is selected from the group consisting of: genes
encoding enzymes mediating luminescence reactions (luxA, luxB, luxAB, luc, rue, nluc ),
genes encoding enzymes mediating colorimetric reactions (lacZ, HRP), genes encoding
fluorescent proteins (GFP, eGFP, YFP, RFP, CFP, BFP, mCherry, near-infrared fluorescent
proteins), nucleic acid molecules encoding affinity peptides (His-tag, 3X-FLAG), and genes
encoding selectable markers (ampC, tet(M), CAT, erm). In another aspect, the reporter
nucleic acid molecule comprises an aptamer. In other aspects, the replicon is packaged into
said non-replicative transduction particle by bacteriophage packaging machinery. In some
embodiments, the reporter nucleic acid molecule comprises a nucleic acid transcript sequence
that is complementary to a second sequence in said reporter nucleic acid molecule. In another
embodiment, the nucleic acid transcript sequence is complementary to a cellular transcript.
In one aspect, the nucleic acid transcript sequence comprises a cis-repressing
sequence. In another aspect, the replica of said reporter nucleic acid molecule comprises a
nucleic acid transcript sequence that is complementary to a second sequence in said replica of
said reporter nucleic acid molecule, wherein said nucleic acid transcript sequence is
complementary to a cellular transcript, and wherein said nucleic acid transcript sequence
. . .
compnses a c1s-repressmg sequence.
In certain aspects, the method for packaging a reporter nucleic acid molecule into a
non-replicative transduction particle, comprising: providing conditions to said bacterial cell
described herein that induce a lytic phase of said bacteriophage to produce non-replicative
transduction particles packaged with said reporter nucleic acid molecule; and isolating said
non-replicative transduction particle comprising said reporter nucleic acid molecule.
In other aspects, the non-replicative transduction particle does not contain a replicated
bacteriophage genome. In one aspect, the induction of said lytic phase triggers excision of
said genomic island nucleic acid molecule from said genome of said bacterial cell.
In another aspect, the invention comprises a composition comprising said non
replicative transduction particle comprising a replica of said reporter nucleic acid molecule
produced from said method described herein.
In one aspect, the invention includes a bacterial cell packaging system for packaging a
reporter nucleic acid molecule into a non-replicative transduction particle, said bacterial cell
comprising: a lysogenized bacteriophage genome comprising a first bacteriophage gene
comprising a deletion of a packaging initiation site sequence of said first bacteriophage gene
that prevents packaging of a bacteriophage nucleic acid molecule into said non-replicative
transduction particle; and a reporter nucleic acid molecule comprising a second bacteriophage
gene comprising a second packaging initiation site sequence that facilitates the packaging a
replica of said reporter nucleic acid molecule into said non-replicative transduction particle,
wherein said second bacteriophage gene encodes a protein, wherein said replica of said
reporter nucleic acid molecule forms a replicon for packaging into said non-replicative
transduction particle.
In another aspect, the reporter nucleic acid molecule is operatively linked to a
promoter. In one aspect, the promoter is selected for contributing to reactivity of a reporter
molecule expressed from said reporter nucleic acid molecule in said bacterial cell. In certain
aspects, the reporter nucleic acid comprises an origin of replication. In another aspect, the
replicon comprises a concatamer amenable to packaging into said non-replicative
transduction particle. In one aspect, the first and said second bacteriophage genes each
comprises a pacA gene of the Enterobacteriaceae bacteriophage Pl and comprise said
packaging initiation site sequence. In another aspect, the first bacteriophage gene comprises
the sequence of SEQ ID N0:6. In certain aspects, the second bacteriophage gene comprises
the sequence SEQ ID N0:7. In one aspect, the replicon comprises an Enterobacteriaceae
bacteriophage P 1 lytic replicon. In yet another aspect, the replicon comprises a C 1
repressor-controlled P53 promoter, a promoter P53 antisense, a repL gene, and an in-frame
deletion of a kilA gene. In another aspect, the replicon comprises the sequence of SEQ ID
N0:3. In other aspects, the first and said second bacteriophage genes each comprises a small
terminase (terS) gene comprising said packaging initiation site sequence. In one aspect, the
terS gene is a S. aureus bacteriophage cpl I or cp80a terS gene. In another aspect, the first
bacteriophage gene comprises the sequence of SEQ ID N0:8. In yet another aspect, the
second bacteriophage gene comprises the sequence of SEQ ID N0:9. In one aspect, the
replicon is derived from a S. aureus pT181 plasmid origin ofreplication. In one embodiment,
the replicon comprises the sequence of SEQ ID N0:5, In another embodiment, the packaging
initiation site sequence of said second bacteriophage gene comprises a pac-site. In yet another
embodiment, the packaging initiation site sequence of said second bacteriophage gene
comprises a cos-site.
In certain embodiments, the packaging initiation site sequence of said second
bacteriophage gene comprises a concatamer junction. In one embodiment, a plasmid
comprises said reporter nucleic acid molecule. In another embodiment, the second
bacteriophage gene is operatively linked to a promoter. In yet another embodiment, the
promoter is an inducible promoter or a constitutive promoter. In certain embodiments, the
bacteriophage comprises the Enterobacteriaceae bacteriophage Pl. In one embodiment, the
bacteriophage comprises the S. aureus bacteriophage cp80a or bacteriophage cp 11. In other
embodiments, the bacterial cell comprises an E. coli cell. In another embodiment, the
bacterial cell comprises an S. aureus cell. In one embodiment, the bacterial cell comprises a
Gram-negative cell. In another embodiment, the bacterial cell comprises a Gram-positive cell.
In another aspect, the reporter nucleic acid molecule comprises a reporter gene. In
one aspect, the reporter gene encodes a detectable and/or a selectable marker. In another
aspect, the reporter gene is selected from the group consisting of genes encoding enzymes
mediating luminescence reactions (luxA, luxB, luxAB, luc, rue, nluc ), genes encoding
enzymes mediating colorimetric reactions (lacZ, HRP), genes encoding fluorescent proteins
(GFP, eGFP, YFP, RFP, CFP, BFP, mCherry, near-infrared fluorescent proteins), nucleic
acid molecules encoding affinity peptides (His-tag, 3X-FLAG), and genes encoding
selectable markers (ampC, tet(M), CAT, erm). In one embodiment, the reporter nucleic acid
molecule comprises an aptamer. In another embodiment, the replicon is packaged into said
non-replicative transduction particle by bacteriophage packaging machinery. In yet another
embodiment, the reporter nucleic acid molecule comprises a nucleic acid transcript sequence
that is complementary to a second sequence in said reporter nucleic acid molecule. In one
embodiment, the nucleic acid transcript sequence is complementary to a cellular transcript.
In another embodiment, the nucleic acid transcript sequence comprises a cis-repressing
sequence. In certain embodiments, the replica of said reporter nucleic acid molecule
comprises a nucleic acid transcript sequence that is complementary to a second sequence in
said replica of said reporter nucleic acid molecule, wherein said nucleic acid transcript
sequence is complementary to a cellular transcript and wherein said nucleic acid transcript
. . .
sequence compnses a c1s-repressmg sequence.
The invention includes a method for packaging a reporter nucleic acid molecule into a
non-replicative transduction particle, comprising: providing conditions to said bacterial cell
of any of the claims 86-125 that induce a lytic phase of said bacteriophage to produce non
replicative transduction particles packaged with said reporter nucleic acid molecule; and
isolating said non-replicative transduction particle comprising said reporter nucleic acid
molecule. In one embodiment, the non-replicative transduction particle does not contain a
replicated bacteriophage genome. In another embodiment, the induction of said lytic phase
triggers excision of said genomic island nucleic acid molecule from said genome of said
bacterial cell.
In some aspects, the invention includes a composition comprising said non-replicative
transduction particle comprising a replica of said reporter nucleic acid molecule produced
from said method described herein.
In another aspect, the invention includes a bacterial cell packaging system for
packaging a reporter nucleic acid molecule into a non-replicative transduction particle, said
bacterial cell comprising: a lysogenized bacteriophage genome lacking a packaging gene and
comprising genes that encode proteins that form said non-replicative transduction particle;
and a genomic island nucleic acid molecule comprising a reporter nucleic acid molecule and
a packaging gene. In one aspect, the packaging gene comprises a small terminase (terS)
gene. terS gene comprises a S. aureus bacteriophage cp80a terS gene or a bacteriophage cpl 1
terS gene.
In one aspect, the terS gene comprises the sequence of SEQ ID N0:9. In another
aspect, the genomic island nucleic acid molecule comprises a SaPibov2 genomic island
nucleic acid molecule. In yet another aspect, the genomic island nucleic acid molecule is
selected from the group consisting of a SaPI, a SaPil, a SaPI2, a SaPibov 1 and a SaPibov2
genomic island nucleic acid molecule. In another embodiment, the reporter nucleic acid
molecule is operatively linked to a promoter. In yet another embodiment, the reporter nucleic
acid molecule comprises an origin of replication. In some embodiments, the bacteriophage
comprises a S. aureus bacteriophage cp80a or bacteriophage cp 11. In other embodiments, the
bacterial cell comprises an S. aureus cell. In one embodiment, the genomic island nucleic
acid molecule comprises an integrase gene and wherein said integrase gene encodes an
integrase protein for excising and integrating said genomic island nucleic acid molecule out
of and into a bacterial genome of said bacterial cell. In another embodiment, the integrase
gene comprises the sequence of SEQ ID NO: 10. In yet another embodiment, the genomic
island nucleic acid molecule is integrated into a bacterial genome of said bacterial cell.
In certain aspects, the genomic island nucleic acid molecule can be replicated and
forms molecule replicon that is amenable to packaging by the bacteriophage packaging
machinery in said bacterial cell. In another aspect, the nucleic acid molecule forms a
concatamer. In yet another aspect, the replicated genomic island nucleic acid molecule is
capable of being packaged into said non-replicative transduction particle. In certain aspects,
the packaging gene comprises a pac site sequence. In another aspect, the packaging gene
comprises a cos-site sequence. In yet another embodiment, the packaging gene comprises a
concatamer junction.
In other embodiments, the reporter nucleic acid molecule comprises a reporter gene.
In some embodiments, the reporter gene encodes a selectable marker and/or a selectable
marker. In another embodiment, the reporter gene is selected from the group consisting of
enzymes mediating luminescence reactions (luxA, luxB, luxAB, luc, rue, nluc ), enzymes
mediating colorimetric reactions (lacZ, HRP), fluorescent proteins (GFP, eGFP, YFP, RFP,
CFP, BFP, mCherry, near-infrared fluorescent proteins), affinity peptides (His-tag, 3X
FLAG), and selectable markers (ampC, tet(M), CAT, erm). In certain embodiments, the
reporter nucleic acid molecule comprises an aptamer. In other embodiments, the genomic
island nucleic acid molecule lacks an integrase gene. In another embodiment, the invention
includes a bacterial gene comprising an integrase gene operatively linked to a promoter and
wherein said integrase gene encodes an integrase protein for excising and integrating said
genomic island nucleic acid molecule out of and into a bacterial genome of said bacterial cell.
In one embodiment, the reporter nucleic acid molecule comprises a nucleic acid transcript
sequence that is complementary to a second sequence in said reporter nucleic acid molecule.
In other embodiments, the nucleic acid transcript sequence is complementary to a cellular
transcript. In yet other embodiments, the nucleic acid transcript sequence comprises a cis
repressing sequence. In another embodiment, the replica of said reporter nucleic acid
molecule comprises a nucleic acid transcript sequence that is complementary to a second
sequence in said replica of said reporter nucleic acid molecule. In other embodiments, the
nucleic acid transcript sequence is complementary to a cellular transcript. In other
embodiments, the nucleic acid transcript sequence comprises a cis-repressing sequence.
The invention includes a method for packaging a reporter nucleic acid molecule into a
non-replicative transduction particle, comprising: providing conditions to said bacterial cell
of any of the claims 130-160 that induce a lytic phase of said bacteriophage to produce non
replicative transduction particles packaged with said reporter nucleic acid molecule; and
isolating said non-replicative transduction particle comprising said reporter nucleic acid
molecule. In some embodiments, the non-replicative transduction particle does not contain a
replicated bacteriophage genome. In one embodiment, the induction of said lytic phase
triggers excision of said genomic island nucleic acid molecule from said genome of said
bacterial cell.
In another embodiment, the invention includes a composition comprising said non
replicative transduction particle comprising a replica of said reporter nucleic acid molecule
produced from said method described herein.
The invention also includes a method for detecting a presence or an absence of a
bacterial cell in a sample, comprising: introducing into a sample a non-replicative
transduction particle comprising a reporter gene encoding a reporter molecule and lacking a
bacteriophage genome under conditions such that said non-replicative transduction particle
can transduce said bacterial cell and wherein said reporter gene can be expressed in said
bacterial cell; providing conditions for activation of said reporter molecule; and detecting for
a presence or an absence of a reporter signal transmitted from said expressed reporter
molecule, wherein a presence of said reporter signal correctly indicates said presence of said
bacterial cell.
In one embodiment, the method achieves at least 80% specificity of detection with
reference to a standard, at least 90% specificity of detection with reference to a standard, or at
least 95% specificity of detection with reference to a standard. In another embodiment, the
method achieves at least 80% sensitivity of detection with reference to a standard, at least
85% sensitivity of detection with reference to a standard, or at least 90% sensitivity of
detection with reference to a standard, or at least 95% sensitivity of detection with reference
to a standard. In yet another embodiment, the method achieves at least 95% specificity of
detection and at least 90% sensitivity of detection with reference to a standard. In another
embodiment, the standard is a Gold standard. In yet another embodiment, the bacterial cell
comprises a Methicillin Resistant Staphylococcus aureus (MRSA) cell. In other
embodiments, the bacterial cell comprises a Methicillin Sensitive Staphylococcus aureus
(MSSA) cell.
In another embodiment, the reporter gene encodes a detectable or selectable marker.
In one embodiment, the reporter gene is selected from the group consisting of genes encoding
enzymes mediating luminescence reactions (luxA, luxB, luxAB, luc, rue, nluc ), genes
encoding enzymes mediating colorimetric reactions (lacZ, HRP), genes encoding fluorescent
proteins (GFP, eGFP, YFP, RFP, CFP, BFP, mCherry, near-infrared fluorescent proteins),
nucleic acid molecules encoding affinity peptides (His-tag, 3X-FLAG), and genes encoding
selectable markers (ampC, tet(M), CAT, erm). In one embodiment, the reporter gene is
operatively linked to a constitutive promoter.
In another aspect, the reporter signal can be detected from a sample at a limit of
detection (LoD) ofless than 1,000 colony forming units (CFU). In other aspects, the reporter
signal can be detected from a sample at a limit of detection (LoD) ofless than 100 colony
forming units (CFU). In one aspect, the reporter signal can be detected from a sample at a
limit of detection (LoD) ofless than 10 colony forming units (CFU). In other aspects, the
reporter signal can be detected from a sample at a LoD less than five CFU. In another aspect,
the reporter signal can be detected from a sample at a LoD of three or less CFU.
In one embodiment, the method includes providing an antibiotic to said sample at a
pre-determined concentration and detecting a presence or absence of said reporter signal to
determine whether said bacterial cell is resistant or sensitive to said antibiotic. In another
embodiment, the method includes providing varying pre-determined concentrations antibiotic
to said sample and detecting the amount of said reporter signal to determine the minimum
inhibitory concentration of said bacterial cell to said antibiotic.
In one aspect, the invention includes a composition comprising a nucleic acid
construct that encodes a nucleic acid reporter transcript that is capable of forming at least two
conformations comprising a first conformation that prevents reporter expression comprising
an intramolecular double stranded region comprising a first subsequence and a second
subsequence, and a second conformation that lacks said intramolecular double-stranded
region and allows reporter gene expression, wherein conversion between said first and second
conformations is mediated by competitive binding of a cellular transcript to said first and/or
said second subsequence.
In another aspect, the invention includes a non-replicative transduction particle
comprising said nucleic acid construct. In yet another aspect, the competitive binding of said
cellular transcript to said first and/or said second subsequence results in said second
conformation of said nucleic acid reporter construct. In one aspect, the first subsequence or
said second subsequence comprises a cis-repressing sequence. In another aspect, the cis
repressing sequence comprises a sequence that is complementary or substantially
complementary to a portion of said cellular transcript. In other aspects, the first subsequence
or said second subsequence comprises a reporter gene sequence. In yet another aspect, the
reporter gene sequence comprises a ribosome binding site. In other aspects, the reporter gene
sequence encodes a detectable molecule. In another aspect, the detectable marker comprises
a fluorescent molecule or an enzyme capable of mediating a luminescence or colorimetric
reaction. In one embodiment, the reporter gene sequence encodes a selectable marker. In
another embodiment, the selectable marker comprises an antibiotic resistance gene.
In other embodiments, the first subsequence and said second subsequence are located
cis to each other on said nucleic acid construct to form said intramolecular double stranded
region. In certain embodiments, the first subsequence and said second subsequence are
complementary or substantially complementary to each other to form said intramolecular
double stranded region. In one embodiment, the first subsequence or said second subsequence
of said first conformation comprises a transcriptional enhancer sequence, and wherein said
transcriptional enhancer sequence is upstream from a coding region of said reporter gene
sequence. In another embodiment, the first conformation of said nucleic acid reporter
transcript is capable of binding to a cleaving enzyme. In other embodiments, the first
conformation of said nucleic acid reporter transcript is a target for degradation by a cellular
enzyme. In other aspects, the first conformation comprises a non-binding intramolecular
region. In another aspect, the non-binding intramolecular region is located 3' of said first
subsequence and 5' of said second subsequence. In other aspects, the non-binding
intramolecular region comprises a sequence YUNR, wherein Y is a pyrimidine, U is a Uracil,
N is any nucleotide, and R is a purine.
In one embodiment the first subsequence or said second subsequence comprises a
modified sequence of said cellular transcript. In another embodiment, the modified sequence
comprises a nucleotide substitution. In yet another embodiment, the modified sequence
comprises a sequence insertion, a deletion or an inversion of said cellular transcript.
The method includes a composition comprising a nucleic acid construct that encodes a
nucleic acid reporter transcript comprising a gene reporter sequence and that is capable of
forming at least two conformations of said nucleic acid reporter transcript, a first unstable
conformation that prevents translation of said reporter gene sequence in said nucleic acid
reporter transcript, and a second stable conformation resulting from binding of said first
unstable conformation with a cellular transcript, said second stable secondary conformation
allowing translation of said reporter gene sequence of said nucleic acid reporter transcript.
In one embodiment, the composition comprises a non-replicative transduction particle
comprising said nucleic acid construct. In another embodiment, the cellular transcript binds
at a 3 'UTR sequence of said nucleic acid reporter transcript. In one embodiment, the second
stable secondary conformation is formed by cleavage of a portion of a sequence of said first
unstable secondary conformation. In another embodiment, the reporter gene sequence
encodes a detectable molecule. In some embodiments, the detectable marker comprises a
fluorescent molecule or an enzyme capable of mediating a luminescence or colorimetric
reaction. In other embodiments, the reporter gene sequence encodes a selectable marker. In
another embodiment, the selectable marker comprises an antibiotic resistance gene.
The invention also includes a composition comprising a nucleic acid construct that
encodes a nucleic acid reporter transcript comprising a reporter gene sequence and that is
capable of forming at least two conformations of said nucleic acid reporter transcript,
comprising a first conformation that prevents further transcription of said nucleic acid
construct, and a second conformation formed upon binding of said first conformation with a
cellular transcript, wherein said second conformation allows transcription of said nucleic acid
construct. In some embodiments, the composition comprises a non-replicative transduction
particle comprising said nucleic acid construct. In another embodiment, the nucleic acid
reporter transcript comprises a cis-repressing sequence.
In one embodiment, the nucleic acid reporter transcript comprises a reporter gene
sequence. In another embodiment, the first conformation forms from a binding of said cis
repressing sequence to said reporter gene sequence. In some embodiments, the first
conformation is a substrate for a cleaving enzyme. In one embodiment, the first
conformation of said nucleic acid reporter transcript comprises a sequence that forms a
transcription termination structure. In other embodiments, the binding of said cellular
transcript to said sequence that forms a transcription termination structure results in cleavage
of a portion of said nucleic acid reporter transcript and formation of said second
conformation.
The invention comprises a vector comprising a regulatory sequence operably linked to
a nucleic acid sequence that encodes said nucleic acid reporter transcript described herein.
The invention includes a method for detecting a target transcript in a cell, comprising:
introducing into said cell said nucleic acid reporter construct described herein; and detecting
the presence or absence of an output signal from said cell, wherein said presence of said
output signal indicates the presence of the target transcript in said cell. The method includes
detecting a presence of a bacterial cell based on detecting said presence of said target
transcript.
In one embodiment, the method for detecting a presence of a bacterial cell in a sample
comprising introducing into said sample said nucleic acid reporter construct described herein;
and detecting the presence or absence of an output signal from said sample, wherein said
presence of said output signal indicates the presence of the bacterial cell in said sample.
The invention comprises a kit, comprising a compartment for holding a sample
comprising a cell and said nucleic acid reporter construct described herein; and instructions
for detecting the presence or absence of an output signal from said sample, wherein the
presence of the output signal indicates the presence of a target transcript in said cell
The invention comprises a composition, comprising a non-replicative transduction
particle comprising a nucleic acid reporter construct, the nucleic acid reporter construct
comprising a first promoter operatively linked a reporter gene, wherein said first promoter is
capable of being induced by an inducer protein endogenous in a bacterial cell.
The invention includes a method for detecting a presence of a bacterial cell in a
sample comprising contacting said sample with a non-replicative transduction particle
comprising nucleic acid reporter construct comprising a first promoter operatively linked to a
reporter gene, wherein said first promoter is capable of being induced by an inducer protein
endogenous to said bacterial cell; and detecting the presence or absence of an output signal
from said reporter gene, wherein said presence of said output signal indicates the presence of
said bacterial cell in said sample.
In one embodiment, the first promoter is the same as an inducible promoter
operatively linked to a target nucleic acid molecule in said bacterial cell.
The invention comprises a composition, comprising a non-replicative transduction
particle comprising a nucleic acid reporter construct, the nucleic acid reporter construct
comprising a reporter gene that encodes a reporter molecule, the non-replicative transduction
particle capable of entering a bacterial cell; and a caged substrate that exogenous to said
bacterial cell that once un-caged is capable of reacting to said reporter molecule in said cell.
The invention comprises a method for detecting a presence of a bacterial cell in a
sample comprising contacting said sample with a caged substrate and a non-replicative
transduction particle comprising a nucleic acid reporter construct, the nucleic acid reporter
construct comprising a reporter gene that encodes a reporter molecule, the caged substrate
exogenous to said cell that once un-caged is capable of binding to said reporter molecule in
said bacterial cell; and detecting the presence or absence of an output signal from said
reporter molecule, wherein said presence of said output signal indicates the presence of said
bacterial cell in said sample.
In one embodiment, a target enzyme in said cell binds said caged substrate to produce
an un-caged substrate. In some embodiments, the un-caged substrate reacts with said reporter
molecule to produce said output signal.
The invention also includes a composition, comprising a non-replicative transduction
particle comprising a nucleic acid reporter construct, the nucleic acid reporter construct
encoding a switchable molecule capable of binding to a target molecule in a bacterial cell to
form a complex; and a substrate capable of penetrating said cell and binding said complex to
produce a detectable signal from said cell.
The invention includes a method for detecting a presence of a bacterial cell in a
sample comprising contacting said sample with a substrate and a non-replicative transduction
particle comprising a nucleic acid reporter construct encoding a switchable molecule, the
switchable molecule capable of binding a target molecule in said cell to form a complex, the
substrate capable of binding said complex to form a substrate-bound complex; and detecting
the presence or absence of an output signal from said substrate-bound complex, wherein said
presence of said output signal indicates the presence of said bacterial cell in said sample. In
one embodiment, the binding of said switchable molecule to said target molecule produces a
conformational change in said switchable molecule. In another embodiment, the
conformational change in said switchable molecule allows said substrate to bind to said
complex.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
These and other features, aspects, and advantages of the present invention will
become better understood with regard to the following description, and accompanying
drawings, where:
Figure 1 illustrates an example of the design and function of the silent
mutation/complementation-based Pl plasmid packaging system, according to an embodiment
of the invention.
Figure 2 illustrates a schematic of the pGWPIOOOl vector, according to an
embodiment of the invention.
Figure 3 illustrates an example of the design and function of a pac-site
deletion/complementation plasmid packaging system, according to an embodiment of the
invention.
Figure 4 illustrates a schematic of the pGW80A0001 vector, according to an
embodiment of the invention.
Figure 5 depicts the process for genomic island (GI) packaging by a bacteriophage,
according to an embodiment of the invention.
Figure 6 depicts an example of the design and function of a GI-based packaging
system, according to an embodiment of the invention.
Figure 7 depicts the design and function of a GI-based packaging system that lacks
the integrase gene, according to an embodiment of the invention.
Figure 8 depicts the design and function of a SaPibov2-based packaging system that
lacks the integrase gene, according to an embodiment of the invention.
Figure 9 depicts a system for the use of NRTPs for the detection of inducers to target
gene promoters within viable cells, according to an embodiment of the invention.
Figure 10 depicts a reporter system that includes a reporter nucleic acid molecule
(e.g., plasmid) that is constructed for detecting VanR, the inducer of the promoter of the
vancomycin resistance (vanA) gene in Enterococcus faecium (or E. faecalis), according to an
embodiment of the invention. The reporter plasmid carries a reporter gene that is operatively
linked to the vanA gene promoter.
Figure 11 depicts a reporter system that includes a reporter nucleic acid molecule
constructed for detecting TcdD, the inducer of the promoters of the toxins A and B genes
(tcdA and tcdB, respectively) of C. difficile, according to an embodiment of the invention.
The reporter nucleic acid molecule includes a reporter gene that is operatively linked to the
tcdA gene promoter.
Figure 12 depicts a reporter system that includes a reporter nucleic acid molecule is
constructed for detecting SarS, the inducer of the promoter of the Protein A gene (spa) in S.
aureus, according to an embodiment of the invention. The reporter nucleic acid molecule
includes the bacterial luciferase genes luxA and luxB operatively linked to the spa gene
promoter (P spa).
Figure 13 shows a reporter system that comprises a system for the detection of
intracellular enzymes within viable cells that employs caged substrate molecules that can be
un-caged by a target intracellular enzyme, according to an embodiment of the invention.
Figure 14 depicts the design and function of a ~-lactamase enzyme detection system,
according to an embodiment of the invention.
Figure 15 shows a reporter system for the detection of intracellular molecules within
viable cells that employs switchable molecules capable of generating a detectable signal upon
their binding to a target molecule, according to an embodiment of the invention.
Figure 16 depicts the design and function of a bacteriophage/switchable-aptamer
(SA)-based intracellular molecule reporter system, according to an embodiment of the
invention.
Figure 17 depicts an example of a system that uses a cis-repression mechanism that
can target the 5' UTR (untranslated region) of a reporter sequence on a reporter transcript,
according to an embodiment of the invention.
Figure 18 shows an example of a system for detecting the presence of a target
transcript in a cell that is based on a cis-repression mechanism targeting the ribosome binding
site (RBS) of a reporter sequence in a reporter transcript, according to an embodiment of the
invention.
Figure 19 illustrates an exemplary system for detecting the presence of a target
transcript in a cell that is based on a cis-repression mechanism targeting the coding region
(“AUG”) of a reporter sequence in a reporter transcript, according to an embodiment of the
invention.
Figure 20 illustrates an example system for detecting the presence of a target
transcript in a cell that is based on a repression mechanism using an unstable reporter
transcript, according to an embodiment of the invention.
Figure 21 shows the results of the transduction assay in which 36 tetracycline-
sensitive MRSA were exposed to transduction particles carrying pGW80A0001 and then
were spotted onto media plates containing 5 ug/mL of tetracycline, according to an
embodiment of the invention.
Figure 22 illustrates the luminescence measured from 80 clinical isolates of MRSA
and 28 clinical isolates of methicillin sensitive S. aureus (MSSA) transduced with the
transduction particle, according to an embodiment of the invention.
Figure 23 shows the results of S. aureus growth at 4, 8, 16, 32, 64, and 128 ug/mL of
cefoxitin.
Figure 24 shows the RLU values obtained by the NRTP assay in the presence of 4, 8,
16, 32, 64, and 128 ug/mL cefoxitin. The x-axis in Figure 24 is set at the MSSA RLU cutoff
value.
Figure 25 shows a secondary structure of the mecA transcript (SEQ ID NO: 16)
generated based on the lowest energy conformation calculated by MFold and visualized with
VARNA.
Figure 26 shows the terminal loop 23 (T23) of the mecA transcript (nucleotides 1,464-
1,519 of SEQ ID NO: 16) that contains a YUNR consensus sequence.
Figure 27 depicts a cis-repressing sequence added to the 5’ terminus of the luxAB
genes and designed to form a stem-loop structure that blocks the RBS sequence
(“AAGGAA”) of the luxA gene (nucleotides 1-61 of SEQ ID NO: 19).
Figure 28 shows a diagram of base pairing between the target transcript (nucleotides
1,464-1,519 of SEQ ID NO: 16) and the cis-repressing sequence of the reporter transcript
(nucleotides 1-61 of SEQ ID NO: 19).
Figure 29 shows an example of a target mecA gene sequence (SEQ ID NO: 15),
according to an embodiment of the invention.
Figure 30 shows an exemplary mecA transcript sequence that can be used for
designing a reporter transcript (SEQ ID NO:16), according to an embodiment of the
invention.
Figure 31 is an example of a luxAB gene loci DNA sequence (SEQ ID NO: 17) that
can be used for designing a reporter transcript, according to an embodiment of the invention.
Figure 32 is an example of a luxAB transcript sequence that can be used for
designing a reporter transcript (SEQ ID NO:18), according to an embodiment of the
invention.
Figure 33 is an example of a luxAB cis-repressed transcript sequence that can be
used in a reporter transcript (SEQ ID NO:19), according to an embodiment of the invention.
Figure 34 shows an example of a cell comprising a vector that encodes a reporter
transcript, where there is no endogenous mecA transcript in the cell, according to an
embodiment of the invention.
Figure 35 shows a vector introduced into a cell, where the vector encodes the
reporter transcript, which includes a cis-repressing sequence and a reporter sequence (luxA
and luxB genes). When the mecA transcript present in the cell binds to the cis-repressing
sequence, the inhibitory hairpin loop opens up and the RBS for the luxA gene is exposed.
Translation of the reporter sequences (luxA and luxB) can occur, resulting in the formation of
a luxAB enzyme. The luxAB enzyme produces a detectable luminescent signal. In this
manner, the transcript reporter vector reports the presence of endogenous mecA transcripts
within a cell.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
Terms used in the claims and specification are defined as set forth below unless
otherwise specified.
As used herein, “reporter nucleic acid molecule” refers to a nucleotide sequence
comprising a DNA or RNA molecule. The reporter nucleic acid molecule can be naturally
occurring or an artificial or synthetic molecule. In some embodiments, the reporter nucleic
acid molecule is exogenous to a host cell and can be introduced into a host cell as part of an
exogenous nucleic acid molecule, such as a plasmid or vector. In certain embodiments, the
reporter nucleic acid molecule can be complementary to a target gene in a cell. In other
embodiments, the reporter nucleic acid molecule comprises a reporter gene encoding a
reporter molecule (e.g., reporter enzyme, protein). In some embodiments, the reporter
nucleic acid molecule is referred to as a "reporter construct" or "nucleic acid reporter
construct."
A "reporter molecule" or "reporter" refers to a molecule (e.g., nucleic acid or
protein) that confers onto an organism a detectable or selectable phenotype. The detectable
phenotype can be colorimetric, fluorescent or luminescent, for exapmle. Reporter molecules
can be expressed from reporter genes encoding enzymes mediating luminescence reactions
(luxA, luxB, luxAB, luc, rue, nluc ), genes encoding enzymes mediating colorimetric
reactions (lacZ, HRP), genes encoding fluorescent proteins (GFP, eGFP, YFP, RFP, CFP,
BFP, mCherry, near-infrared fluorescent proteins), nucleic acid molecules encoding affinity
peptides (His-tag, 3X-FLAG), and genes encoding selectable markers (ampC, tet(M), CAT,
erm). The reporter molecule can be used as a marker for successful uptake of a nucleic acid
molecule or exogenous sequence (plasmid) into a cell. The reporter molecule can also be
used to indicate the presence of a target gene, target nucleic acid molecule, target intracellular
molecule, or a cell, as described herein. Alternatively, the reporter molecule can be a nucleic
acid, such as an aptamer or ribozyme.
In some aspects of the invention, the reporter nucleic acid molecule is operatively
linked to a promoter. In other aspects of the invention, the promoter can be chosen or
designed to contribute to the reactivity and cross-reactivity of the reporter system based on
the activity of the promoter in specific cells (e.g., specific species) and not in others. In
certain aspects, the reporter nucleic acid molecule comprises an origin of replication. In other
aspects, the choice of origin of replication can similarly contribute to reactivity and cross
reactivity of the reporter system, when replication of the reporter nucleic acid molecule
within the target cell contributes to or is required for reporter signal production based on the
activity of the origin of replication in specific cells (e.g., specific species) and not in others.
In some embodiments, the reporter nucleic acid molecule forms a replicon capable of being
packaged as concatameric DNA into a progeny virus during virus replication.
As used herein, a "target transcript" refers to a portion of a nucleotide sequence of
a DNA sequence or an mRNA molecule that is naturally formed by a target cell including
that formed during the transcription of a target gene and mRNA that is a product of RNA
processing of a primary transcription product. The target transcript can also be referred to as a
cellular transcript or naturally occurring transcript.
As used herein, the term "transcript" refers to a length of nucleotide sequence
(DNA or RNA) transcribed from a DNA or RNA template sequence or gene. The transcript
can be a cDNA sequence transcribed from an RNA template or an mRNA sequence
transcribed from a DNA template. The transcript can be protein coding or non-coding. The
transcript can also be transcribed from an engineered nucleic acid construct.
A transcript derived from a reporter nucleic acid molecule can be referred to as a
"reporter transcript." The reporter transcript can include a reporter sequence and a cis
repressing sequence. The reporter transcript can have sequences that form regions of
complementarity, such that the transcript includes two regions that form a duplex (e.g., an
intermolecular duplex region). One region can be referred to as a "cis-repressing sequence"
and has complementarity to a portion or all of a target transcript and/or a reporter sequence.
A second region of the transcript is called a "reporter sequence" and can have
complementarity to the cis-repressing sequence. Complementarity can be full
complementarity or substantial complementarity. The presence and/or binding of the cis
repressing sequence with the reporter sequence can form a conformation in the reporter
transcript, which can block further expression of the reporter molecule. The reporter
transcript can form secondary structures, such as a hairpin structure, such that regions within
the reporter transcript that are complementary to each other can hybridize to each other.
"Introducing into a cell," when referring to a nucleic acid molecule or exogenous
sequence (e.g., plasmid, vector, construct), means facilitating uptake or absorption into the
cell, as is understood by those skilled in the art. Absorption or uptake of nucleic acid
constructs or transcripts can occur through unaided diffusive or active cellular processes, or
by auxiliary agents or devices including via the use of bacteriophage, virus, and transduction
particles. The meaning of this term is not limited to cells in vitro; a nucleic acid molecule
may also be "introduced into a cell," wherein the cell is part of a living organism. In such
instance, introduction into the cell will include the delivery to the organism. For example, for
in vivo delivery, nucleic acid molecules, constructs or vectors of the invention can be injected
into a tissue site or administered systemically. In vitro introduction into a cell includes
methods known in the art, such as electroporation and lipofection. Further approaches are
described herein or known in the art.
A "transduction particle" refers to a virus capable of delivering a non-viral nucleic
acid molecule into a cell. The virus can be a bacteriophage, adenovirus, etc.
A "non-replicative transduction particle" refers to a virus capable of delivering a
non-viral nucleic acid molecule into a cell, but does not package its own replicated viral
genome into the transduction particle. The virus can be a bacteriophage, adenovirus, etc.
A "plasmid" is a small DNA molecule that is physically separate from, and can
replicate independently of, chromosomal DNA within a cell. Most commonly found as small
circular, double-stranded DNA molecules in bacteria, plasmids are sometimes present in
archaea and eukaryotic organisms. Plasmids are considered replicons, capable of replicating
autonomously within a suitable host.
A "vector" is a nucleic acid molecule used as a vehicle to artificially carry foreign
genetic material into another cell, where it can be replicated and/or expressed.
A "virus" is a small infectious agent that replicates only inside the living cells of
other organisms. Virus particles (known as virions) include two or three parts: i) the genetic
material made from either DNA or RNA molecules that carry genetic information; ii) a
protein coat that protects these genes; and in some cases, iii) an envelope of lipids that
surrounds the protein coat.
"MRSA" refers to Methicillin-resistant Staphylococcus aureus.
"MSSA" refers to Methicillin-sensitive Staphylococcus aureus.
The term "ameliorating" refers to any therapeutically beneficial result in the
treatment of a disease state, e.g., a disease state, including prophylaxis, lessening in the
severity or progression, remission, or cure thereof.
The term "in situ" refers to processes that occur in a living cell growing separate
from a living organism, e.g., growing in tissue culture.
The term "in vivo" refers to processes that occur in a living organism.
The term "mammal" as used herein includes both humans and non-humans and
include but is not limited to humans, non-human primates, canines, felines, murines, bovines,
equines, and porcines.
"G," "C," "A" and "U" each generally stand for a nucleotide that contains
guanine, cytosine, adenine, and uracil as a base, respectively. "T" and "dT" are used
interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine,
e.g., deoxyribothymine. However, it will be understood that the term "ribonucleotide" or
"nucleotide" or "deoxyribonucleotide" can also refer to a modified nucleotide, as further
detailed below, or a surrogate replacement moiety. The skilled person is well aware that
guanine, cytosine, adenine, and uracil may be replaced by other moieties without
substantially altering the base pairing properties of an oligonucleotide comprising a
nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide
comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or
uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the
nucleotide sequences of the invention by a nucleotide containing, for example, inosine.
Sequences comprising such replacement moieties are embodiments of the invention.
As used herein, the term "complementary," when used to describe a first
nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an
oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and
form a duplex structure under certain conditions with an oligonucleotide or polynucleotide
comprising the second nucleotide sequence, as will be understood by the skilled person.
Complementary sequences are also described as binding to each other and characterized by
binding affinities.
For example, a first nucleotide sequence can be described as complementary to a
second nucleotide sequence when the two sequences hybridize (e.g., anneal) under stringent
hybridization conditions. Hybridization conditions include temperature, ionic strength, pH,
and organic solvent concentration for the annealing and/or washing steps. The term stringent
hybridization conditions refers to conditions under which a first nucleotide sequence will
hybridize preferentially to its target sequence, e.g., a second nucleotide sequence, and to a
lesser extent to, or not at all to, other sequences. Stringent hybridization conditions are
sequence dependent, and are different under different environmental parameters. Generally,
stringent hybridization conditions are selected to be about 5°C lower than the thermal melting
point (Tm) for the nucleotide sequence at a defined ionic strength and pH. The Tm is the
temperature (under defined ionic strength and pH) at which 50% of the first nucleotide
sequences hybridize to a perfectly matched target sequence. An extensive guide to the
hybridization of nucleic acids is found in, e.g., Tijssen (1993) Laboratory Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes part I, chap.
2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays,"
Elsevier, N.Y. ("Tijssen"). Other conditions, such as physiologically relevant conditions as
may be encountered inside an organism, can apply. The skilled person will be able to
determine the set of conditions most appropriate for a test of complementarity of two
sequences in accordance with the ultimate application of the hybridized nucleotides.
This includes base-pairing of the oligonucleotide or polynucleotide comprising the
first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second
nucleotide sequence over the entire length of the first and second nucleotide sequence. Such
sequences can be referred to as "fully complementary" with respect to each other herein.
However, where a first sequence is referred to as "substantially complementary" with respect
to a second sequence herein, the two sequences can be fully complementary, or they may
form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon
hybridization, while retaining the ability to hybridize under the conditions most relevant to
their ultimate application. However, where two oligonucleotides are designed to form, upon
hybridization, one or more single stranded overhangs, such overhangs shall not be regarded
as mismatches with regard to the determination of complementarity. For example, a dsRNA
comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23
nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21
nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to
as "fully complementary" for the purposes described herein.
"Complementary" sequences, as used herein, may also include, or be formed
entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and
modified nucleotides, in as far as the above requirements with respect to their ability to
hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but not limited to, G:U
Wobble or Hoogstein base pairing.
The terms "complementary," "fully complementary" and "substantially
complementary" herein may be used with respect to the base matching between two strands
of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, between
complementary strands of a single stranded RNA sequence or a single stranded DNA
sequence, as will be understood from the context of their use.
As used herein, a "duplex structure" comprises two anti-parallel and substantially
complementary nucleic acid sequences. Complementary sequences in a nucleic acid
construct, between two transcripts, between two regions within a transcript, or between a
transcript and a target sequence can form a "duplex structure." In general, the majority of
nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both
strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a
modified nucleotide. The two strands forming the duplex structure may be different portions
of one larger RNA molecule, or they may be separate RNA molecules. Where the two
strands are part of one larger molecule, and therefore are connected by an uninterrupted chain
of nucleotides between the 3 '-end of one strand and the 5 '-end of the respective other strand
forming the duplex structure, the connecting RNA chain is referred to as a "hairpin loop."
Where the two strands are connected covalently by means other than an uninterrupted chain
of nucleotides between the 3 '-end of one strand and the 5 '-end of the respective other strand
forming the duplex structure, the connecting structure is referred to as a "linker." The RNA
strands may have the same or a different number of nucleotides. The maximum number of
base pairs is the number of nucleotides in the shortest strand of the duplex minus any
overhangs that are present in the duplex. Generally, the duplex structure is between 15 and
or between 25 and 30, or between 18 and 25, or between 19 and 24, or between 19 and 21,
or 19, 20, or 21 base pairs in length. In one embodiment the duplex is 19 base pairs in length.
In another embodiment the duplex is 21 base pairs in length. When two different siRNAs are
used in combination, the duplex lengths can be identical or can differ.
As used herein, the term "region of complementarity" refers to the region on the
antisense strand that is substantially complementary to a sequence, for example a target
sequence, as defined herein. Where the region of complementarity is not fully
complementary to the target sequence, the mismatches are most tolerated in the terminal
regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or
2 nucleotides of the 5' and/or 3' terminus.
The term percent "identity," in the context of two or more nucleic acid or
polypeptide sequences, refer to two or more sequences or subsequences that have a specified
percentage of nucleotides or amino acid residues that are the same, when compared and
aligned for maximum correspondence, as measured using one of the sequence comparison
algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to
persons of skill) or by visual inspection. Depending on the application, the percent "identity"
can exist over a region of the sequence being compared, e.g., over a functional domain, or,
alternatively, exist over the full length of the two sequences to be compared.
For sequence comparison, typically one sequence acts as a reference sequence to
which test sequences are compared. When using a sequence comparison algorithm, test and
reference sequences are input into a computer, subsequence coordinates are designated, if
necessary, and sequence algorithm program parameters are designated. The sequence
comparison algorithm then calculates the percent sequence identity for the test sequence(s)
relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the
local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981 ), by the
homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the
search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444
(1988), by computerized implementations of these algorithms (GAP, BESTFIT, PASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).
One example of an algorithm that is suitable for determining percent sequence
identity and sequence similarity is the BLAST algorithm, which is described in Altschul et
al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology Information
(www.ncbi.nlm.nih.gov/).
The term "sufficient amount" means an amount sufficient to produce a desired
effect, e.g., an amount sufficient to produce a detectable signal from a cell.
The term "therapeutically effective amount" is an amount that is effective to
ameliorate a symptom of a disease. A therapeutically effective amount can be a
"prophylactically effective amount" as prophylaxis can be considered therapy.
It must be noted that, as used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless the context clearly dictates
otherwise.
II. Lysogenic and Lytic Cycle of Viruses
Viruses undergo lysogenic and lytic cycles in a host cell. If the lysogenic cycle is
adopted, the phage chromosome can be integrated into the bacterial chromosome, or it can
establish itself as a stable plasmid in the host, where it can remain dormant for long periods
of time. If the lysogen is induced, the phage genome is excised from the bacterial
chromosome and initiates the lytic cycle, which culminates in lysis of the cell and the release
of phage particles. The lytic cycle leads to the production of new phage particles which are
released by lysis of the host.
Certain temperate phage can exhibit lytic activity, and the propensity for this may
vary with varying host bacteria. To illustrate this phenomenon, the lytic activity of two
temperate S. aureus phages on ten MRSA clinical isolates was examined via plaque assay
(Table 1). The phage cpl 1 exhibited lytic activity on 10 out of 10 clinical MRSA isolates and
cp80a exhibited lytic activity on six of the 10 clinical MRSA isolates. Thus, reporter assays
relying on the natural lysogenic cycle of phages can be expected to exhibit lytic activity
sporadically.
Table 1: Lytic activity (denoted by the letter "x") of the S. aureus temperate
phages pll and p80a on ten clinical MRSA isolates
MRSA isolate
<1>11 <l>80a
3 x x
4 x x
x x
7 x x
9 x x
x x
In addition, virus-based reporter assays, such as phage-based reporters, can suffer
from limited reactivity (i.e., analytical inclusivity) due to limits in the phage host range
caused by host-based and prophage-derived phage resistance mechanisms. These resistance
mechanisms target native phage nucleic acid that can result in the degradation or otherwise
inhibition of the phage DNA and functions. Such resistance mechanisms include restriction
systems that cleave phage DNA and CRISPR systems that inhibit phage-derived transcripts.
Both lytic activity and phage resistance can be inhibitory to assays based on
reporter phages. Lytic activity can inhibit signal by destroying or otherwise inhibiting the
cell in its ability to generate a detectable signal and thus affecting limits of detection by
reducing the amount of detectable signal or preventing the generation of a detectable signal.
Phage resistance mechanisms can limit the host range of the phage and limit the inclusivity of
the phage-based reporter, similarly affecting limits of detection by reducing the amount of
detectable signal or preventing the generation of a detectable signal. Both lytic activity and
phage resistance caused by the incorporation of phage DNA in a reporter phage can lead to
false-negative results in assays that incorporate these phage reporters.
III. Methods for Producing Non-Replicative Transduction Particles (NRTP)
A. Disruption/Complementation-Based Methods For ProducingNon-
Replicative Transduction Particles
1) Silent Mutation/Complementation Packaging System
The invention includes methods for producing NRTPs using a silent
mutation/complementation-based method.
This non-replicative transduction particle packaging system is based on
introducing a silent mutation into a component of the genome of a virus that is recognized by
the viral packaging machinery as the element from which genomic packaging is initiated
during viral production. Examples of such an element include the pac-site sequence of pac
type bacteriophages and the cos-site sequence of cos-type bacteriophages.
Because these packaging initiation sites are often found within coding regions of
genes that are essential to virus production, the silent mutation is introduced such that the
pac-site is no longer recognized as a site of packaging initiation by the viral packaging
machinery. At the same time, the mutation does not disrupt the gene in which the site is
encoded. By disrupting the packaging site sequence, the mutated virus is able to undergo a
lytic cycle, but is unable to package its genomic DNA into its packaging unit.
An exogenous reporter nucleic acid molecule, such as plasmid DNA, can be
introduced into a host cell that has been lysogenized with a viral genome with a mutated
packaging initiation site sequence. The exogenous reporter nucleic acid molecule can include
a native packaging initiation site sequence. The exogenous reporter nucleic acid molecule
can be introduced into the cell and replicated in the cell. When the mutated virus is
undergoing a lytic cycle, the expressed viral packaging machinery packages the exogenous
reporter nucleic acid molecule with the native packaging initiation site sequence into the viral
packaging unit. The viral genome is not packaged into the packaging unit because its
packaging initiation site sequence has been mutated. In certain embodiments, the mutation in
the packaging initiation site sequence comprises a silent mutation that prevents cleavage of
the packaging initiation sequence, but does not disrupt the expression of the gene product that
encompasses the packaging initiation site sequence. This produces non-replicative
transduction particles, e.g., viral structural components carrying the replicated exogenous
nucleic acid molecule.
An example of such a system is based on the bacteriophage Pl, a pac-type phage.
In an embodiment, a plasmid including a native P 1 pac site is transformed into a cell. The
cell is lysogenized with a Pl prophage genome. The Pl prophage genome includes a silent
mutation in the pac-site sequence encoded within the pacA gene of P 1. When the lytic cycle
of the prophage is induced, the system results in the production of Pl-based transduction
particles carrying the plasmid DNA. An example of a silent mutation that is suitable for this
system is described in U.S. Pub. No. 2005/0118719, filed on November 7, 2002, which is
incorporated by reference in its entirety. An example is also found in SEQ ID NO: 2, listed
below (Pl pac-site with silent mutations, lower case letters signify mutated bases).
Figure 1 illustrates an example of the design and function of the silent
mutation/complementation-based Pl plasmid packaging system 100, according to an
embodiment of the invention. In this system, an E.coli cell 101 is lysogenized with a Pl
prophage 102 that includes a silent mutation in its packaging initiation site sequence (e.g.,
pac-site). The cell is transformed with a plasmid containing the native pac-site 103, and the
plasmid is replicated in the cell to form plasmid concatamers 104. The plasmid can also
include a reporter gene that encodes a reporter molecule. When the lytic cycle of the Pl
prophage is induced, the Pl prophage is excised from the bacterial genome and the Pl
structural components, such as capsid proteins, 105 are expressed. The Pl structural
components only package DNA that contains a native pac-site (e.g., plasmid DNA), thus
producing non-replicative transduction particles carrying plasmid DNA 106 (e.g., a reporter
gene).
An example vector for use in the silent mutation/complementation-based Pl
plasmid packaging system is shown in Figure 2. Details about how to construct the strains
and vectors of the silent mutation/complementation-based Pl plasmid packaging system are
described in detail in Example 1 below.
2) Deletion/Complementation-Based Packaging System
The invention includes methods for producing NRTPs using a
deletion/ complementation-based method.
This non-replicative transduction particle packaging system is based on deletion
of a component of the genome of a virus that is recognized by the viral packaging machinery
as the element from which genomic packaging is initiated during viral production. Examples
of such an element include the pac-site sequence of pac-type bacteriophages and the cos-site
sequence of cos-type bacteriophages. These packaging initiation sites are often found within
coding regions of genes that are essential to virus production. In some embodiments, the
packaging initiation site alone is deleted, which allows the mutated virus to undergo a lytic
cycle but does not allow the virus to package its genomic DNA. For example, SEQ ID NO: 6
is an example of a P 1 pacA gene with a deleted pac-site sequence (lower case letters indicate
the deleted pac-site sequence). In other embodiments, the entire gene comprising the
packaging initiation site is deleted. For example, SEQ ID NO: 8 shows the deletion of the
terS gene (lower case characters show the deleted sequence).
In one example, a cell's genome is lysogenized with a viral genome where the
packaging initiation site has been deleted. A complementing plasmid is introduced into the
cell, and the plasmid DNA includes a gene with a packaging initiation site sequence that
complements the deleted packaging initiation site sequence in the viral genome. When the
mutated virus is undergoing a lytic cycle, the viral packaging proteins package a replicon of
the plasmid DNA into the packaging unit because of its packaging initiation site, and non
replicative transduction particles are produced carrying the replicated plasmid DNA.
In some embodiments, it is preferable that the deletion/complementation is
designed such that there is no homology between the mutated virus DNA and the
complementing exogenous DNA. This is because lack of homology between the mutated
virus DNA and the complementing exogenous DNA avoids the possibility of homologous
recombination between the two DNA molecules that can result in re-introduction of a
packaging sequence into the virus genome. To accomplish a lack of homology, one strategy
is to delete the entire gene that contains the packaging initiation site sequence from the virus
genome and then complement this gene with an exogenous DNA molecule that contains no
more than exactly the DNA sequence that was deleted from virus. In this strategy, the
complementing DNA molecule is designed to express the gene that was deleted from the
virus.
Another example of such a system is provided using the bacteriophage cp80a, a
pac-type phage. The phage genome is lysogenized in a host bacterial cell, and the phage
genome includes a small terminase gene where the pac-site of a pac-type prophage cp80a has
been deleted. A plasmid including a complementary small terminase gene with a native pac
site is transformed into the cell. When the lytic cycle of the lysogenized prophage is induced,
the bacteriophage packaging system packages plasmid DNA into progeny bacteriophage
structural components, rather than packaging the native bacteriophage DNA. The packaging
system thus produces non-replicative transduction particles carrying plasmid DNA.
Figure 3 illustrates an example of the design and function of a pac-site
deletion/complementation plasmid packaging system 300, according to an embodiment of the
invention. A bacterial cell 301 is lysogenized with a pac-type phage 302 that has its small
terminase (terS) gene deleted. The cell is transformed with a rolling circle replication
plasmid 303 that includes a small terminase gene that complements the terS gene deletion in
the phage. The small terminase gene contains the packaging initiation site sequence, e.g., a
pac-site. The plasmid 303 can also include a reporter gene that encodes a reporter molecule.
A protein complex comprising the small terminase and large terminase proteins is
able to recognize and cleave a double-stranded DNA molecule at or near the pac-site, and this
allows the plasmid DNA molecule to be packaged into a phage capsid. When the prophage
in the cell is induced, the lytic cycle of the phage produces the phage's structural proteins 304
and the phage's large terminase protein 305. The complementing plasmid is replicated, and
the small terminase protein 306 is expressed. The replicated plasmid DNA 307 containing
the terS gene (and the reporter gene) are packaged into phage capsids, resulting in non
replicative transduction particles carrying only plasmid DNA 308.Figure 4 shows an example
of a resulting vector used in the pac-site deletion/complementation plasmid packaging
system. Further details about the components and construction of pac-site
deletion/complementation plasmid packaging system are in Example 2 below.
B. Pathogenicity Island-Based Packaging System
Pathogenicity islands (PTis) are a subset of horizontally transferred genetic
elements known as genomic islands. There exists a particular family of highly mobile PTis
in Staphylococcus aureus that are induced to excise and replicate by certain resident
prophages. These PTis are packaged into small headed phage-like particles and are
transferred at frequencies commensurate with the plaque-forming titer of the phage. This
process is referred to as the SaPI excision replication- packaging (ERP) cycle, and the high
frequency SaPI transfer is referred to as SaPI-specific transfer (SPST) to distinguish it from
classical generalized transduction (CGT). The SaPis have a highly conserved genetic
organization that parallels that ofbacteriophages and clearly distinguishes them from all other
horizontally acquired genomic islands. The SaPil-encoded and SaPibov2-encoded integrases
of the corresponding elements, and it is
are required for both excision and integration
assumed that the same is true for the other SaPis. Phage 80a can induce several different
SaPis, including SaPI 1, SaPI2, and SaPibov 1, whereas cp 11 can induce SaPibov 1 but neither
of the other two SaPis.
Figure 5 depicts the natural process for genomic island (GI) packaging 500 by a
bacteriophage. In nature, a bacterial cell 501 lysogenized with a suitable prophage 503 and
carrying a GI 504 can produce phage particles carrying GI concatamers 512. In this process,
when the phage is induced into its lytic cycle, the phage genome is excised (not shown) from
the bacterial genome 502, which then expresses bacteriophage proteins including capsid
constituents 505 and the large terminase protein (TerL) 506. Prophage induction also triggers
GI excision via the expression of the GI integrase protein (int) 507. In a similar manner to
the excised phage genome (not shown), the GI circularizes 508, expresses its own small
terminase protein (TerS) 509, and begins to replicate forming a GI concatamer 510. The
phage TerL gene and GI TerS gene can then combine bind and cleave the GI concatamer via
a pac-site sequence in the GI genome, and the GI concatamer can then be packaged into
phage capsids 511 resulting in phage particles carrying GI concatamers 512.
In natural systems, as depicted in Figure 5, the resulting lysate produced from
phage production includes both native phage particles, as well as GI-containing phage
particles. The native phage particles are a result of packaging of the native phage genome
due to recognition of the pac-site within phage genome concatamers.
1) Genomic Island (GI) Packaging System Design and
Function
Methods of the invention for producing NRTPs include a GI based-packaging
system.
Compared to a plasmid packaging system, the natural GI-packaging system
benefits from the fact that the DNA that is packaged is derived from a genomic region within
the bacterial genome and thus does not require the maintenance of a plasmid by the bacterial
host.
In some embodiments, the invention includes a bacterial cell packaging system for
packaging a reporter nucleic acid molecule into a non-replicative transduction particle,
wherein the bacterial cell comprises a lysogenized bacteriophage genome lacking a
packaging gene, and a genomic island, cryptic phage, or other nucleic acid molecule
requiring a bacteriophage (e.g., a heper phage) for mobilization of the nucleic acid molecule
and comprising a reporter nucleic acid molecule and a packaging gene. Genomic island
based systems can be based on S. aureus Pathogenicity Islands (SaPis ), the E. coli criptic
phage P4 and helper phage P2, and the Enterococci criptic phage P7 and helper phage Pl, for
example.
GI-packaging systems can be exploited such that exogenous nucleic acid
sequences are packaged by the bacteriophage. This can be accomplished by incorporating
such exogenous nucleic acids sequences into the GI.
In order to eliminate the native phage from this process, the small terminase gene
of the prophage can be deleted. The small terminase gene sequence contains the pac-site
sequence of the native phage, and this deletion has the effect of preventing the packaging of
native phage DNA. In other embodiments, only the pac site of the small terminase gene can
be deleted. The GI that will be packaged includes its own pac-site and a small terminase
gene that expresses a suitable small terminase protein, and only GI DNA will be amenable for
packaging in this system.
Figure 6 depicts an example of the design and function of a GI-based packaging
system 600, according to an embodiment of the invention. In this system, a bacterial cell 601
has its genome lysogenized with a suitable prophage 603 that has its small terminase gene
deleted, and the cell's genome 602 carries a GI 604. When the phage is induced into its lytic
cycle, the phage genome is excised (not shown) from the bacterial genome 602. The phage
genome expresses bacteriophage proteins, including capsid constituents 605 and the large
terminase protein (TerL) 606. Prophage induction also triggers GI excision via the
expression of the GI integrase protein (int) 607. In a similar manner to the excised phage
genome (not shown), the GI circularizes 608 and expresses its own small terminase protein
(TerS) 609 and is replicated forming a GI concatamer 610. The phage TerL gene and GI
TerS gene can then combine, bind and cleave the GI concatamer via a pac-site sequence in
the GI DNA. The GI concatamer can then be packaged into phage capsids 611 resulting in
phage particles carrying GI concatamers 612. In this system, phage DNA will not be
packaged into phage particles, since it lacks the terS gene that contains the phage's pac-site
sequence, and thus cannot be recognized by the expressed GI TerS and phage TerL proteins.
When phage particles containing packaged GI DNA are administered to a
recipient cell, the phage will bind to the recipient cell's surface and then introduce the
packaged GI DNA concatamer into the cell. Once inside the cell, the GI can again express its
integrase protein, and the GI can then integrate into its specific site in the recipient cell's
genome. If exogenous DNA sequences are included in the GI prior to packaging, the
packaging system thus allows for delivering exogenous DNA sequences to a recipient cell
and integrating these exogenous DNA sequences into the recipient cell's genome.
2) GI-Based Packaging System Lacking Integrase
In another embodiment, the packaging system described above is designed such
that packaged GI DNA cannot integrate into a recipient cell's genome. This can be
accomplished by deleting the integrase gene in the GI and complementing the deletion by
causing the expression of the integrase gene in trans from the GI. In this manner, the
integrase protein is available for excision of the GI in the packaging host cell, and the GI
DNA that has been packaged in a bacteriophage does not contain the integrase gene and
cannot express the integrase protein, thus preventing integration of the delivered GI.
Figure 7 depicts the design and function of a GI-based packaging system that
lacks the int gene 700, according to an embodiment of the invention. In this system, a
bacterial cell 701 is lysogenized with a suitable prophage that has had its small terminase
gene deleted 703. The cell's genome 702 carries a GI that has its integrase (int) gene deleted
704 and also carries the deleted int gene operatively linked to a suitable promoter 705. The
int gene can thus express the integrase protein (Int) in trans from the GI 706. When the
phage is induced into its lytic cycle, the phage genome is excised (not shown) from the
bacterial genome 702, which then expresses bacteriophage proteins including capsid
constituents 707 and the large terminase protein (TerL) 708. Prophage induction also triggers
GI excision via the expression of the integrase protein 707. In a similar manner to the
excised phage genome (not shown), the excised GI circularizes 709, expresses its own small
terminase protein (TerS) 710, and begins to replicate forming a GI concatamer 711. The
phage TerL gene and GI TerS gene can then combine, bind and cleave the GI concatamer via
a pac-site sequence in the GI DNA, and the GI concatamer can then be packaged into phage
capsids 712 resulting in phage particles carrying GI concatamers 713. In this system, phage
DNA will not be packaged since it lacks the terS gene that contains the phage's pac-site
sequence and thus cannot be recognized by the expressed GI TerS and phage TerL proteins.
When phage particles containing packaged GI DNA lacking the int gene are
administered to a recipient cell, the phage will bind to the recipient cell's surface and then
introduce the packaged GI DNA concatamer into the cell. Once inside the cell, the GI cannot
express its integrase protein due to the lack of the integrase gene and the GI cannot then
integrate into its specific site in the recipient cell's genome. If exogenous DNA sequences
are included in the GI prior to packaging, the packaging system thus allows for delivering
exogenous DNA sequences to a recipient cell and the delivered DNA sequences do not
integrate into the recipient cell's genome at the specific site for GI integration.
3) Design and Function of SaPlbov2-Based Packaging Lacking
Integrase
In some embodiments, the method of producing NRTPs employ a GI SaPibov2
and a bacteriophage cp 11 in a GI-based packaging system. Alternative embodiments can
employ other SaPI GI' s and other suitable bacteriophages, including the SaPI' s SaPil, SaPI2,
SaPibovl, and SaPibov2 along with the bacteriophage 80a, and the SaPI's SaPibovl and
SaPibov2 along with the bacteriophage cp 11. Based on the description below, one of skill in
the art would know how to develop a GI-based packaging system that does not lack the int
gene, as described in Section II A.
Figure 8 depicts the design and function of a SaPibov2-based packaging system
800 that lacks the int gene, according to an embodiment of the invention. In this system, a S.
aureus cell 801 is lysogenized with cp 11 that has its small terminase gene deleted 803. The
cell's genome 802 carries SaPibov2 that has its integrase (int) gene deleted 804 and also
carries the deleted int gene operatively linked the constitutively expressed PclpB gene
promoter 805. The int gene can express the integrase protein (Int) in trans from SaPibov2
806. When the phage is induced into its lytic cycle, the phage genome is excised (not shown)
from the bacterial genome 802, which then expresses bacteriophage proteins including capsid
constituents 807 and the large terminase protein (TerL) 808. Prophage induction also triggers
SaPibov2 excision via the expression of the integrase protein 806. In a similar manner to the
excised phage genome (not shown), the excised SaPibov2 circularizes 809, expresses its own
small terminase protein (TerS) 810 and begins to replicate forming a SaPibov2 concatamer
811. The phage TerL gene and SaPibov2 TerS gene can then combine bind and cleave the
SaPibov2 concatamer via a pac-site sequence in the SaPibov2 DNA and the SaPibov2
concatamer can then be packaged into phage capsids 812 resulting in phage particles carrying
SaPibov2 concatamers 813. In this system, phage DNA will not be packaged since it lacks
the terS gene that contains the phage's pac-site sequence and thus cannot be recognized by
the expressed SaPibov2 TerS and phage TerL proteins.
IV. Reporters
In some embodiments, the NRTPs and constructs of the invention comprise a
reporter nucleic acid molecule including a reporter gene. The reporter gene can encode a
reporter molecule, and the reporter molecule can be a detectable or selectable marker. In
certain embodiments, the reporter gene encodes a reporter molecule that produces a
detectable signal when expressed in a cell.
In certain embodiments, the reporter molecule can be a fluorescent reporter
molecule, such as, but not limited to, a green fluorescent protein (GFP), enhanced GFP,
yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein
(BFP), red fluorescent protein (RFP) or mCherry, as well as near-infrared fluorescent
proteins.
In other embodiments, the reporter molecule can be an enzyme mediating
luminescence reactions (luxA, luxB, luxAB, luc, rue, nluc, etc.). Reporter molecules can
include a bacterial luciferase, a eukaryotic luciferase, an enzyme suitable for colorimetric
detection (lacZ, HRP), a protein suitable for immunodetection, such as affinity peptides (His
tag, 3X-FLAG), a nucleic acid that function as an aptamer or that exhibits enzymatic activity
(ribozyme), or a selectable marker, such as an antibiotic resistance gene (ampC, tet(M), CAT,
erm). Other reporter molecules known in the art can be used for producing signals to detect
target nucleic acids or cells.
In other aspects, the reporter molecule comprises a nucleic acid molecule. In
some aspects, the reporter molecule is an aptamer with specific binding activity or that
exhibits enzymatic activity (e.g., aptazyme, DNAzyme, ribozyme).
Reporters and reporter assays are described further in Section V herein.
V. NRTPs and Reporter Assays
A. Inducer Reporter Assay
The invention comprises methods for the use ofNRTPs as reporter molecules for
use with endogenous or native inducers that target gene promoters within viable cells. The
NRTPs of the invention can be engineered using the methods described in Section III and
below in Examples 1-6.
In some embodiments, the method comprises employing a NRTP as a reporter,
wherein the NR TP comprises a reporter gene that is operably linked to an inducible promoter
that controls the expression of a target gene within a target cell. When the NR TP that
includes the reporter gene is introduced into the target cell, expression of the reporter gene is
possible via induction of the target gene promoter in the reporter nucleic acid molecule.
Figure 9 depicts a genomic locus of a target cell 900 with two genes, a gene
encoding an inducer 902 and a target gene 903. Also depicted is a reporter nucleic acid
molecule 904 that includes a reporter gene 905 that is operatively linked to the promoter 906
of the target gene of the target cell. The reporter nucleic acid molecule 904 can be introduced
into the cell via a NR TP. In the native cell, when the inducer gene 902 is expressed and
produces the inducer protein 907, the inducer protein 907 is able to induce the target gene
promoter 906 that is operatively linked to the target gene, thus causing the expression of the
target gene and the production of the target gene product 908.
When the reporter nucleic acid molecule 904 is present within the target organism,
the inducer 907 is also able to induce the target gene promoter 906 present within the reporter
nucleic acid molecule 904, thus causing the expression of the reporter gene 905 resulting in
the production of a reporter molecule 909 capable of generating a detectable signal.
Thus, the production of a detectable signal from the reporter molecule 909 is
indicative of the presence of the cell, based on the presence of the inducer protein 907 within
a target cell.
1) VanR Reporter System
In one embodiment, the reporter system includes NRTP comprising a reporter
nucleic acid molecule (e.g., plasmid). The reporter nucleic acid molecule can be constructed
for detecting VanR, the inducer of the promoter of the vancomycin resistance (vanA) gene in
Enterococcus faecium (or E. faecal is). The reporter plasmid carries a reporter gene that is
operatively linked to the vanA gene promoter.
Figure 10 outlines the design and function of a VanR reporter system. Figure 10
depicts a region of the transposon Tn1546 1001 that may be present in E. faecium. The
Tn1546 transposon can include the vanR inducer gene 1002 and the vanA target gene 1003.
Also depicted in the figure is a reporter nucleic acid molecule 1004 that can be packaged in a
NRTP and introduced into the cell. The reporter nucleic acid molecule 1004 includes a
reporter gene 1005 that is operatively linked to a promoter PH 1006 that controls the
expression of the vanHAX operon that includes the vanA gene. In the native cell, when the
vanR gene 1002is expressed and produces the VanR protein 1007, VanR is able to induce PH
1006 in the Tnl 546 transposon, thus causing the expression of the vanA gene and thus
producing the VanA protein 1008. When the reporter nucleic acid molecule 1003 (vector) is
present within the target organism, VanR is also able to induce PH 1006 within the reporter
nucleic acid molecule 1003, thus causing the expression of a reporter molecule 1009. Thus,
the production of a reporter molecule is indicative of the presence of VanR within a target
cell.
Examples of promoters that are suitable for the development of a VRE assay
include: the vanA gene promoter and a vanB gene promoter. Arthur, M., et al., The VanS
sensor negatively controls VanR-mediated transcriptional activation of glycopeptide
resistance genes of Tnl 546 and related elements in the absence of induction. J. Bacteriol.,
1997. 179(1):p. 97-106.
2) TcdD reporter system
In another embodiment of this system, a reporter nucleic acid molecule is
introduced into a cell using a NRTP. The reporter nucleic acid molecule can be constructed
for detecting TcdD, the inducer of the promoters of the toxins A and B genes (tcdA and tcdB,
respectively) of C. difficile. The reporter nucleic acid molecule includes a reporter gene that
is operatively linked to the tcdA gene promoter.
Figure 11 outlines the design and function of a TcdD reporter system, according to
an embodiment of the invention. Figure 11 depicts a region of the transposon PaLoc 1101
that may be present in C. difficile. The PaLoc transposon may contain the tcdD gene 1102
and the tcdA target gene 1103. Also depicted in the figure is a reporter nucleic acid molecule
1104 (e.g., vector) that is introduced into the cell using a NRTP. The reporter nucleic acid
molecule 1104 includes the reporter gene 1105 that operatively linked to the tcdA gene
promoter (PtcdA) 1106.
In the native cell, when the tcdD gene is expressed and produces the TcdD protein
1107, TcdD is able to induce PtcdA 1106 in the PaLoc transposon 1101, thus causing the
expression of the tcdA gene 1103 and thus producing the toxin A protein 1108.
When the reporter nucleic acid molecule 1104 is present within the target
organism, TcdD is also able to induce PtcdA 1106 within the reporter vector, thus causing the
expression of a reporter molecule 1109. Thus, the production of a reporter molecule 1109 is
indicative of the presence ofTcdD within a target cell.
Examples of promoters suitable for the development of a C. difficile assay
include: the tcdA gene promoter and the tcdB gene promoter. Karlsson, S., et al., Expression
ofClostridium difficile Toxins A and Band Their Sigma Factor TcdD Is Controlled by
Temperature. Infect. Immun., 2003. 71(4): p. 1784-1793.
Target cells and inducers: Target cells can include eukaryotic and prokaryotic cell
targets and associated inducers.
Vector delivery systems: The delivery of the vector containing the recombinant
DNA can by performed by abiologic or biologic systems. Including but not limited to
liposomes, virus-like particles, transduction particles derived from phage or viruses, and
conjugation.
3) Bacteriophage-based SarS reporter system
In another embodiment of the invention, a reporter nucleic acid molecule is
constructed for detecting SarS, the inducer of the promoter of the Protein A gene (spa) in S.
aureus. The reporter nucleic acid molecule can be introduced into the cell in a NRTP and
includes the bacterial luciferase genes luxA and luxB operatively linked to the spa gene
promoter (P spa). The reporter nucleic acid molecule is delivered to S. aureus via a NR TP, for
example. If SarS is present in the cell, it will induce the expression of the luxAB genes, thus
producing luciferase enzyme that is capable of generating a luminescent signal.
Figure 12 outlines the design and function of a SarS reporter system, according to
one embodiment of the invention. Figure 12 depicts a region of the S. aureus genome 1201
that contain the sarS gene 1202 and spa gene 1203. Also depicted in the figure is a reporter
nucleic acid molecule (e.g., vector) 1204 delivered by NRTP to the cell and that includes the
luxAB reporter genes 1205 that operatively linked to the promoter Pspa 1206 that controls the
expression of the spa gene 1203.
In the native cell, when the sarS gene 1202 is expressed, producing SarS protein
1207, the protein is able to induce Pspa 1206 in the S. aureus genome transposon, thus causing
the expression of the spa gene 1203 and producing the Protein A 1208.
When the reporter nucleic acid molecule 1204 is present within the target
organism, SarS 1207 is also able to induce Pspa 1206 within the reporter nucleic acid molecule
1204, thus causing the expression ofluxAB resulting in the production of the luciferase
enzyme 1209 that can generate a luminescent signal. Thus, the production of luciferase is
indicative of the presence of SarS within a target cell.
B. Enzyme Reporter Assay
The invention comprises a system for the detection of intracellular enzymes
within viable cells that employs caged substrate molecules that can be un-caged by a target
intracellular enzyme, according to an embodiment of the invention.
Figure 13 depicts the design and function of an intracellular enzyme detection
system. A reporter molecule-expressing vector 1301 is delivered to a target cell 1302 with a
NRTP (not shown). The reporter molecule-expressing vector 1301 is able to penetrate the
target cell 1302 via the NRTP and deliver a reporter molecule gene 1303 into the target cell
1302, and a reporter molecule 1304 can then be expressed from the reporter molecule gene
1303. A caged substrate 1305 is also added to the target cell 1302 and is able to penetrate
into the target cell 1302. If a target intracellular enzyme 1307 is present in the target cell
1306, the enzyme 1307 is able to remove the caging component of the caged substrate 1305,
thus producing an un-caged substrate 1308. The un-caged substrate 1308 can then react with
the reporter molecule 1304 inside of the cell 1302, and the product of this reaction results in a
detectable signal 1309.
Target cells and enzymes: Target cells can include eukaryotic and prokaryotic
cell targets and associated enzymes, including, for example, ~-lactamases in S. aureus.
Vector delivery systems: The delivery of the vector containing the recombinant
DNA can by performed by abiologic or biologic systems. Including but not limited to
liposomes, virus-like particles, transduction particles derived from phage or viruses, and
conjugation.
Reporter molecules and caged substrates: Various reporter molecules and
caged substrates can be employed as those described in Daniel Sobek, J.R., Enzyme detection
system with caged substrates, 2007, Zymera, Inc.
1) Bacteriophage-based P-lactamase Reporter
In one embodiment, a reporter molecule-expressing vector can be carried by a
NRTP, such that the vector can be delivered into a bacterial cell. The reporter molecule to be
expressed can be Renilla luciferase, and the caged substrate can be Renilla luciferin that is
caged, such that a ~-lactamase enzyme that is endogenous to the target cell is able to cleave
the caging compound from the caged luciferin and release un-caged luciferin.
Figure 14 depicts the design and function of a ~-lactamase enzyme detection
system, according to an embodiment of the invention. A Renilla luciferase-expressing vector
carried by a bacteriophage-based NRTP 1401 is added to a target S. aureus cell 1402. The
Renilla luciferase-expressing vector is able to penetrate the target cell 1402 using a NRTP
comprising the vector. The NRTP delivers the Renilla luciferase gene 1403 into the target
cell 1402, and Renilla luciferase 1404 can then be expressed from its gene. Caged Renilla
luciferin 1405 is also added to the target cell 1402 and is able to penetrate into the target cell
1402. If an intracellular ~-lactamase 1407 is present in the target cell 1402, the enzyme is
able to remove the caging component of the caged luciferin 1406, thus producing an un
caged luciferin 1408. The un-caged luciferin1408 can then react with the Renilla luciferase
1404 inside of the cell 1402, and the product of this reaction results in luminescence 1409.
In this manner, when a target cell that contains the ~-lactamase is exposed to the
NR TP and caged luciferin, the cell will exhibit a luminescent signal that is indicative of the
presence of the ~-lactamase present in the cell.
C. Intracellular Molecule Reporter
The invention includes a system for the detection of intracellular molecules within
viable cells that employs switchable molecules capable of generating a detectable signal upon
their binding to a target molecule.
Figure 15 depicts the design and function of a switchable molecule (SM)-based
intracellular molecule detection system. A SM-expressing vector 1501 is delivered to a target
cell 1502 in a NRTP. The SM-expressing vector 1501 is able to penetrate the target cell 1502
and deliver a SM gene 1503 into the target cell 1502. A SM protein 1504 can then be
expressed from the SM gene 1503. The SM protein 1504 can then bind to a target molecule
1505 inside of the cell and thus forms an SM-target molecule complex 1506. The binding of
the SM 1504 to the target molecule 1505 results in a conformational change in the SM 1504
that makes the bound SM amenable to binding of a substrate. A substrate 1508 is added to
the cell 1507 and is able to penetrate into the cell 1502. Bound SM inside of the cell 1502 is
able to also bind the substrate, thus forming a SM-target molecule-substrate complex 1509.
Finally, the binding of the substrate 1508 by the target molecule-bound SM has the effect of
producing a detectable signal 1510. Thus a detectable signal generated by the system is
indicative of the presence of a target molecule inside of a cell.
Target cells and molecules: Various eukaryotic and prokaryotic cell targets can
be employed and switchable aptamer-based SM's can be designed to target various nucleic
acid and amino acid-based intracellular molecular targets as described in Samie Jaffrey, J.P.,
Coupled recognition/detection system for in vivo and in vitro use, 2010, Cornell University.
Vector delivery systems: The delivery of the vector containing the recombinant
DNA can by performed by abiologic or biologic systems. Including but not limited to
liposomes, virus-like particles, transduction particles derived from phage or viruses, and
conjugation.
1) Non-Replicative Transduction Particle/Switchable Aptamer-
Based Intracellular Molecule Reporter System
In one example of this method, a switchable molecule-expressing vector can be
carried by a bacteriophage-based transduction particle such that the vector can be delivered
into a bacterial cell. The switchable molecule to be expressed can be a switchable aptamer
that is designed to undergo a conformational change upon its binding to an intracellular target
molecule. The conformational change allows the aptamer to then bind a fluorophore that
exhibits enhanced fluorescence when bound by the aptamer.
Figure 16 depicts the design and function of a bacteriophage/switchable-aptamer
(SA)-based intracellular molecule reporter system. A SA-expressing vector carried by a
NRTP 1601 is added to a target cell 1602. The NRTP 1601 is able to deliver the SA
expressing vector and the SA-expressing gene 1603 into the target cell 1602. An SA protein
1604 can then be expressed from the SA gene 1603. The SA protein 1604 can then bind to a
target molecule 1605 inside of the cell and thus form an SA-target molecule complex 1606.
The binding of the SA 1604 to the target molecule 1605 results in a conformational change in
the SA that makes the bound SA amenable to binding of a fluorophore 1608. A fluorophore
1607 is added to the cell and is able to penetrate into the cell 1608. Bound SA inside of the
cell is able to also bind the fluorophore thus forming an SA-target molecule-fluorophore
complex 1609. Finally, the binding of the fluorophore by the target molecule-bound SA has
the effect of enhancing the fluorescence of the fluorophore 1610. Thus, a detectable
fluorescent signal generated by the system is indicative of the presence of a target molecule
inside of a cell.
D. Transcript Reporter Assay
The invention comprises a reporter assay comprising an antisense RNA-based
method for detecting target transcripts within viable cells by causing the expression of a
reporter molecule if a target transcript is present within a cell.
Certain intracellular methods in the art for inhibiting gene expression employ
small interfering RNA, such as double-stranded RNA ( dsRNA), to target transcribed genes in
cells. The dsRNA comprise antisense and sense strands that are delivered into or expressed
in cells, and the strands of the dsRNA act via a trans-acting inhibition mechanism, where one
strand (typically the antisense strand) binds to a target gene sequence (RNA transcript) and
prevents expression of the target gene sequence. Double-stranded RNA molecules have been
shown to block (knock down) gene expression in a highly conserved regulatory mechanism
known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a
dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans.
dsRNA has also been shown to degrade target RNA in other organisms, including plants (see,
e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see,
e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,
Limmer; and DE 101 00 586.5, Kreutzer et al.). However, binding of a strand of the dsRNA
to the target gene can be non-specific. If a similar mechanism were to be applied to a
detection system, this non-specific binding can result in high false positive rates, which make
it unsuitable for the development of clinically useful detection systems.
Previous trans-acting inhibition mechanisms have been shown to be unsuitable for
development of clinically useful detection systems. For example, some methods result in
high levels of non-specific signals and up to 90% false positive rate, when achieving a 90%
sensitivity of the assay. See U.S. Patent No. 8,329,889. Certain methods for post
transcriptional regulation of gene expression have been developed that use a cis-repressed
marker transcript, such as a green fluorescent protein marker, where the ribosomal binding
site of the marker is blocked by the cis-repressing sequence, along with a trans-activating
RNA transcript. When the trans-activating RNA transcript binds to the cis-repressed marker
transcript, the hairpin structure of the cis-repressed marker transcript is altered, and the
upstream ribosome binding site of the marker gene is exposed, allowing transcription and
expression of the marker gene. However, these methods have not previously been used for
the detection of endogenous transcripts, nor successful beyond a basic switching mechanism
for controlling expression of genes in cells.
1) Nucleic Acid Molecule Interactions and Mechanisms
The methods of the invention take advantage of the transcript-level regulation
mechanisms, including antisense RNA (asRNA) mechanism in cells, to deliver nucleic acid
molecules into cells. The antisense mechanism includes all forms of sequence-specific
mRNA recognition leading to reduced, eliminated, increased, activated, or otherwise altered
expression of a target transcript. See Good, L., Translation Repression By Antisense
Sequences. Cellular and Molecular Life Sciences, 2003. 60(5): p. 854-861, and Lioliou, E.,
RNA-mediated regulation in bacteria: from natural to artificial systems, New Biotechnology.
2010. 27(3): p. 222-235. Naturally occurring asRNAs are found in all three kingdoms oflife,
and they affect messenger RNA (mRNA) destruction, repression and activation, as well as
RNA processing and transcription. See Sabine, B., Antisense-RNA regulation and RNA
Inteiference. Biochimica et Biophysica Acta (BBA)- Gene Structure and Expression, 2001.
1575(1-3): p. 15-25. This mechanism has been exploited in inhibiting protein synthesis for
therapeutic applications.
Antisense RNA is a single-stranded RNA that is complementary to a messenger
RNA (mRNA) strand transcribed within a cell. asRNA may be introduced into a cell to
inhibit translation of a complementary mRNA by base pairing to it and physically obstructing
the translation machinery. Antisense RNA anneal to a complementary mRNA target
sequence, and translation of the mRNA target sequence is disrupted as a result of steric
hindrance of either ribosome access or ribosomal read through.
The antisense RNA mechanism is different from RNA interference (RNAi), a
related process in which double-stranded RNA fragments ( dsRNA, also called small
interfering RNAs (siRNAs)) trigger catalytically mediated gene silencing, most typically by
targeting the RNA-induced silencing complex (RISC) to bind to and degrade the mRNA.
Annealing of a strand of the dsRNA molecule to mRNA or DNA can result in fast
degradation of duplex RNA, hybrid RNA/DNA duplex, or duplex RNA resembling precursor
tRNA by ribonucleases in the cell, or by cleavage of the target RNA by the antisense
compound itself.
The RNAi pathway is found in many eukaryotes and is initiated by the enzyme
Dicer, which cleaves long double-stranded RNA ( dsRNA) molecules into short double
stranded fragments of ~20 nucleotides that are called siRNAs. Each siRNA is unwound into
two single-stranded RNAs (ssRNA), namely the passenger strand and the guide strand. The
passenger strand is degraded, and the guide strand is incorporated into the RNA-induced
silencing complex (RISC). In post-transcriptional gene silencing, the guide strand base pairs
with a complementary sequence in a messenger RNA molecule, and cleavage is induced by a
protein called Argonaute, the catalytic component of the RISC complex.
In regards to the nucleic acid interactions of the mechanisms of the invention,
interactions between a reporter transcript and a target transcript can rely on base pairings
between loops present in both transcripts (e.g., "kissing complexes"), or between a loop and a
single-stranded (ss) region. In some cases, the kissing complex formation suffices for
mediating the desired effect of the interaction, and in other cases, propagation of the primary
contacts will lead to an interaction resulting in the desired effect.
2) Mechanisms for Cis-Repression and Trans-Activation of
Translation of a Reporter Construct via Transcript-Level
Regulation
The following description illustrates transcript reporter systems based on various
repression/activation mechanisms that can be used, according to embodiments of this
invention. In each of Figures 17-20, a vector includes a reporter construct comprising a
reporter sequence, and the regions on the reporter construct are shown in each of the figures,
including regions that can be targeted for repression by a cis-repressing sequence. The
description below provides non-limiting examples of various inhibition mechanisms,
including transcription attenuation, translation attenuation, and destabilization of the
transcript, and various activation mechanisms including conformational changes and
cleavage.
Figure 17 depicts an example of a system 1700 that uses a cis-repression
mechanism that can target the 5' UTR (untranslated region) 1701 of the reporter sequence
1702 on a reporter transcript 1703. The regions within the reporter sequence 1702 (5'UTR
(1701), RBS, Coding Region and 3'UTR) are also shown. The cis-repressing sequence 1705
is upstream of the reporter sequence and up to the 5' UTR 1701 of the reporter sequence. An
RNA polymerase 1704 transcribes the sequence of the reporter construct 1703 from the
vector 1706.
At some point during transcription, the transcription process is stopped by the
formation of a transcription termination (TT) stem-loop structure 1707 in the reporter
transcript 1703, due to an interaction within the transcribed cis-repressing sequence 1705.
The transcription termination 1707 structure stops 1708 the RNA polymerase 1704 from
transcribing the vector 1706. In some embodiments, a transcription termination protein (e.g.,
NusA in E. coli) binds to RNA polymerase and/or to the transcription termination 1707
structure to cease transcription of the reporter construct.
When a target transcript 1709 is present in the cell, the target transcript 1709 binds
to the reporter transcript 1703. In some embodiments, the binding between the target
transcript and the reporter transcript is by base pairing of the nucleotides in each sequence.
The interaction between the target transcript 1709 and the reporter transcript 103 causes the
transcription termination (TT) stem-loop structure 1707 to be cleaved 1710. Cleavage of the
reporter transcript 1703 can occur by a cellular enzyme, such as RNase III, for example. In
this case, the secondary structure of a target transcript is analyzed for the presence of an
RNAse III consensus sequence among the ssRNA regions of the secondary structure, for
example 5’-nnWAWGNNNUUN-3’ (SEQ ID NO: 20) or 5’-NAGNNNNCWUWnn-3’ (SEQ
ID NO: 21) where “N” and “n” are any nucleotide and “W” is A or U and “N” indicates a
relatively strict requirement for Watson–Crick base pairing, while “n” indicates a minimal
requirement for base pairing. When such a consensus sequence is found on a target
transcript, the loop of the transcription termination structure 1707 can be designed to be
complementary to said RNAse III consensus sequence such that when the ssRNA in each
RNA molecule hybridize, the RNAse III cleavage site is formed allowing for cleavage of the
transcription termination structure 1707. In the mecA transcript, loop T23, starting at
nucleotide 1,404, has the sequence CAGAUAACAUUUU (SEQ ID NO: 22) that is suitable
for such an approach.
In some embodiments, a cleavage site is engineered in the reporter construct, such
that the reporter transcript is cleaved after transcription. The cleavage, in the example
provided, can occur immediately adjacent to the location of the loop in the transcription
terminator structure. Transcription is re-initiated 1711 by the RNA polymerase 104.
Cleavage of the transcription termination (TT) stem-loop structure 1707 allows the remainder
of the reporter sequence 1702 to be transcribed and subsequently translated. This results in
the production of a detectable or selectable marker from the translated reporter molecule.
In prokaryotes, the transcription termination structure 1707 involves a Rho-
independent mechanism with a stem-loop structure that is 7-20 base pairs in length, rich in
cytosine-guanine base pairs and is followed by a chain of uracil residues. NusA binds to the
transcription termination stem-loop structure 1707 causing RNA polymerase to stall during
transcription of the poly-uracil sequence. Weak Adenine-Uracil bonds lower the energy of
destabilization for the RNA-DNA duplex, allowing it to unwind and dissociate from the RNA
polymerase. In eukaryotes, the transcription termination structure 1707 is recognized by
protein factors and involves cleavage of the new transcript followed by polyadenylation.
Figure 18 shows an example of a system 1800 for detecting the presence of a
target transcript in a cell that is based on a cis-repression mechanism targeting the ribosome
binding site (RBS) 1801 of the reporter sequence 1702 in the reporter transcript 1703. The
RBS 1801 is a sequence of mRNA that is bound by the ribosome 1802 when initiating protein
translation. The cis-repressing sequence 1705 is designed to bind to the RBS 1801 (e.g., the
cis-repressing sequence 1705 is complementary to the RBS sequence 1801). The RBS 1801
binds to the cis-repressing sequence 1705 and becomes sequestered (inaccessible by a
ribosome 1802), preventing the translation of the reporter transcript 1703. When a target
transcript 109 from the cell binds to the reporter transcript 1703, the target transcript 1709 has
a higher binding affinity for the RBS sequence 1801, and a conformational change occurs in
the reporter transcript 1703 in a manner that releases the binding between the cis-repressing
1705 sequence and the RBS sequence 1801. This allows the ribosome 1802 to bind to the
RBS 1801, thereby allowing for translation of the reporter transcript 1703.
Figure 19 illustrates an exemplary system 1900 for detecting the presence of a
target transcript in a cell that is based on a cis-repression mechanism targeting the coding
region ("AUG") 1901 of the reporter sequence 1702 in the reporter transcript 1703. The cis
repressing sequence 1705 is constructed such that it binds with (e.g., complementary to) the
coding region 1901 of the reporter sequence 1702. The "AUG" start codon is shown as part
of the coding region 1901. The binding of the cis-repressing sequence 1705 and the coding
region 1901 results in a conformation that leads to cleavage 1902 of the reporter construct
1703. Cleavage of the reporter transcript 1703 prevents translation.
When a target transcript 1709 is present in the cell, the target transcript 1709 binds
to the cis-repressing sequence 1705 in a manner that causes a conformational change in the
reporter transcript 1703. This conformational change prevents or removes the interaction
between the cis-repressing sequence 1705 and the coding region 1901 of the reporter
sequence 1702, thereby allowing for translation of the reporter sequence 1702.
Figure 20 illustrates an example system 2000 for detecting the presence of a target
transcript in a cell that is based on a repression mechanism using an unstable reporter
transcript 2001. The reporter transcript 2001 is designed to be unstable such that it forms an
unstable conformation that prevents the translation of the reporter transcript 2001. A reporter
transcript 2001 is defined to be unstable if it is prone to rapid degradation due to a variety of
factors, such as activity of exosome complexes or a degradosome. A target transcript 1709 in
the cell binds to a portion of the unstable reporter transcript 2001. In this example, the
portion responsible for destabilizing the transcript is located in the 3' UTR 2005 of the
reporter sequence, and the 3' UTR 2005 acts like the cis-repressing sequence of the reporter
construct 1703. The binding of the target transcript 1709 with the 3' UTR 2005 of the
reporter sequence results in a cleaving event 2003 that stabilizes the reporter transcript 2001
and allows for translation 2004 of the reporter transcript 2001. Cleavage occurs upon binding
of the target transcript 1709 and serves to remove the portion of sequence that is responsible
for destabilizing the transcript. In this example, the target transcript 1709 binds to the 3' UTR
405 of the reporter sequence, but the system 400 can also be designed such that binding and
cleavage occurs in the 5' UTR, upstream of the 5' UTR, or downstream of the 3' UTR.
Binding and cleavage can occur anywhere outside of regions necessary for translation of the
reporter sequence 1702.
In some embodiments, the cis-repressing sequence itself comprises two sequences
that can bind to each other (e.g., complementary to each other), and the conformation of the
reporter transcript that results from the binding of the two sequences of the cis-repressing
sequence prevents translation of the reporter sequence in the reporter transcript.
3) Naturally Occurring and Synthetic Systems for
Repression/ Activation Mechanisms
Several naturally occurring and synthetically produced transcript-level
mechanisms have been described that demonstrate the individual mechanisms (i.e.,
conformational change and cleavage) employed in each of the examples illustrated in Figures
17-20.
Transcription termination has been observed in antisense RNA (asRNA)-mediated
transcriptional attenuation. In one example, two loop-loop interactions between
RNAIII/repR mRNA are subsequently followed by the formation of a stable duplex. This
complex stabilizes a Rho-independent terminator structure to arrest elongation by RNA
polymerase (RNAP).
The RBS sequestration mechanism has been described via the development of a
synthetic riboswitch system. In this system, a sequence complementary to a RBS is placed
upstream of the RBS, allowing the presence of a linker sequence between the two regions.
After transcription of the mRNA, the two complementary regions hybridize. creating a
hairpin that prevents docking of the ribosome. To activate translation, a synthetic trans
activating RNA carrying the RBS sequence binds to the hybridized RNA, allowing the RBS
to be exposed and available for translation.
The prevention of translation due to the cleaving of RNA has also been described
in a natural system where the asRNA MicC targets a sequence inside the coding region of
ompD mRNA. The interaction, which is promoted by Hfq, causes the cleaving of the mRNA
byRNaseE.
Yet another natural mechanism demonstrates a cleaving event to activate
translation rather than inhibiting it. The E. coli GadY asRNA targets the intergenic region
between two genes of the gadXW operon. Following the formation of a stable helix between
GadY and the 3 'UTR of gadX, an RNase cleavage occurs in the transcript and stabilizes
gadX transcript allowing for its translation.
4) Mechanism of Conformational Change by Cis-Repression of
the Reporter Sequence and by Binding of a Target Transcript
The general mechanisms employed in the invention are intermolecular nucleic
acid molecule interactions that may result in two subsequent mechanisms: (1) a
conformational change in the secondary structure of the nucleic acid molecules, and (2) a
cleaving event. Described herein are methods for designing reporter transcripts that can
undergo a conformational change between a cis-repressed conformation and a de-repressed
conformation, such that the conformational change is induced by binding of a target transcript
to the reporter transcript.
As described above, a reporter transcript can comprise a reporter sequence and be
designed such that translation of the reporter gene sequence is blocked by cis-repression of
the ribosome binding site (RBS) of the reporter gene.
In some embodiments, the following tools can be used for designing the reporter
transcripts of the invention.
1) RNA secondary structure is calculated using secondary structure program,
such as Mfold available at a server maintained by The RNA Institute College of Arts and
Sciences, University at Albany, State University of New York (Mfold web server for nucleic
acid folding and hybridization prediction. Nucleic Acids Res. 31 (13), 3406-15, (2003))
(http://rnfold.rna.albany.edu/'?q=mfold/RNA-Folding~Form).
2) Intermolecular RNA interactions are calculated using a software program such
as RNA-RNA InterACTion prediction using Integer Programming (RactIP) available at a
server maintained by the Graduate School of Information Science, Nara Institute of Science
and Technology (NAIST), Department of Biosciences and Informatics, Keio University
Japan (http://ma.naist.jp/ractip/).
3) RNA secondary structure is visualized using Visualization Applet for RNA
(VARNA) (http://vama.lri.fr/), which is a Java lightweight Applet dedicated to drawing the
secondary structure of RNA.
A secondary structure of the target transcript can be generated based on the lowest
energy conformation calculated by MFold and visualized with VARNA.
ssRNA regions or target regions can be identified within the target transcript that
can be ideal for binding to a reporter transcript. In some instances, the secondary structure of
the target transcript includes a consensus sequence or loop sequence that can bind to a portion
of the reporter sequence. For example, in the mecA transcript of methicillin-resistant S.
aureus, there is a terminal loop that includes a consensus YUNR sequence ("UUGG") that
can be used to bind to a cis-repressing sequence of a reporter transcript. Analysis of the
secondary structure of the target transcript can reveal these one or more ssRNA regions that
can be suitable for binding to a cis-repressing sequence. The cis-repressing sequence of the
reporter transcript can then be designed to bind to these one or more ssRNA regions.
In some embodiments, the cis-repressing sequence can be designed to bind to the
RBS of the reporter sequence in the reporter transcript and form a stem-loop structure within
the reporter transcript, such that the cis-repressing sequence blocks binding of an RNA
polymerase to the RBS of the reporter sequence. Upon binding of the cis-repressing
sequence to the ssRNA region of the target transcript, the RBS of the reporter sequence can
be exposed and translation of the reporter sequence can be initiated.
In some embodiments, the cis-repressing sequence of the reporter transcript can be
designed to be positioned at the 5' terminus of the reporter sequence and designed to generate
a stem-loop structure in the reporter sequence, such that the RBS sequence of the reporter
sequence is blocked. The cis-repressing stem-loop structure can be designed to block the
RBS sequence based on the lowest energy conformation of the reporter transcript, as
calculated by MFold and visualized with VARNA. The predicted inter-molecular
interactions between the target transcript and the cis-repressing sequence of the reporter
transcript can be calculated by RactIP and visualized by VARNA. A diagram can be drawn
to visualize the base pairing between the target transcript and the cis-repressing sequence of
the reporter transcript, as shown in Figure 28 below.
The interaction can include base pairing between 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
, 16, 17, 18, 19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more nucleotides in the target sequence and cis
repressing sequence. The complementary binding between the two sequences can be fully
complementary, substantially complementary or partially complementary. The base pairing
can be across contiguous nucleotide sequences or regions within the target and cis-repressing
sequences, for example, as shown in Figure 28.
) Cleavage Mechanisms for Cis-repressed Transcripts or
Reporter Transcripts
The general mechanisms employed in the invention are intermolecular nucleic
acid molecule interactions that may result in two subsequent mechanisms: (1) a
conformational change in the secondary structure of the nucleic acid molecules, and (2) a
cleaving event. Described herein are methods and systems for designing reporter transcripts
that employ a cleaving event.
In some embodiments, a cleaving mechanism can be employed in the system and
methods of the invention for cis-repression or for trans-activation. For example, as described
above in Figures 17, 19 and 20, a system can be designed to take advantage of a cleaving
mechanism by exposing a nucleic acid sequence of the reporter transcript to a cleaving
enzyme (RNase) or sequestering a single-stranded sequence that is recognized by a sequence
specific RNAase.
In one example, an ribonuclease E (RNAse E) site can be designed in the reporter
transcript("*" indicates the cleaving site):
(G,A)N(C,A)N(G)(G,U,A)*(A,U)(C,U)N(C,A)(C,A). See Kaberdin et al., Probing the
substrate specificity of E. coli RNase E using a novel oligonucleotide-based assay. Nucleic
Acids Research, 2003, Vol. 31, No. 16 (doi: 10.1093/nar/gkg690).
In a cis-repression system, a cis-repressing sequence can be incorporated in the
design of a reporter transcript, such that when transcribed, the conformation of the reporter
transcript exposes a single stranded region containing a sequence RNAse E recognition motif
at the desired site to be cleaved. In some embodiments, the cleavage site can be involved in
repression of the transcription of the reporter transcript, for example, if the cleavage site is
within the coding region of the reporter gene.
For a trans-derepression system, the cis-repressed transcript can be engineered to
bind to a target transcript, such that the interaction causes a conformational change in the
reporter transcript that sequesters the single-stranded region containing the RNAse E site.
The system can be designed such that the cis-repressing mechanism is due to a
specific secondary structure generated by a conformation of the cis-repressing sequence, such
as the transcription termination structure described above. In this example, a cleaving event
serves to de-repress the reporter sequence. This can be accomplished by designing the cis
repressing sequence to interact with (bind to) a naturally-occurring plasmid or other cellular
transcript, such that the interaction results in the generation of a single-stranded region
containing the RNAse E site that can be cleaved and thus removes the cis-repressing
sequence from the reporter transcript.
In some embodiments, when a cleavage event is employed for expression of the
reporter, the RN Ase E site is designed to be outside of the coding region of a reporter
sequence with enough sequence length in the 5' and 3' UTR in order to allow for a viable
reporter transcript. In this case, the RNAse E site is designed to be at least 0, 1, 2, 3, 4, 5, 10,
, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or more base pairs upstream of the
start codon in prokaryotic systems and at least 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,
300, 400, 500, or more base pairs upstream of the start codon in eukaryotic systems or at least
, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or more base pairs downstream of
the stop codon. In other embodiments, when a cleavage event is employed for repression of
the reporter, the RN Ase E site is designed to be within the coding region of the reporter
sequence or otherwise placed in order to inhibit expression of the reporter.
6) Transcripts
As described above, a transcript is a length of nucleotide sequence (DNA or RNA)
transcribed from a DNA or RNA template sequence or gene. The transcript can be a cDNA
sequence transcribed from an RNA template or an mRNA sequence transcribed from a DNA
template. The transcript can be transcribed from an engineered nucleic acid construct. The
transcript can have regions of complementarity within itself, such that the transcript includes
two regions that can form an intra-molecular duplex. One region can be referred to as a "cis
repressing sequence" that binds to and blocks translation of a reporter sequence. A second
region of the transcript is called a "reporter sequence" that encodes a reporter molecule, such
as a detectable or selectable marker.
The transcripts of the invention can be a transcript sequence that can be 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In
other embodiments, the transcript can be at least 25, 30, 40, 50, 60, 70, 80, 90, 100, 500,
1000, 1500, 2000, 3000, 4000, 5000 or more nucleotides in length. The cis-repressing
sequence and the reporter sequence can be the same length or of different lengths.
In some embodiments, the cis-repressing sequence is separated from the reporter
sequencebyl,2,3,4,5,6, 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20,21,22,23,24,
, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, or more spacer nucleotides.
7) Vectors
In another aspect, the transcripts (including antisense and sense sequences) of the
invention are expressed from transcription units inserted into DNA or RNA vectors (see, e.g.,
Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication
No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad,
U.S. Pat. No. 6,054,299). These sequences can be introduced as a linear construct, a circular
plasmid, or a viral vector, including bacteriophage-based vectors, which can be incorporated
and inherited as a transgene integrated into the host genome. The transcript can also be
constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al.,
Proc. Natl. Acad. Sci. USA (1995) 92:1292).
The transcript sequences can be transcribed by a promoter located on the
expression plasmid. In one embodiment, the cis-repressing and reporter sequences are
expressed as an inverted repeat joined by a linker polynucleotide sequence such that the
transcript has a stem and loop structure.
Recombinant expression vectors can be used to express the transcripts of the
invention. Recombinant expression vectors are generally DNA plasmids or viral vectors.
Viral vectors expressing the transcripts can be constructed based on, but not limited to,
adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol.
(1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998)
6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell
68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used
to introduce a variety of genes into many different cell types, including epithelial cells, in
vitro and/or in vivo (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and
Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl.
Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA
87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al.,
1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-
1805; van Beusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay et al., 1992,
Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-
10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S.
Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT
Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral
vectors capable of transducing and expressing genes inserted into the genome of a cell can be
produced by transfecting the recombinant retroviral genome into suitable packaging cell lines
such as PA317 and Psi-CRIP (Cornette et al., 1991, Human Gene Therapy 2:5-10; Cone et
al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be
used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog,
and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the
advantage of not requiring mitotically active cells for infection.
Any viral vector capable of accepting the coding sequences for the transcript(s) to
be expressed can be used, for example, vectors derived from adenovirus (AV); adeno
associated virus (AA V); retroviruses (e.g., lentiviruses (L V), Rhabdoviruses, murine
leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by
pseudotyping the vectors with envelope proteins or other surface antigens from other viruses,
or by substituting different viral capsid proteins, as appropriate.
For example, lentiviral vectors featured in the invention can be pseudotyped with
surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like.
AA V vectors featured in the invention can be made to target different cells by engineering
the vectors to express different capsid protein serotypes. Techniques for constructing AA V
vectors which express different capsid protein serotypes are within the skill in the art; see,
e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein
incorporated by reference.
Selection of recombinant viral vectors suitable for use in the invention, methods
for inserting nucleic acid sequences for expressing the transcripts into the vector, and
methods of delivering the viral vector to the cells of interest are within the skill in the art.
See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis MA (1988),
Biotechniques 6: 608-614; Miller AD (1990), Hum Gene Therap. 1: 5-14; Anderson W F
(1998), Nature 392: 25-30; and Rubinson DA et al., Nat. Genet. 33: 401-406, the entire
disclosures of which are herein incorporated by reference.
Viral vectors can be derived from AV and AA V. A suitable AV vector for
expressing the transcripts featured in the invention, a method for constructing the
recombinant AV vector, and a method for delivering the vector into target cells, are described
in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010. Suitable AA V vectors for expressing
the transcripts featured in the invention, methods for constructing the recombinant AV vector,
and methods for delivering the vectors into target cells are described in Samulski R et al.
(1987), J. Virol. 61: 3096-3101; Fisher K Jet al. (1996), J. Virol, 70: 520-532; Samulski R
et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941;
International Patent Application No. WO 94/13788; and International Patent Application No.
WO 93/24641, the entire disclosures of which are herein incorporated by reference.
The promoter driving transcript expression in either a DNA plasmid or viral
vector featured in the invention may be a eukaryotic RNA polymerase I (e.g., ribosomal RNA
promoter), RNA polymerase II (e.g., CMV early promoter or actin promoter or Ul snRNA
promoter) or generally RNA polymerase III promoter (e.g., U6 snRNA or 7SK RNA
promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression
plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter.
The promoter can also direct transgene expression to the pancreas (see, e.g., the insulin
regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA
83 :2511-2515)).
In addition, expression of the transcript can be precisely regulated, for example,
by using an inducible regulatory sequence and expression systems such as a regulatory
sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels,
or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems,
suitable for the control of trans gene expression in cells or in mammals include regulation by
ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and
isopropyl-beta-Dthiogalactopyranoside (IPTG). A person skilled in the art would be able
to choose the appropriate regulatory/promoter sequence based on the intended use of the
dsRNA transgene.
Generally, recombinant vectors capable of expressing transcript molecules are
delivered as described below, and persist in target cells. Alternatively, viral vectors can be
used that provide for transient expression of transcript molecules. Such vectors can be
repeatedly administered as necessary. Once expressed, the transcript binds to target RNA
and modulates its function or expression. Delivery of transcript expressing vectors can be
systemic, such as by intravenous or intramuscular administration, by administration to target
cells ex-planted from the patient followed by reintroduction into the patient, or by any other
means that allows for introduction into a desired target cell.
Transcript expression DNA plasmids are typically transfected into target cells as a
complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers
(e.g., Transit-TKO™). Multiple lipid transfections for dsRNA-mediated knockdowns
targeting different regions of a single PROC gene or multiple PROC genes over a period of a
week or more are also contemplated by the invention. Successful introduction of vectors into
host cells can be monitored using various known methods. For example, transient
transfection can be signaled with a reporter, such as a fluorescent marker, such as Green
Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers
that provide the transfected cell with resistance to specific environmental factors (e.g.,
antibiotics and drugs), such as hygromycin B resistance.
The delivery of the vector containing the recombinant DNA can by performed by
abiologic or biologic systems. Including but not limited to liposomes, virus-like particles,
transduction particles derived from phage or viruses, and conjugation.
8) Reporters for Transcript Assay
In some embodiments, the nucleic acid construct comprises a reporter sequence
(e.g., a reporter gene sequence). The reporter gene encodes a reporter molecule that produces
a signal when expressed in a cell. In some embodiments, the reporter molecule can be a
detectable or selectable marker. In certain embodiments, the reporter molecule can be a
fluorescent reporter molecule, such as a green fluorescent protein (GFP), yellow fluorescent
protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), or red
fluorescent protein (RFP). In other embodiments, the reporter molecule can be a
chemiluminescent protein.
Reporter molecules can be a bacterial luciferase, an eukaryotic luciferase, a
fluorescent protein, an enzyme suitable for colorimetric detection, a protein suitable for
immunodetection, a peptide suitable for immunodetection or a nucleic acid that function as an
apatamer or that exhibits enzymatic activity.
Selectable markers can also be used as a reporter. The selectable marker can be
an antibiotic resistance gene, for example.
9) Cells and Target Genes for Transcript Reporter Assay
Examples of cells that can be used for detection include Gram-positive and Gram
negative bacteria, such as S. aureus, E. coli, K. pneumoniae, etc., fungi such as Streptomyces
coelicolor, and other eukaryotic cells, including cells from humans, other mammals, insects,
invertebrates, or plants.
Target transcripts can include any endogenous transcript, whether coding or non-
coding. Target transcripts can be derived from eukaryotic and prokaryotic cells, including,
for example, mecA transcript in S. aureus cells (indicative of MRSA), the tcdB transcript in
C. difficile (indicative of toxigenic C. diff), and HPV E6/E7 transcripts in cervical epithelial
cells (indicative of cervical cancer). Genes associated with infectious agents, such as viruses,
can be targets as well, including HIV, HPV, etc. Other examples of target genes include non
coding RNA such as transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as RNAs
such as snoRNAs, microRNAs, siRNAs, snRNAs, exRNAs, and piRNAs and ncRNAs.
EXAMPLES
Below are examples of specific embodiments for carrying out the present
invention. The examples are offered for illustrative purposes only, and are not intended to
limit the scope of the present invention in any way. Efforts have been made to ensure
accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some
experimental error and deviation should, of course, be allowed for.
The practice of the present invention will employ, unless otherwise indicated,
conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and
pharmacology, within the skill of the art. Such techniques are explained fully in the
literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H.
Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current
addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989);
Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.);
Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing
Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press)
Vols A and B(l 992).
Example 1: Silent Mutation/Complementation Packaging System
The following is an example of the design and construction of a silent
mutation/complementation-based packaging system for producing non-replicative
transduction particles.
The materials used for developing the packaging system are listed below:
Bacterial Strains:
• NI 706, an E.coli K-12 Pl cl-100 Tn9 lysogen
Vectors:
• Y14439 (pBHRl backbone)
The following GenBank accession numbers (N.B., the sequences referred to by
accession number are those listed in the database as of the priority date of this application) or
SEQ ID NOs. can be used for the vector backbone and cassette sequences:
• X06758 (bacterial luciferase genes luxAB)
• SEQ ID NO:l (Native Pl pac-site)
• SEQ ID N0:3 (Pl lytic replicon containing the Cl repressor-controlled P53 promoter,
the promoter P53 antisense, the repL genes, and an in-frame deletion of the kilA gene)
• SEQ ID N0:4 (Pblast promoter driving luxAB expression)
Construction of NI 706(pac): pacA mutated strain: An exemplary sequence of a
pacA mutated sequence is shown in SEQ ID NO: 2, shown in the informal sequence listing
below. The mutation can be accomplished by constructing the mutated sequence via gene
synthesis and then replacing the native sequence in NI 706 with the mutated sequence via an
allelic exchange approach.
Construction of the GWPIOOOI reporter vector: The GWPIOOOI vector contains
the pBHRl origin of replication exhibiting broad Gram-negative activity, two selectable
markers for kanamycin and chloramphenicol, the native bacteriophage P 1 pac-site sequence,
the luxA and luxB genes are from Vibrio harveyi operatively linked to the constitutive
blasticillin promoter (Pblast), and the Pl lytic replicon containing the Cl repressor-controlled
P53 promoter, the promoter P53 antisense, the repL genes, and an in-frame deletion of the
kilA gene.
Figure 2 shows the resulting vector (GWPIOOOI, SEQ ID NO:l 1), which can be
constructed in a variety of manners that are known to one of skill in the art including
obtaining the cassettes via PCR from their native sources or via gene synthesis and assembly
of the vector via traditional restriction enzyme-based cloning or alternative techniques such
as Gibson assembly.
Silent/Complementation Packaging System: The packaging system includes the
pacA mutant strain NI 706(pac) complemented with the vector pGWPIOOOl. As known to
one of skill in the art, the manner of constructing this system can be accomplished by
transformation NI 706(pac) with vector pGWPIOOOl. The vector pGWPIOOOl can be
maintained in cultures of the transformed NI 706(pac) by growing the transformant in the
presence of 50 ug/mL of kanamycin.
Production of Transduction Particles Carrying Plasmid DNA: Non-replicative
transduction particles carrying vector pGWPIOOOl can be produced from NI 706(pac)
transformants via thermal induction at 42°C. Incubation at 42°C results in induction of the
Pl lytic cycle in which the prophage excises from the NI 706 genome, produces phage
structural elements, and packages pGWPIOOOl concatameric DNA formed by the lytic
replicon in progeny phage particles, as depicted in Figure 1. The resulting cell lysate is then
collected and contains non-replicative transduction particles, each consisting of bacteriophage
P 1 particles carrying a linear concatamer of pGWP 10001 DNA.
Example 2: Deletion/Complementation Packaging System
The following is an example of the design and construction of a
deletion/complementation-based packaging system for producing non-replicative
transduction particles.
The materials used for developing the packaging system are listed below:
Bacterial Strains:
RN4220 is a restriction defective S. aureus strain that is a non-lysogenic
derivative ofNCTC 8325 and is an efficient recipient for E. coli DNA. It was first described
in Kreiswirth, B.N. et al., The toxic shock syndrome exotoxin structural gene is not detectably
transmitted by a prophage. Nature, 1983. 305(5936): p. 709-712.
RN10616 is derived by lysogenizing RN4220 with bacteriophage cp80a. Ubeda, C.
et al., Specificity of staphylococcal phage and SaPI DNA packaging as revealed by integrase
and terminase mutations. Molecular Microbiology, 2009. 72(1): p. 98-108.
ST24 is derived from deleting the small terminase gene terS from the lysogenized
bacteriophage cp80a in RN10616. Ubeda, C. et al., Specificity of staphylococcal phage and
SaPI DNA packaging as revealed by integrase and terminase mutations. Molecular
Microbiology, 2009. 72(1): p. 98-108.
Vectors:
Examples of plasmids that can be used as source plasmids for cassettes, in some
embodiments of the invention are described in Charpentier, E., et al., Novel Cassette-Based
Shuttle Vector System for Gram-Positive Bacteria. Appl. Environ. Microbiol., 2004. 70(10):
p. 6076-6085.
The following GenBank accession numbers can be used for cassette sequences:
• SEQ ID N0:5 (S. aureus pT181 plasmid origin or replication copy number variant
pT181cop-623 repC)
• M21136 (tetA(M))
• SEQ ID N0:12 (Pc1pB promoter sequence)
• SEQ ID N0:9 (cpll small terminase (terS) gene sequence)
• L09137 (amp ColEl ori)
• X06758 (luxAB)
• M62650 (Transcription Termination)
terS Deletion: The construction of the terS knockout strain ST24 can be
accomplished via an allelic-exchange-based strategy resulting in an in-frame deletion
removing most of the coding sequence of the cp80a small terminase gene. The details of this
strategy are described in Ubeda, C. et al., Specificity of staphylococcal phage and SaPI DNA
packaging as revealed by integrase and terminase mutations. Molecular Microbiology, 2009.
72(1): p. 98-108.
An exemplary sequence of a terS knockout strain is shown in SEQ ID N0:13,
(shown in the sequence listing below). SEQ ID N0:13 is a RN10616 genomic sequence loci
showing the cp80a terS deletion and complementation.
Vector Construction: The GW80A0001 vector is an E. coli/S. aureus shuttle
vector. The vector contains S. aureus (pT181cop-623 repC) and E.coli (ColElori) origins of
replication, the selectable markers for ampicillin (amp) and tetracycline ( tet(M)) resistance
for selection in E. coli and S. aureus, respectively, the cpl 1 small terminase (terS) gene
sequence that includes its own promoter, the luxA and luxB genes are from Vibrio harveyi
operatively linked to the constitutive S. aureus P clpB promoter, and a transcription termination
sequence (TT).
Figure 4 shows the resulting vector (pGW80A0001, SEQ ID N0:14), which can
be constructed in a variety of manners that are known to one of skill in the art. In one
example, the tet(M) cassette and luxAB genes can be obtained via PCR amplification from
the publically available pCN36 and pCN58 vectors (Charpentier, E., et al.). Pc1pB can be
obtained from PCR amplification from S. aureus RN4220 and terS can be obtained via PCR
amplification from RN10616. A vector backbone can be obtained by removing the ermC
gene from the publically available vector pCN48 (Charpentier, E., et al.), and the various
components of the final vector pGW80A0001 can be assembled onto this vector backbone
via appropriately designed restriction enzyme-based cloning.
Deletion/Complementation Packaging System: The packaging system can include
the terS knockout strain ST24 complemented with the vector pGW80AOOO 1 to generate strain
GW24. As known to one of skill in the art, the manner of constructing this system can be
accomplished by transformation ST24 with vector pGW80AOOO 1. The vector pGW80AOOO 1
can be maintained in cultures of the transformed ST24 by growing the transformant in the
presence of 5 ug/mL of tetracycline.
Production of Transduction Particles Carrying Plasmid DNA: Non-replicative
transduction particles carrying vector pGW80A0001 can be produced from GW24 via a
Mitomycin C-induction method that was first demonstrated in E.coli and is now a standard
technique for obtaining prophages from lysogenized bacteria. Otsuji, N. et al., Induction of
Phage Formation in the Lysogenic Escherichia coli K-12 by Mitomycin C. Nature, 1959.
184( 4692): p. 1079-1080. This prophage induction method results in induction of the cp80a
lytic cycle in which the prophage excises from the GW24 genome, produces phage structural
elements, and packages pGW80AOOO 1 concatameric DNA in progeny phage particles, as
depicted in Figure 2. The resulting cell lysate is then collected and contains non-replicative
transduction particles, each consisting of bacteriophage cp80a particles carrying a linear
concatamer of pGW80AOOO 1 DNA.
Example 3: SaPlbov2-Based Packaging System Lacking Integrase
The following is an example of the design and construction of a SaPibov2-based
packaging system for producing non-replicative transduction particles.
The materials used for developing the packaging system are listed below:
The following materials can be used to develop a SaPibov2-based packaging
system lacking integrase.
Bacterial Strains:
RN451 is a S. aureus strain lysogenized with bacteriophage cp 11.
JP2131 is RN451 that has been lysogenized with SaPibov2. See Maiques, E. et al.,
Role of Staphylococcal Phage and SaPI Integrase in Intra- and Interspecies SaPI Transfer. J.
Bacteriol., 2007. 189(15): p. 5608-5616.
JP2488 is strain JP2131 in which the int gene has been deleted from Sapibov2
(SaPibov2~int). Maiques, E. et al., Role of Staphylococcal Phage and SaPI Integrase in
Intra- and Interspecies SaPI Transfer. J. Bacteriol., 2007. 189(15): p. 5608-5616.
Bacteriophage:
Bacteriophage cp 11 can be obtained from S. aureus strain RN0451 via a
Mitomycin C-induction method that was first described in E. coli and is now a standard
technique for obtaining prophages from lysogenized bacteria. Otsuji, N. et al., Induction of
Phage Formation in the Lysogenic Escherichia coliK-12 by Mitomycin C. Nature, 1959.
184(4692): p. 1079-1080.
Promoters:
P clpB can be used as a promoter in this example. The clpB gene promoter is a
constitutive promoter used for controlling the expression of the int gene. The S. aureus clpB
(PcZpB) gene promoter sequence was first described in 2004. Frees, D., et al., Clp ATPases are
required for stress tolerance, intracellular replication and biofilm formation in
Staphylococcus aureus. Molecular Microbiology, 2004. 54(5): p. 1445-1462. It was also first
employed for controlling the gene expression in a plasmid in 2004. Arnaud, M., A.
Chastanet, and M. Debarbouille, New Vector for Efficient Allelic Replacement in Naturally
Nontransformable, Low-GC-Content, Gram-Positive Bacteria. Appl. Environ. Microbiol.,
2004. 70(11): p. 6887-6891. The promoter can be obtained from S. aureus RN4220 using
primers described in 2004. Id.
Production of cpl I/ SaPibov2~int co-lysogen (RN45 l(cpl l SaPibov2~int)): The
strain JP2488( cpl 1 SaPibov2~int) can be produced by lysogenizing JP2488 with cpl 1.
Deletion of cpll terS (RN45 l(cpl LMerS SaPibov2~int)): The strain
RN451( cpl l~terS SaPibov2~int) can be produced by deleting the cpl 1 terS gene from
RN451 ( cp 11 SaPibov2~int), as described in Tormo, M.A. et al., Staphylococcus aureus
Pathogenicity Island DNA Is Packaged in Particles Composed of Phage Proteins. J.
Bacteriol., 2008. 190(7): p. 2434-2440.
Incorporation of P~-int into S. aureus genome (RN451(cpl l~terS SaPibov2~int
Pc1 B-int)): RN451( cpl l~terS SaPibov2~int Pc1pB-int) can be produced by first fusing PclpB and
int via standard molecular biology techniques then inserting the P clpB-int fusion into the
genome of RN451 (cpl l~terS SaPibov2~int) and then selecting clones that have P cZpB-int
inserted outside of the cpl 1 and SaPibov2 regions.
Production of cpl 1 particles carrying only SaPibov2~int Pc1 B-int concatamers:
cp 11 particles carrying only SaPibov2~int P clpB-int concatamers can be produced via
mitomycin-C induction of RN451 (cpl l~terS SaPibov2~int P clpB-int), as described by Otsuji,
N. et al., Induction of Phage Formation in the Lysogenic Escherichia coliK-12 by Mitomycin
C. Nature, 1959. 184(4692): p. 1079-1080. The cell lysate contains non-replicative
transduction particles, each consisting of bacteriophage cpl 1 structural proteins carrying a
linear concatamer of GI-derived DNA.
One of skill in the art will understand how to construct the NRTPs of the
invention using the above-referenced materials and well-known molecular biology and
genetic techniques in the art.
Example 4: terS Deletion/Complementation-based SarS Reporter Transduction
Particles
The following is an example of an inducer reporter-based SarS reporter system
that employs a terS deletion/complementation-based non-replicative transduction particle.
Reporter gene: Bacterial luciferase (luxAB). The luxA and luxB genes are from
Vibrio harveyi. They lack a transcriptional promoter and each contains their own ribosomal
binding site.
Spa gene promoter (Pspa): The spa gene promoter will be used for controlling the
expression of the luxAB genes.
Construction of Pspa-luxABfusion: The luxAB genes can be fused to the Pspa
promoter sequence such that the luxAB genes are operatively linked to the Pspa promoter.
Construction of the luxAB-expressing reporter vector
The luxAB-expressing reporter vector can be constructed via standard molecular
biological techniques by incorporating the Pspa-luxAB fusion product into the MCS of the
shuttle vector depicted below.
-"""-2~!_g~~-£f:£:~~~1_~~:!~S2----~- ~a~: Ml ~ P c1ps ters [>l ~1mp Co£ion )I TT ]
E. coli/S. aureus shuttle vector that carries a S. aureus (pT181cop-623 repC) and
E.coli (ColElori) origins ofreplication, genes for ampicillin (amp) and tetracycline (tet(M))
resistance, the cpl 1 small terminase (terS) gene under the control of a constitutive promoter
(P clpB), a multiple cloning site (MCS), and a transcription termination sequence (TT).
GenBank accession numbers for cassette sequences:
JOl 764 (pT181 replicons)
M21136 (tetA(M))
Accession number not yet available (P clpB)
AF424781 REGION: 16526 .. 16966 (terS)
L09137 (amp ColEl ori)
M62650 (TT)
Propagation of the vector for conducting in vitro manipulations and for
verification of manipulations can be accomplished via the E. coli Top 10 and the final
modified vector can then be introduced into S. aureus RN045 LMerS. Transduction particles
carrying shuttle vector can be produced from the RN045 LMerS transformants via a
Mitomycin C-induction method that was first described in E.coli 1959 and is now a standard
technique for obtaining prophages from lysogenized bacteria. Otsuji, N., et al., Induction of
Phage Formation in the Lysogenic Escherichia coliK-12 by Mitomycin C. Nature, 1959.
184( 4692): p. 1079-1080. The cell lysate is then collected and contains non-replicative
transduction particles each consisting of bacteriophage cpl 1 structural proteins carrying a
linear concatamer of plasmid DNA capable of reporting on the presence of SarS in target S.
aureus cells.
Example 5: terS Deletion/Complementation-based B-lactamase Reporter
Transduction Particles
The following is an example of an intracellular enzyme reporter-based ~
lactamase reporter system that employs a terS deletion/complementation-based non
replicative transduction particle.
Reporter gene: Renilla luciferase (rue)
Promoter: The promoter can be Pbtaz. The constitutive beta-lactamase promoter
can be used for driving the expression of the rue gene.
Caged substrate: Caged coelenterazine-phosphate as described in Daniel Sobek,
J.R., Enzyme detection system with caged substrates, 2007, Zymera, Inc.
Construction of Pblaz-ruc fusion: The rue genes can be fused to the Pbzaz promoter
sequence such that the rue genes are operatively linked to the Pblaz promoter.
Construction of the rue-expressing reporter vector: The rue-expressing reporter
vector can be constructed via standard molecular biological techniques by incorporating the
PbtaZ-ruc fusion product into the MCS of the shuttle vector depicted in Section V, A, 3), i)
above.
Propagation of the vector for conducting in vitro manipulations and for
verification of manipulations can be accomplished via the E. coli Top 10 and the final
modified vector can then be introduced into S. aureus RN045 l~terS. Transduction particles
carrying shuttle vector can be produced from the RN045 l~terS transformants via a
Mitomycin C-induction method that was first described in E.coli 1959 and is now a standard
technique for obtaining prophages from lysogenized bacteria. Otsuji, N., et al., Induction of
Phage Formation in the Lysogenic Escherichia coliK-12 by Mitomycin C. Nature, 1959.
184( 4692): p. 1079-1080. The cell lysate is then collected and contains NRTPs each
consisting of bacteriophage cp 11 structural proteins carrying a linear concatamer of plasmid
DNA capable of expressing Renilla luciferase within viable S. aureus cells within the cpl 1
host range.
Example 6: terS Deletion/Complementation-based Intracellular Molecule
Reporter Transduction Particles
The following is an example of an intracellular molecule reporter-based_reporter
system that employs a terS deletion/complementation-based non-replicative transduction
particle.
Promoter: The promoter can be PbtaZ· The constitutive beta-lactamase promoter
can be used for driving the expression of the rue gene.
Switchable aptamer: Switchable aptamers can be designed and constructed as
described in Samie Jaffrey, J.P., Coupled recognition/detection system for in vivo and in vitro
use, 2010, Cornell University.
Fluorophore substrate: Corresponding fluorophore substrates in conjunction with
the above switchable aptamers can be designed and constructed as described in Samie
Jaffrey, J.P., Coupled recognition/detection system for in vivo and in vitro use, 2010, Cornell
University.
Construction of Pblaz-SAfusion: The SA gene can be fused to the Pblazpromoter
sequence such that the SA gene is operatively linked to the Pbzaz promoter.
Construction of the SA-expressing reporter vector: The SA-expressing reporter
vector can be constructed via standard molecular biological techniques by incorporating the
PbtaZ-SA fusion product into the MCS of the shuttle vector depicted in Example 4 above.
Propagation of the vector for conducting in vitro manipulations and for verification of
manipulations can be accomplished via the E. coli Top 10 and the final modified vector can
then be introduced into S. aureus RN045 LMerS. Transduction particles carrying shuttle
vector can be produced from the RN045 LMerS transformants via a Mitomycin C-induction
method that was first described in E. coli 1959 and is now a standard technique for obtaining
prophages from lysogenized bacteria. Otsuji, N. et al., Induction of Phage Formation in the
Lysogenic Escherichia coli K-12 by Mitomycin C. Nature, 1959. 184(4692): p. 1079-1080.
The cell lysate is then collected and contains non-replicative transduction particles each
consisting of bacteriophage cp 11 structural proteins carrying a linear concatamer of plasmid
DNA capable of expressing the SA within viable S. aureus cells within the cpl 1 host range.
Example 7: Non-Replicative Transduction Particle-Based Reporter System
The non-replicative transduction particles described above can be used in a
reporter system for detecting the presence of viable bacteria via the expression of a reporter
molecule (e.g. luxAB). When this transduction particle introduces a reporter vector (e.g.
pGW80AOOO 1) into a cell within the host range of the transduction particle, cells in which the
promoter (e.g. P clpB) is recognized by the cells transcription machinery are able to drive the
expression of the reporter molecule within that cell.
To test the functionality of non-replicative transduction particles as reporters for
detecting the presence of S. aureus cells, various MSSA/MRSA reporter assays were
developed. In an embodiment, a non-replicative transduction particle was developed from a
S. aureus-specific bacteriophage, and the bacterial luciferase genes luxAB under the control
of a constitutive promoter were incorporated. When the non-replicative transduction particle
delivered the reporter nucleic acid into S. aureus, the constitutive promoter expressed luxAB
suitable for reporting on the presence of a viable S. aureus.
In addition, the antibiotic cefoxitin was added prior to, simultaneously with, or
after the addition of the transduction particles to a sample containing S. aureus cells. If the
cells were not phenotypically resistant to cefoxitin (i.e., were not MRSA), luminescence was
decreased or eliminated, indicating that the cells were MSSA. If, however, the cells were
phenotypically resistant to cefoxitin (i.e., were MRSA), increased or detectable luminescence
was observed, indicating that the cells were MRSA.
Non-Replicative Transduction Particle-Based Viable Cell Reporter Assay
Function
The function of the non-replicative transduction particle as a reporter was assayed.
The transduction host range of the bacteriophage cp80a-based non-replicative transduction
particle was examined in 101 clinical MRSA isolates. The transduction assay was conducted
by exposing cultures of each bacterial isolate grown in modified TSB to GW24 cell lysate
containing the non-replicative transduction particles and culturing the mixture on solid media
containing tetracycline.
In this example, the non-replicative transduction particle carried a tetracycline
selectable marker. Cells transduced with the non-replicative transduction particles were
expected to be resistant to tetracycline. In addition, transduction was examined via
luminescence assay by exposing each bacterial isolate in liquid culture to cell lysate
containing the non-replicative transduction particles and evaluating the mixture for bacterial
luciferase luminescence activity after an incubation period.
The transduction assay showed that the cp80a-based non-replicative transduction
particle was able to transduce all of the 101 clinical isolates ofMRSA and none of the non-S.
aureus Staphylococci.
Figure 21 shows the results of the transduction assay in which 36 tetracycline-
sensitive MRSA were exposed to transduction particles carrying pGW80AOOO 1 and then
were spotted onto media plates containing 5 ug/mL of tetracycline. The results show that all
36 MRSA strains grew on the media containing tetracycline due to transduction with
pGW80AOOO 1. Control experiments in which MRSA isolates were spotted onto tetracycline
containing media without exposure to transduction particles showed no growth (not shown).
Furthermore, plasmid isolation from transduced MRSA strains demonstrated recovery of the
pGW80AOOO 1 plasmid as confirmed via sequencing of the isolated plasmid. The
transduction results thus demonstrated that the origin of replication of the reporter plasmid
exhibits activity on all of the MRSA isolates tested.
Figure 22 illustrates the luminescence measured from 80 clinical isolates of
MRSA and 28 clinical isolates of methicillin sensitive S. aureus (MSSA) transduced with the
transduction particle. In the experiment, cultures ofMRSA and MSSA were grown to an
optical density at 600nm of0.1 and then 100 uL of the cultures grown in Modified TSB were
mixed with 10 uL of GW24 cell lysates containing transduction particles and further
incubated at 3 7°C for a period of 4 hours prior to assaying for luminescence. Luminescence
measurements were conducted by adding 10 uL of a 1 mM solution of Decanal, an aldehyde
that triggers a luminescent reaction within cells expressing bacterial luciferase. As expected,
luminescence was observed from both MRSA and MSSA transduced with the S. aureus
specific non-replicative transduction particle. Furthermore, when cefoxitin was added to the
cell cultures at the same time as the addition of transduction particles, luminescence was
observed from MRSA but not from MSSA, thus demonstrating the ability for the transduction
particles to report on both the presence ofMSSA and ofMRSA. The luminescence results
thus demonstrate that the promoter driving luxAB expression exhibits activity on all of the S.
aureus isolates tested.
Optimization of Non-Replicative Transduction Particle-Based Viable Cell
Reporter MRSA Assay - Transduction Particle Reagent Formulation
The production and formulation of the non-replicative transduction particle
reagent was optimized to a final formulation. In summary, a 15 L scale fermentation was
performed using TSB media including peroxide induction of GW24. The 15 L fermenter
batch was inoculated from a 200 mL overnight seed culture (an inoculum ratio of 1.3%
(v/v)). The culture was induced at an O.D. of 0.8 with hydrogen peroxide and cooled to 25°C
post induction without pH or DO control. Culture supernatant was harvested by tangential
flow filtration (TFF) the following morning for the purpose of clarifying the phage
transduction particles from the cell debris. The material was then further concentrated and
diafiltered into SM Buffer without gelatin and stored at 2-8°C prior to final sterile filtration
and storage.
A detailed summary of the process is outlined below:
Seed flask growth
(1) Inoculate 200 mL ofTSB containing 5 ug/mL tetracycline with GW24
(2) Incubate at 37°C, 200 RPM for 10-18 hours.
Fermentation Innoculation (15L TSB with 5 ug/mL tetracycline)
(1) Prepare the fermenter skid with the following fermentation conditions: 37°C,
agitation at 250 RPM, airflow at 15 LPM, and backpressure at 3 psig.
(2) Inoculate the fermentor using the 200 mL overnight seed culture.
Induce Culture
(1) Once the OD600nm reaches 0.8 (0.6-0.9), induce the culture with 0.5 mM
H202
(2) Increase temperature setpoint of fermenter to 42°C
Post-Induction Conditions and Monitoring
(1) Once 30 minute induction is complete, reset temperature target for fermenter
to 25 °C
(2) One hour post cooling, tum off air feed to fermenter and set agitation to zero
(3) Monitor the fermentation culture at hour intervals, or more frequently as
necessary, until the OD600nm has decreased to or below 0.40.
Harvest/Clarification
(1) After the fermentation culture OD600 has reached a minimum less than or
equal to 0.40, take a 20mL aseptic sample and add 30µL ofBenzonase to the
fermentor.
(2) Reset agitation to 250 rpm. Allow 60 minutes with agitation for Benzonase
incubation.
(3) Clarify the EOF sample with a 15 minute centrifugation at 3000g.
( 4) Pass the clarified material through a 0.45uM membrane filter
Concentration and Buffer Exchange
(1) Concentrate the clarified culture by TFF using 500 kDa flat sheet membrane
IO-fold.
(2) Diafilter the concentrated culture at a constant volume against SM Buffer
without gelatin using the 500kDa TFF membrane used for concentration
Final Filtration
( 1) Filter the concentrated buff er exchanged material through a 0 .2 µm filter.
(2) Store the final filtered phage material at 2-8 °C.
Various other reagents and formulations can be used as known to those of skill in
the art to derive the formulation.
Optimization of Non-Replicative Transduction Particle-Based Viable Cell
Reporter MRSA Assay - Growth Media Formulation
A growth media formulation was optimized for the NRTP-based viable cell
reporter MRSA assay. In order to produce luminescence in the NRTP-based MRSA Assay,
the media needs to be balanced for Staphylococcus aureus growth and have adequate
concentration of cations and additives to favor NRTP transduction. The TSBmod media used
in assays prior to this development study was known to have precipitation issues that would
affect the stability of the media. Growth media formulation required stability in the final
formulation with a goal of 1 year at room temperature.
Methods/Procedures: Cells preparation for MRSA Assay
(1) Ten unique strains ofMRSA for the Subset Assay and one unique strains
ofMSSA were tested in the MRSA Assay.
(2) Overnight cultures were started in a deep 96 well plate at a 1 :50 dilution
in TSB from a frozen one-time use stock and incubated at 37°C on an
orbital shaker for> 15 hours. MRSA/MSSA (8µ1) in TSB (392µ1)
(3) The next day, a day culture at a 1 :50 dilution from the overnight culture
was started in TSB in 96 well deep well plate (392µ1 TSB + Sul cells) and
incubated at 37°C on an orbital shaker for 4 hours.
(4) Cells were spun in a centrifuge for 5 minutes at 1800g force and 10°C,
spent media was aspirated without disturbing the pellet.
(5) Spun cells were washed in 50mM Tris-HCl pH 7.2, centrifuged, buffer
aspirated without disturbing pellet and re-suspended in 400µ1 RPMI.
RPMI is used in order to reduce variability in the metabolic state of cells
and to mimic low metabolism as found in clinical samples.
(6) Plate was covered with airpore seal and incubated on bench for 48hrs.
(7) OD was read by transferring 200µ1 ofRPMI culture in a shallow well OD
plate and blank well with RPMI media alone was used to subtract blank
(8) Cells were normalized to OD 0.1 in 100µ1
(9) Another dilution was made 1: 10 in RPMI to yield an OD of 0.005
Assay base media was prepared to be tested as shown in Table 2 and a
representative set of media modifications in preparation for MRSA assay are shown in Table
Table 2: Base Media for Growth Media Formulation Development
Components TSB B2 BSS-2 Notes
Enzymatic Digest of Soybean Meal
(g) 3 0 3
Adjust pH to
Enzymatic Digest of Casein (g) 17 10 10
7.2 with ION
Yeast Extract (g) NIA 25 25
NaOH.
Sodium Chloride (g) 25 25
Autoclave or
Dipotassium Phosphate (g) 2.5 1 0
filter sterilize
alpha-D Glucose (g) 2.5 5 5
Volume (litre) 1 1 1
Table 3: Base Media Modifications for Growth Media Formulation
Development
Concentration of salt/
additives for modification
Mod CaCl2 MgCl2 BGP Tris-HCI A HEPES
Base media (30ml) number (mM) (mM) (mM) pH7.0 (mM) (mM) (mM)
B2 M53 5.0 2.0 0.0 50.0 10.0 0.0
BSS-2 M50 10.0 2.0 60.0 50.0 10.0 0.0
BSS-2 M54 6.7 3.3 60.0 50.0 0.0 0.0
BSS-2 M55 5.0 5.0 60.0 50.0 0.0 0.0
BSS-2 M56
6.7 3.3 60.0 0.0 0.0 10.0
BSS-2 M57 5.0 5.0 60.0 0.0 0.0 10.0
TSB (original)
.0 10.0 60.0 0.0 0.0 0.0
TSB M58 5.0 10.0 60.0 0.0 11.1 0.0
To each media preparation, NRTP and Cefoxitin was added according to Table 4
below to make the NRTP media reagent:
Table 4: MRSA Assay Growth Medial Transduction particle reagent
combination
Final
30ml media Concentration
Cefoxitin 5ug/ml
GW24 Lysate 30X
The MRSA Assay was run with the following steps:
(1) Assay Plate Setup: Add 198 µl of Phage Media Reagent and 2.0 µl of
each dilution of bacteria 0.05 OD and 0.005 OD in RPMI (roughly
equivalent to 20,000 and 2,000 CFU/mL, respectively) or 2.0 uL ofRPMI
as a blank.
(2) Incubate Assay Plate: Incubate Assay plate on orbital shaker at ~ 100 rpm
for 4 hours at 3 7°C.
(3) Prepare Luminometer (Molecular Devices SpectraMax L): Wash reagent
line with 70% ethanol followed by DI water then prime with the substrate
reagent. Set up software as Fast Kinetic with injection of 50 µL of
substrate reagent at 250 µl/sec after 10 baseline points and read at 40
points every 0.25 seconds.
(4) Run Assay: Test each bacterial dilution plate, after letting plate
equilibrate to room temperature for 5 minutes.
Analysis
(1) Determine cutoff by averaging blank RLU across all replicates and time
points and adding three standard deviations.
(2) Determine maximum RLU for each sample using SoftMaxPro.
(3) Determine ifthe maximum RLU was greater than the cutoffRLU, and if
so, then the sample data was used for comparisons of media performance.
(4) Normalize all max RLU values to the Max RLU in TSB Ml (media in use
until development started) for the strain being analyzed at the specific
dilution.
(5) Average the normalized RLU values across all MRSA strains for a
particular media and its modification
( 6) Average the averages for the two dilution plates, ultimately leading to a
single numerical value representing the fold increase in performance
based on RLU of a particular media across 10 different MRSA strains in 2
cell dilutions tested.
Results o[NRTP-Based Viable Cell Reporter MRSA Assay
Determination of CutoffRLU: The average and standard deviation of the RLU
was calculated across all time points (25) for each blank replicate (4). The cutoff was
calculated for each plate as the average blank RLU plus three standard deviations.
Determination of Relative Improvements: The maximum RLU was exported for
each sample (blanks, MSSA and MRSA at all dilutions) from SoftMaxPro and compared to
the cutoff RLU. If the sample had 2 data points greater than the cutoff for phage
concentration, then the max RLU value was utilized for analysis.
The values were normalized by dividing a particular max RLU by the max RLU
of its control condition (that strain in TSB Ml-origianl media, at the dilution being analyzed).
The ratios obtained were averaged across 10 MRSA for each media condition and each
dilution, as shown in Table 5. The average across the two dilutions is also shown in the table.
Table 5: MRSA Assay Results from Various Growth Media Formulations
Average for
Media Plate 1 Plate 2 both dilutions
B2 M53 1.89 1.88 1.89
BSS-2 M50 1.37 1.47 1.42
BSS-2 M54 1.50 1.76 1.63
BSS-2 M55 1.82 2.90 2.36
BSS-2 M56 2.38 4.19
6.00
BSS-2 M57 2.00 3.92 2.96
TSB Ml 1.00 1.00 1.00
TSB M58 1.18 0.96 1.07
Conclusions
BSS2-M56 exhibited the best performance on average across the various media
tested. HEPES buffer based media performed better than Tris-HCl buffered media. HEPES
is known to be a biologically favorable buffering system as opposed to Tris-HCl. B2 based
base/broth had better performance than TSB based broth.
Various other reagents and formulations can be used as known to those of skill in
the art to derive the formulation. Other suitable formulations were developed via similar
experiments as described above. Examples of other suitable formulations are included below
in Tables 6, 7, and 8.
Table 6: BSC Media Formulation
BSC Amount
Components
Enzymatic 14.5g
Digest of Casein
Yeast Extract 35.5g
Sodium 35.5g
Chloride
alpha-D Glucose 7g
Total Vol 1 L
Table 7: BSC Media Modification
BSC-M64
Chemical Name Final (Assay) Cone
BGP (mM) 60.0
HEPES (mM) 10.0
LiCl(mM) 84.0
BSC To 1 L
Table 8: Transduction Particle Media Modification
Transduction Particle Formulation
(PM4)
Chemicals Final (Assay ) cone
CaC12 (M)
0.00667
MgC12 (M) 0.00335
HEPES (M) 0.01000
GW24 lysate stock 0.01250
Sodium Azide (%) 0.0006
Water To 1 mL
Optimization of Non-Replicative Transduction Particle-Based Viable Cell
Reporter MRSA Assay - Substrate Reagent Formulation
In order to produce luminescence in the MRSA Assay, the Substrate Reagent must
include an aldehyde as a substrate for luciferase. An initially developed aliphatic aldehyde
formulation (4.2 mM Tridecanal in TSB) was not stable and formed a heterogeneous
emulsion rather than a solution. This example outlines the development of a Substrate
Reagent formulation that addresses these issues with a goal of 6 months at room temperature
or 2-8°C stability.
This example describes the steps that were taken to develop the Substrate Reagent
to a final formulation.
Methods/Procedures
All screening and stability experiments were tested using a "Model System" that
consists of S. aureus strain RN4220 harboring a LuxAB-expressing plasmid. The typical
preparation and testing method was as follows.
(1) Overnight Culture: 2mLTSB+1 uL of 10 mg/mL Tetracycline+ 1 colony
of Model System Bacteria from TSA plate, shaking at 225 rpm overnight
at 37°C
(2) Day Culture: Diluted overnight culture 1 :50 or 1: 100 into TSB+5 ug/mL
Tetracycline, shaking at 225 rpm for 1.5-2 hours at 37°C.
(3) Normalize Day Culture: Measured 1 mL of day culture on Nanodrop
with cuvette at 600 nm, blanking with TSB+5 ug/mL Tetracycline.
Diluted to 0.1 OD with TSB+ 5ug/mL Tetracycline.
(4) Dilute Culture for Testing: Diluted 0.1 OD Culture with TSB+ 5ug/mL
Tetracycline to a 1 :200, 1 :2000 and 1 :20000 dilution which was roughly
equivalent to 100000, 10000 and 1000 CFU/mL.
(5) Plate Bacteria: Added 200 uL of each dilution and a blank (TSB+5
ug/mL Tetracycline with no bacteria) in three replicates to a Greiner Bio
one white assay plate for each substrate to be tested.
(6) Prepare Luminometer (SpectraMax L): Wash reagent line with 70%
ethanol followed by DI water then prime with the substrate. Set up
software as Fast Kinetic with injection of 50 uL substrate at 250 ul/sec
after 10 baseline points and read at 40 points every 0.25 seconds.
(7) Run Assay: Test each formulation of Substrate Reagents with washing
and priming SpectraMax L between each substrate. Bring all Substrate
Reagents to room temperature before testing.
All confirmation experiments were tested using the MRSA Assay in order to
ensure similar results on the actual assay as the Model System used to screen new
formulations.
(1) Prepare Culture: Ten MRSA low performing strains and one MSSA strain
were grown to log-phase in TSB in a 2 mL deep well block. Cells were
spun down, washed with Ix PBS then resuspended in RPMI media.
(2) Normalize Bacteria: Measure 200 uL ofRPMI culture and RPMI blank in
Greiner Bio-one clear plate on VersaMax at 600 nm. Subtract blank OD
from each strain. Normalize each strain to 0.05 OD in RPMI media.
(3) Dilute Bacteria: Dilute 0.05 OD culture 1: 10 in RPMI media to 0.005 OD.
(4) Prepare Phage Media Reagent: Add Phage, Cefoxitin and Sodium
Pyruvate to BSS-M56 including:
a. Cefoxitin ( 5 ug/mL)
b. GW24 lysate stock (0.03X)
c. Sodium Pyruvate (0.025M)
(5) Set up Assay Plate= Add 198 uL of Phage Media Reagent and 2 uL of
each dilution of bacteria (0.05 OD and 0.005 OD in RPMI, roughly
equivalent to 20000 or 2000 CFU/mL) or 2 uL ofRPMI as a blank in two
replicates.
( 6) Incubate Assay Plate = Incubate Assay plate on orbital shaker at ~ 100
rpm (speed 3) for 4 hours at 37°C.
(7) Prepare Luminometer (SpectraMax L) =Wash reagent line with 70%
ethanol followed by DI water then prime with the substrate. Set up
software as Fast Kinetic with injection of 50 uL substrate at 250 ul/sec
after 10 baseline points and read at 40 points every 0.25 seconds.
(8) Run Assay= Test each formulation of Substrate Reagents with washing
and priming SpectraMax L between each substrate.
Experiments for the development of Substrate Reagent formulation were designed
to improve the following:
(1) Improve solubility via adding surfactants (Tween 20, Triton X-100, NP-
40, Brij-35, SNS, etc.), adding solvents (Ethanol, Methanol, DMSO, etc.),
adding non-volatile oils (Castor Oil)
(2) Improve stability via adding stabilizers (Triethanolamine, Cyclodextrin
etc.), adding antioxidants (Vitamin E, Vitamin E Acetate, Vitamin E PEG
1000, Oxyrase, etc.), adjust method ofTridecanal addition (with
surfactant, with solvent, into final solution, with antioxidant, etc.), storing
Tridecanal and Substrate Reagent under nitrogen to reduce oxidation of
aldehyde, and reducing possibility of microbial contamination by adding
preservatives such as ProClin and by sterile filtration of the Substrate
Reagent.
(3) Improve Assay Performance via adjustment of the pH of the formulation
and the pH buffer system
(4) Improve overall performance via determining the aldehyde with highest
RLU output (tested aldehydes from 6-14 carbons in multiple formulations
to determine if an improvement in solubility, stability and assay
performance was observed).
(5) Improve overall performance via adding antifoam in order to reduce
foaming during preparation of reagent and addition of reagent to sample
during the assay.
Analysis and Results
The kinetic reaction was plotted for each sample and a line fit to the average at
each read point of three replicates. Typically results showed at 1 :2000 dilution of 0.1 OD
model system bacteria, roughly equivalent to 10,000 CFU/mL or 2,000 CPU/assay.
The normalized maximum RLU to that of the reference substrate reagent was
analyzed for stability experiments. At each stability time point, maximum RLU for each
sample was normalized to the reference substrate maximum RLU. Normalized Maximum
RLU was plotted over time points and linear regression with 95% CI was plotted.
Conclusions
The key parameters adjusted from the reference formulation for producing a final
Substrate Reagent formulation are summarized in Table 9.
Table 9: Summary o(Reagent Formulation Development Results
Modification to Substrate Reagent Reason
4.2 mM Tridecanal+TSB Original Substrate Reagent
Reduce possibility of
Remove TSB contamination
Add 1 % Tween 20 Improve Solubility
Adjust to pH3 with 79.45% 0.1 M
Citric Acid-19.55% 0.2 M Sodium
Phosphate Dibasic Buffer Improve Assay Performance
Add Tridecanal directly to
concentrated surfactant Improve Stability
Add Filtering of Substrate Reagent
through 0.2 um PES membrane Improve Stability
Add 0.05% ProClin 300 Improve Stability
Add Triethanolamine Improve Stability
Change 1 % Tween 20 to 0.5% Triton Improve Stability, Improve
X-100 Solubility
Change from 79.45% 0.1 M Citric
Acid-19.55% 0.2 M Sodium Phosphate
Dibasic Buffer to 82% 0.1 M Citric Improve Assay Performance,
Acid-18% 0.1 M Sodium Citrate reduce possibility of precipitation
Buffer, remain at pH3 with removal of phosphate buffer
Add 100 ppm Antifoam Y30 Improve Assay Performance
Improve Stability, reduce
Add 0.5% Vitamin E Acetate precipitation
Change Primary Tridecanal
Manufacturer from Alfa Aesar to
Sigma/OmegaChem Improve Assay Performance
Improve Assay Performance,
Change 0.5% Vitamin E Acetate to 1- Improve Solubility,
2% Vitamin E PEG 1000 Improve Stability
Two Substrate Reagent Formulations were prepared for two different storage
temperatures, one for storage at 2-8°C and one at 18-24°C.
Final Substrate Reagent Formulations stored at 2-8°C. Formulation: 0.5% Triton
X-100+ 4.2mM Tridecanal+ 0.5% Vitamin E Acetate+ 100 ppm Antifoam Y30+ 0.5%
Triethanolamine+ 82% 0.1 M Citric Acid+ 18% 0.1 M Sodium Citrate @pH3+ 0.05%
ProClin 300. The formulation did not precipitate after 1 month at 2-8°C and was able to
detect MRSA strains the same as on Day 0.
Final Substrate Reagent Formulations stored at 18-24°C. Formulation: 0.5%
Triton X-100+ 6.3mM Tridecanal+ 100 ppm Antifoam Y30+ 0.5% Triethanolamine+ 82%
0.1 M Citric Acid+ 18% 0.1 M Sodium Citrate @pH3+ 2% a-Tocopherol-PEG 1000
Succinate+ 0.05% ProClin 300. The formulation did not precipitate after 1 month at 18-24°C
and was able to detect MRSA strains the same as on Day 0.
Various other reagents and formulations can be used as known to those of skill in
the art to derive the formulation.
Analytical Performance of Non-Replicative Transduction Particle-Based Viable
Cell Reporter MRSA Assay
The analytical performance of the optimized NRTP MRSA assay was examined,
including an analysis of the assay's limit of detection and an analysis of the cross-reactivity
and microbial interference of the assay when challenged with non-target organisms.
A) Limit o(Detection Assay
The Limit of Detection of the NRTP assay was assessed via determining the
lowest amount of MRSA cells representing various strains that could produce a relative light
unit (RLU) signal above that of a threshold determined from blank samples. MRSA strains
included the SCCmec Types I, II, and IV as well as a MRSA strain carrying the mecA gene
variant mecC - a strain ofMRSA that conventional FDA-cleared MRSA PCR assays have
failed to detect.
The following key materials were used in the clinical performance study:
Growth Media Reagent: BSS-M56
Substrate Reagent: Final Substrate Reagent Formulations to be stored at 18-24°C
as described above.
Transduction Particle Reagent: BSS-M56 base with lOug/mL (i.e. 2X
concentration) cefoxitin and transduction particle reagent as described above at 2X
concentration.
LoD Study Protocol:
Overnight Culture: For each MRSA strain and a MSSA negative control strain, 2
mL of TSB were inoculated with a colony of the strain previously grown on TSA plates.
Overnight MRSA cultures included 5ug/mL cefoxitin. All samples were incubated overnight
at 3 7°C in a shaking incubator.
Day Culture: 20 uL of each of the overnight cultures were transferred into a new
culture tube containing 2 mL of Growth Media Reagent. The inoculums were then incubated
at 37C with shaking for approximately lhr 45min, until the OD(600nm) reached 0.1.
Serial dilutions:
a) lOOOuL of each of the samples were dispensed into row A of 2mL deep
well 96-well plate.
b) The remaining rows (B-H) were then filled with 900 uL of Growth
Media Reagent.
c) 10-fold serial dilutions were then prepared taking 1 OOuL from row A
and mixing in row B, etc., such that row H contained samples of row A
material at 10- dilution.
Enumeration of bacterial load: 5 uL of each well of row E was spotted onto a TSA
plate which was then tilted to allow the spot of liquid to spread onto the plate (in order to
later facilitate colony counting). (Row Eis a 10- dilution of row A). Plates were then
incubated overnight at 37°C.
Assay preparation:
a) Wells of a white 96 well assay plate were filled with 100 uL of 2x
Transduction Particle Reagent.
b) Rows F and G (i.e., 10- and 10- -fold dilutions of row A, respectively) were
then used to fill wells of the 96 well assay plate containing Transduction Particle Reagent
such that each sample was added to the plate in four-replicates.
c) The plate was then sealed with a breathable seal and incubated for 4 hours at
37°C with moderate shaking, 50rpm.
At the end of 4 hours, the plate was remove from incubator and immediately
measured for luminescence on a SpectraMax L that injected 50 µl of the Substrate Reagent
and measured luminescence for a period of 1 minute.
Analysis:
The luminescence data from each sample was plotted as RLU vs. time. Blank
samples were used to determine a Cutoff calculated from all time points of the blank samples
using the following formula: (Mean Blank RLU + 3* SD Blank RLU)
The average peak RLU post-substrate injection was then obtained for each sample
in order to determine the sample of highest dilution for which an RLU value was generated
that was above the blank samples Cutoff. The colony forming unit (CFU) counts at the
highest dilution for which an RLU value was generated that was above the blank samples
Cutoff was determined from the enumeration study, and this CFU count was reported as the
LoD in the study.
Results:
The LoD for all MRSA samples tested was determined to be below 10 CFU.
Table 11 summarizes the results of the lowest LoDs obtained in the study.
Table 11: Results of the lowest LoDs obtained in the LoD study.
SCCmec LoD
Type (CFU)
II 2
IV 3
mecC 1
All MRSA strains tested resulted in fewer than 10 CFU detected with the NRTP
assay above a Cutoff calculated from blank samples. MSSA did not generate RLU values
above the blank samples Cutoff.
RLU values are shown at the highest dilution for which an RLU value was
generated that was above the blank samples Cutoff were plotted as the average RLU value
and standard deviation for the four replicates tested for each sample. The horizontal axis is
set at the blank samples Cutoff and the CFU counts for the sample that generated each RLU
data point is superimposed with the data. All MRSA samples generated RLU values above
the Cutoff while MSSA did not.
Cross-Reactivity and Microbial Interference Study
A cross-reactivity and microbial interference study was performed. The purpose
of the study was to test a set of bacterial strains commonly encountered in clinical samples
and known to potentially be in the host range of the bacteriophage cp80a in the MRSA Assay
to see if there was cross reactivity or interference of these strains with phage or substrate used
in the test.
Previous experiments with clinical samples had resulted in false positive results
with a presence of Enterococci faecal is and Staphylococcus epidermidis as indicated from the
presence of blue and white colonies when plating on BBL™ CHROMagar™ Staph aureus
plates. In addition, Listeria monocytogenes and Listeria innocua may be within the infective
or penetrative host range of the phage cp80a which may also contribute to cross-reactivity in
the MRSA assay. The study tested Enterococci faecalis, Staphylococcus epidermidis,
Listeria monocytogenes and Listeria innocua for cross reactivity/ interference with Viability
6 7 8
MRSA assay. Each strain was tested at high cell numbers in the order of 10 , 10 or 10 cells
in the assay volume. Tests were done without the addition of GW24 lysate to address
potential autoluminescence of strains.
Experiment 1 tested various strains (MSSA-S121, NRS# 9-
Staphylococcus haemolyticus, NRS # 6- Staphylococcus epidermidis, ATCC 12228-
Staphylococcus epidermidis, ATCC 15305- Staphylococcus saprophyticus, ATCC 29212-
Enterococcus.faecalis, ATCC 60193-Candida albicans, ATCC 12453-Proteus mirabilis) for
luminescence at high cell numbers under normal assay conditions.
Experiment 2: A subset of strains that were luminescent from Experiment 1 were
re-assayed in the presence of various antibiotics at various concentrations to quench
background luminescence.
Experiment 3: E. faecalis and S32 (MRSA) were tested with various substrate
formulations developed as described above without GW24 lysate and without incubation.
Experiment 4: ATCC 33090-Listeria innocua and ATCC 191 l l-Listeria
monocytogenes were tested for background signal and non-specific luminescence and retested
with various substrate formulations developed as described above along with E. faecal is and
S. epidermidis.
Experiment 5: E. faecalis was retested with a final substrate formulation
developed as described above.
Substrate Reagent formulations tested in this study are summarized in Table 9.
Table 10: Substrate Reagent Formulations
Experiment Substrate Description
1 Original Substrate
1% Tween20 + 4.2 mM Tridecanal, pH3.0
2 Original Substrate
Substrate 1 6.3 mM Tridecanal + 0.5% Vitamin E Acetate, pH 3.0
Substrate 2 20 mM Nonanal + 0.5% Vitamin E Acetate, pH 3.0
Substrate 3 8.4 mM Tridecanal + 0.5% Vitamin E Acetate, pH 3.0
6.3 mM Tridecanal + 1% a-Tocopherol-PEG 1000 Succinate,
Substrate 4
pH 3.0
4 Original substrate 1 % Tween20 + 4.2 mM Tridecanal, pH 3.0
0.5% Triton+ 4.2 mM Tridecanal (Sigma) + 0.5% Vitamin E
Substrate 5
Acetate, pH 3.0
Substrate 6 6.3mM Tridecanal + 2% VitE PEG, pH 3.0
Methods/Procedures:
The following were steps performed for the MRSA Assay.
A) Strains Grown for Experiments 1-5
On the day before the assay, an overnight culture was started in a deep 96 well
plate at a 1 :50 dilution in TSB from a frozen one-time use stock and incubated at 37°C on an
orbital shaker for> 15 hours. Bacteria (8 uL) in TSB (392 uL ).
The absorbance of culture was measured on Versamax. The TSB was set as blank
in template on SoftmaxPro. Optical density (OD) was measured at 600nm.
On the day of the assay, cells were re-suspended to an OD 0.5 to set up the assays.
Prepared BSS-M56 for Experiments 1-5.
B) Transduction particle media reagent was prepared for all Experiments l, 2, 4
and 5 (no transduction particle reagent used in Experiment 3): 15 ug/mL cefoxitin + GW24
lysate stock from as described above at 30X.
C) Sample Preparation: Various dilutions were made from overnight cultures of
strains. All strains were diluted BSS M56.
D) MRSA Assay was run for Experiments 1-5
Media was loaded with or without phage and cefoxitin at 5 µg/ml to assay plate.
2.5ul cells were added. The assay plate was incubated with a plate lid at 37°C on an orbital
shaker with the speed set to approximately 100 rpm for 4 hours.
Next, the assay plates were measured on the SpectraMax L with the following
standard assay parameters:
Fast Kinetic Luminescence
Read for 20 time points at 0.5 second intervals. Substrate was injected with M
injector with 50 uL/well at 250 ul/sec including 5 baseline reads. No incubation temperature
was set and was read at room temperature.
The SpectraMax L was primed with Substrate Reagent before running the assay.
The results were analyzed with the following:
A) Determined cutoff by averaging blank RLU across all replicates and time
points and adding three standard deviations.
B) Determined maximum RLU for each sample using SoftMaxPro.
C) Determined if the maximum RLU was greater than the cutoffRLU, and if so,
then the sample data was used for analysis.
Results Summary
Experiment 1: Various strains were tested for cross reactivity and interference
using the Original Substrate formulation, out of those tested, NRS# 9- S. haemolyticus, NRS
# 6- S. epidermidis and E. faecalis tested false positive in MRSA assay.
Experiment 2: Out of the three strains tested, NRS #9 and E.faecalis tested
MRSA positive with all Cefoxitin conditions tested. All three strains (NRS #9, E.faecalis,
NRS #6) tested positive when no transduction particle reagent was used in the assay,
indicating that non-specific luminescence was not transduction particle reagent-dependent but
rather strain and substrate reagent dependent. Carb (Carbencillin) at all concentrations tested
was effective in removing the false positive signal.
Experiment 3: E. faecalis gave a positive signal without transduction particle
reagent. MRSA strain S32 also gave a positive signal without transduction particle reagent.
This result was indicative of the substrate reagent causing background luminescence.
Substrate 4 was effective in eliminating background signal in the assay.
Experiment 4: Strains ATCC 33090 -Listeria innocua, ATCC 191 I I-Listeria
monocytogenes, were tested for luminescence with transduction particle reagent and substrate
reagent as Listeria sp. can be within the host range of the bacteriophage used in the MRSA
assay. Luminescence was observed from L. innocua with and without transduction particle
reagent using Original Substrate formulation indicating that the luminescence was due to
non-specific reaction potentially with the substrate. Substrate 5 was effective in eliminating
luminescence from Listeria but not E. faecal is.
Experiment 5: Retested E. faecalis with Substrate 6. In two independent runs on
two different days with high load of cells at 0.5 OD, the assay yielded negative results.
Conclusions
The cross-reactivity study demonstrated background luminescence from several
bacterial species at high loads. The light output did not require transduction particle reagent
and certain substrate formulations utilizing phosphate ions contributed to non-specific signal.
Because no light output from cross-reactive species was observed from the use of
transduction particle reagent, in the case that cp80a penetrates cross-reactive species, light
output is prevented from the lack of activity of the S. aureus PclpB promoter that is
operatively linked to the bacterial luciferase genes and/or the lack of activity of the S. aureus
pT181 origin ofreplication within these species.
Replacing the buffer from sodium phosphate dibasic in the formulation with
sodium citrate and citric acid eliminated background luminescence from all cross-reactive
species tested except for E. faecal is. Substrate 6 with the added ingredient of a tocopherol
PEG 1000 Succinate eliminated the remaining non-specific signal from E. faecalis.
Clinical Performance of Non-Replicative Transduction Particle-Based Viable
Cell Reporter MRSA Assay - Results with Reference to Direct Plating onto
CHROMAgar MRSA II
A MRSA screening assay was developed employing cp80a-based luxAB
expressing non-replicative transduction particles (NRTP). The assay consisted of adding
NRTP to a clinical sample suspected of containing MRSA, incubating the sample for a period
of 4 hours at 37°C, and then assaying the incubated sample by injecting an aldehyde into the
sample while measuring for luminescence with a photomultiplier tube. The results of the
assay were compared to that of commercially available chromogenic media designed for the
detection of MRSA as a reference in order to determine the sensitivity and specificity of the
assay. The NRTP-based assay was expected to correlate well with the culture-based
reference since both require the presence of viable MRSA cells and both rely on the
expression of the MRSA phenotype. The results showed excellent correlation with the
reference.
The purpose of the study was to determine the performance of the NRTP-based
MRSA Assay with reference to CHROMAgar MRSA II from testing remnant nasal swab
samples collected for the purpose of MRSA screening.
Scope:
De-identified nasal swab samples collected from patients for the purpose of
MRSA surveillance by a clinical institution were tested for the presence of MRSA using the
NRTP-based MRSA Assay, CHROMAgar MRSA II, CHROMAgar SA and Blood Agar TSA
via direct plating and via enriched culture followed by plating. The results of the NRTP
based MRSA Assay were compared with the results of the CHROMAgar MRSA II assay in
order to calculate the sensitivity and specificity of the NRTP-based MRSA Assay with
reference to CHROMAgar MRSA II.
The following key materials were used in the clinical performance study:
Growth Media Reagent: BSS-M56
Substrate Reagent: Final Substrate Reagent Formulations to be stored at 18-24°C
as described as described above
Transduction Particle Reagent: BSS-M56 base with lOug/mL (i.e. 2X
concentration) cefoxitin and transduction particle reagent as described above at 2X
concentration.
Methods/Procedures
Clinical Sample Description: Sample transport tubes containing liquid Amies
(220093 - BD BBLTM CultureSwabTM Liquid Amies) were provided to a clinical institution
for collecting de-identified remnant nasal swabs collected by the clinical institution. Prior to
placing the nasal swabs into the provided sample transport tube, the clinical institution used
the swab for performing their own direct culture MRSA screening by streaking the swab onto
a culture plate. More specifically, anterior nares specimens were collected at the clinical
institution internal standard procedures and using the clinical institution's standard collection
swab. The clinical institution then performed direct culture screening with the swab. The
remnant swab was then added to the sample transport tube in which the swab tip was
submerged in the Amies buffer in the sample transport tube. Samples were then kept at room
temperature for 2-24 hours prior to further processing.
Sample Handling: Upon receipt, samples were stored overnight at room
temperature in a biosafety cabinet upright to ensure swab immersion in the sample transport
tube Amies buffer. After overnight storage, samples were further processed as follows.
Clinical Sample Preparation
Using a 1 mL Pipette, 300 µl of Growth Media Reagent was added to 15 mL
falcon tubes.
The swabs from remnant nasal swabs were removed from the original transport
tube and immersed into the Growth Media Reagent in a corresponding falcon tube. The swab
contents were then eluted into the Growth Media Reagent in the falcon tube by rolling it back
and forth in the Growth Media Reagent 4-6 times. The swab was then placed back into the
original transport tube and stored at 2-8 °C until the end of the study while the eluted clinical
samples in the falcon tube were transferred to 1.5 mL tubes and kept at room temperature
until further processing.
Running the NRTP MRSA Assay: The following samples were loaded directly
into a white 96 well assay plate.
Clinical Samples: 100 µl of the eluted material of each clinical sample in singlet.
MRSA positive control: 2 µl of a thoroughly mixed 0.1 OD culture of a known
MRSA isolate into 98 uL of Growth Media Reagent in triplicate.
MSSA negative control: 2 µl of a thoroughly mixed 0.1 OD culture of a known
MSSA isolate into 98 uL of Growth Media Reagent in triplicate.
Blanks: 100 µl of Growth Media Reagent in triplicate.
To each sample, 100 µL of Transduction Particle Reagent was added. The assay
plate was then placed in an incubator set at 3 7°C, shaking on orbital shaker for 4 hours. At
the end of 4 hours, the plate was removed from incubator and immediately measured for
luminescence on a SpectraMax L that injected 50 µl of the Substrate Reagent and measured
luminescence for a period of 1 minute.
Bacteria Plating for clinical sample CFU enumeration: Each eluted clinical sample
was plated in order to determine bacterial colony counts on CHROMAgar MRSA II,
CHROMAgar SA and Blood Agar (TSA II) via direct and enriched culture as follows.
Organism CFU counts were determined by direct plating. MRSA CFU counts were
determined by plating on CHROMAgar MRSA II. S. aureus CFU counts were determined
by plating on CHROMAgar SA plate. CFU counts of any organism whose growth is
supported by Blood Agar TSA were determined by plating on Blood Agar TSA. In the case
that direct plating did not produce colonies due to the load of organisms being below the limit
of detection of the plates used, sample enrichment was also performed by incubating a
portion of the eluted clinical sample in TSB overnight at 3 7°C with shaking and then again
plating the enriched culture on CHROMAgar MRSA II. All plates were incubated for 20-24
hours at 37°C. After incubation, the CFU counts of any colonies appearing on each plate
were recorded.
Analysis: The presence and CFU load ofMRSA, S. aureus, and total organisms
per eluted clinical sample were calculated based on the CFU counts obtained on
CHROMAgar MRSA II, CHROMAgar SA, and Blood Agar TSA, respectively.
NRTP Assay analysis: Data from each sample were plotted as RLU vs. time.
Cutoff Determination: The Assay Cutoff was calculated from all time points of
the blank samples using the following formula: (Mean Blank RLU + 3* SD Blank RLU).
MRSA Positive Determination: The RLU of each time point after substrate
injection was determined to be above or below the Assay Cutoff. If two or more data points
after injection were above the Assay Cutoff then the sample was designated as "MRSA
Positive."
Results: The MRSA positive results of the NRTP Assay were compared to those
of the direct and enriched culture plating onto CHROMAgar MRSA II. The following
calculations were conducted in order to determine the NTRP Assay Sensitivity and
Specificity with reference to CHROMAgar MRSA II.
True Positive (TP)
0 Sample that produced a MRSA positive result on both the NR TP
Assay and CHROMAgar MRSA II
True Negative (TN)
0 Sample that produced a MRSA negative result on both the NRTP
Assay and CHROMAgar MRSA II
False Positive (FP)
0 Sample that produced a MRSA positive result on the NR TP Assay and
a MRSA negative result on CHROMAgar MRSA II
False Negative (FN)
Sample that produced a MRSA negative result on the NR TP Assay and
a MRSA positive result on CHROMAgar MRSA II
Sensitivity= TP/(TP+FN)
Specificity= TN/(TN+FP)
Results with Reference to Direct Plating onto CHROMAgar MRSA II
Table 11 shows following results were obtained comparing the NRTP Assay with
reference to direct plating on CHROMAgar MRSA II.
Table 11. NRTP Assay Results vs. Direct Plating on CHROMAgar MRSA II
Results
Total CHROMAgar CHROMAgar NRTP NRTP True True False False
Samples MRSAII MRSAII ASSAY ASSAY Positive Negative Positive Negative
Positive Negative Positive Negative
62 12
69 7 57 7 57 5 0
Based on the above data, the sensitivity and specificity of the assay with reference
to direct plating onto CHROMAgar MRSA II were calculated to be:
Sensitivity = 100%
Specificity= 92%
Clinical Performance of Non-Replicative Transduction Particle-Based Viable
Cell Reporter MRSA Assay - Results with Reference to Enriched Culture,
followed by Plating onto CHROMAgar MRSA II
Based on the results with reference to direct plating on CHROMAgar MRSA II,
all clinical samples were re-tested with reference to enriched culture, followed by plating on
CHROMAgar MRSA II. The rationale for the follow-on testing was based on the possibility
that false positive results when compared to direct plating may indeed be true positives that
were detected by the NR TP assay but may have been missed by direct plating. A portion of
the remaining eluted swab samples were re-tested via the NRTP assay as described above.
Another portion of the remaining eluted swab samples were also tested via enriched culture,
followed by plating onto CHROMAgar MRSA II. Enriched culture testing consisted of
adding 100 uL of the remaining eluted swab material to 2 mL ofTSB and incubating at 37C
with shaking for a period of 18-24 hours. The resulting culture was then streaked onto
CHROMAgar MRSA II in order to determine the presence ofMRSA in the culture. Table 12
summarizes the data from both the direct plating and enrichment followed by plating assays -
only the samples that produced a MRSA positive result on either NRTP Assay or
CHROMAgar MRSA II are shown.
Table 12: NRTP Assay Results vs. Direct Plating and Enriched Culture
followed by Plating on CHROMAgar MRSA II
Only the samples that produced a MRSA positive result on either NRTP Assay or
CHROMAgar MRSA II are shown.
Direct Enrichment +
Sample Enrichment +
NRTP Assay CHROMagar MRSA CHROMagar MRSA
# NRTP Asssay
II II
1 + + + +
2 + + + +
+ + + +
+ + + +
+ + + +
6 + + + +
7 + + + +
+ + +
9 + - + +
+ - + +
11 -
+ + +
12 - -
Table 13 shows following results were obtained comparing the NRTP Assay with
reference to enriched culture of clinical samples, followed by plating on CHROMAgar
MRSAII.
Table 13. NRTP Assay Results vs. Enriched Culture Followed By Plating on
CHROMAgar MRSA II Results
Total CHROMA CHROMA NRTP NRTP True True False False
Sam pl garMRSA garMRSA ASSA ASSA Positiv Negati Positiv Negati
es II Positive II Negative e ve e ve
Positiv Negati
e ve
69 11 58 12 57 11 57 1 0
Based on the above data, the sensitivity and specificity of the assay with reference
to enriched culture followed by plating onto CHROMAgar MRSA II was calculated to be:
• Sensitivity = 100%
• Specificity= 98.3%
Example 8: NRTP-Based Assay For Antimicrobial Susceptibility Testing -
Correlation Of Minimum Inhibitory Concentration To Luminescence Output
In another example, a S. aureus cefoxitin susceptibility assay was developed to
determine the minimum inhibitory concentration of cefoxitin required to inhibit the growth of
cefoxitin resistant S. aureus. Unlike a MRSA cefoxitin resistance assay as described above,
which differentiates cefoxitin sensitive from cefoxitin resistant S. aureus, the MRSA
cefoxitin susceptibility assay in this example describes the development of an assay to
determine the minimum amount of cefoxitin needed to inhibit the grown of S. aureus in the
presence of cefoxitin.
The following key materials were used in the clinical performance study:
Growth Media Reagent: BSS-M56
Substrate Reagent: Final Substrate Reagent Formulations to be stored at 18-24°C
as described in Example 7.
Transduction Particle Reagent: BSS-M56 base with lOug/mL (i.e. 2X
concentration) cefoxitin and transduction particle reagent as described in Example 7 at 2X
concentration MIC Study Protocol.
Overnight Culture: For each MRSA strain (NRS35 and S7) and a MSSA negative
control strain (MSSA121), 2 mL ofTSB were inoculated with a colony of the strain
previously grown on TSA plates. Overnight MRSA cultures included 5ug/mL cefoxitin. All
samples were incubated overnight at 3 7°C in a shaking incubator.
Day Culture: 20 uL of each of the overnight cultures were transferred into a new
culture tube containing 2 mL of Growth Media Reagent. The inoculums were then incubated
at 37C with shaking for approximately lhr 45min, until the OD(600nm) reached 0.1.
MIC determination via plating:
a) Each of the day cultures was streaked onto TSA plates containing cefoxitin at 4,
8, 16, 32, 64, and 128 ug/mL.
b) Plates were incubated for 18 hours at 37C to determine growth.
NRTP Assay preparation:
a) Wells of a white 96 well assay plate were filled with 100 uL of 2x
Transduction Particle Reagent.
b) For each of the day cultures, five wells were then filled with 100 uL of day
culture.
c) For each of the day cultures, cefoxitin was added to one well each such that
the cefoxitin concentration in the well was at 4, 8, 16, 32, 64, and 128 ug/mL.
d) The plate was then sealed with a breathable seal and incubated for 4 hours at
37°C with moderate shaking, 50rpm.
At the end of 4 hours, the plate was remove from incubator and immediately
measured for luminescence on a SpectraMax L that injected 50 µl of the Substrate Reagent
and measured luminescence for a period of 1 minute.
Analysis:
The maximum luminescence value after Substrate Reagent addition from each
sample was plotted. MSSA sample RLU values were used to determine a Cutoff calculated
using the following formula: (Mean MSSA RLU + 3* SD MSSA RLU).
Results:
Figure 23 shows the results of S. aureus growth at 4, 8, 16, 32, 64, and 128 ug/mL
of cefoxitin. Figure 24 shows the RLU values obtained by the NRTP assay in the presence of
4, 8, 16, 32, 64, and 128 ug/mL cefoxitin. The x-axis in Figure 24 is set at the MSSA RLU
cutoff value.
As can be seen in Figure 23, MRSA NRS25 exhibited a MIC of 128 ug/mL
cefoxitin while MRSA S7 exhibited a MIC of 64 ug/mL cefoxitin. Correspondingly, MRSA
NRS25 exhibited appreciable luminescence above the MSSA RLU cutoff to a cefoxitin
concentration up to 64 ug/mL cefoxitin while MRSA S7 exhibited luminescence above the
MSSA RLU cutoff to a cefoxitin concentration up to 32 ug/mL.
Based on the above data, the NRTP assay demonstrates that RLU values obtained
from the assay correlate with MIC results and thus the NRTP assay may be used to develop
antibiotic susceptibility assays.
Example 9: Transcript Reporter Assay: Mechanism of Conformational Change
by RBS-Blocking Cis-Repression Of Luxab Translation Activated By The mecA
Gene Transcript Of Mrsa
As described above, a reporter transcript can be designed such that translation of
the reporter gene sequence is blocked by cis-repression of the ribosome-binding site (RBS) of
the reporter gene.
The following tools were used for designing the reporter transcripts of the
invention.
1) RNA secondary structure was calculated using secondary structure program,
such as Mfold (http://mfold.rna.albanv.edu/?q=rnfold/RNA-Folding-Form).
2) Intermolecular RNA interactions were calculated using a software program
such as RNA-RNA InterACTion prediction using Integer Programming (RactIP)
(httn://ma.naisLjn/ractin/).
3) RNA secondary structure was visualized using Visualization Applet for RNA
(VARNA) (hH12;l/.y~rn.~Jr.L.fr!.).
Figure 25 shows a secondary structure of the mecA transcript generated based on
the lowest energy conformation calculated by MFold and visualized with VARNA. The
terminal loop 23 (T23) contains a YUNR sequence UUGG consisting of bases 1,487-1,490 of
the mecA transcript sequence. Analysis of the secondary structure of the mecA gene
transcript revealed several ssRNA regions that were suitable for designing a cis-repressed
luxAB reporter that can be de-repressed via interactions between the reporter and an ssRNA
region.
As shown in detail in Figure 26, the terminal loop 23 (T23) of the mecA transcript
contains a YUNR consensus sequence. A YUNR (pYrimidine-Uracil-Nucleotide-puRine)
consensus sequence has been shown to be a critical target for intermolecular RNA complexes
in natural systerns, A cis-repressing sequence was designed to form a stem-loop structure
with the RBS of the reporter sequence, such that the cis-repressing sequence blocks binding
of an RNA polymerase to the RBS of the reporter sequence. The reporter sequence was
exposed upon binding of the loop of the cis-repressing stem-loop structure with T23 of the
mecA transcript.
As shown in Figure 27, a cis-repressing sequence 2701 was added to the 5'
terminus of the luxAB genes and designed to form a stem-loop structure that blocks the RBS
sequence ("AAGGAA") 2702 of the luxA gene. The cis-repressing stem-loop structure was
predicted to block the luxA RBS ("AAGGAA") sequence, based on the lowest energy
conformation of the luxAB transcript including the cis-repressing sequence at the 5' terminus
of the luxAB transcript, as calculated by MF old and visualized with VARNA.
The first 61 nucleotides of the cis-repressed luxAB genes are shown in up
to the start codon AUG of the luxA gene. The RBS sequence "AAGGAA" includes bases 47-
52. This terminal loop of the reporter transcript was designed to interact with (bind to) the
terminal loop 23 (T23) of the mecA transcript, which contains a YUNR sequence.
The terminal loop of the cis-repressing sequence was designed to interact with
T23 of the mecA transcript, such that hybridization of the cis-repressed luxAB transcript and
the mecA transcript via the interaction of the loop from the cis-repressing stem-loop structure
and T23 of the mecA transcript results in exposure of the RBS of the luxA gene. Figure 28
shows the predicted inter-molecular interactions between the mecA T23 sequence and the cis
repressing sequence on the luxAB transcript calculated by RactIP and visualized by VARNA.
Lines indicate base pairing between the mecA transcript and the cis-repressed luxAB
transcript. The interaction between the two sequences results in exposure of the luxA RBS
sequence AAGGAA and thus de-repression of the luxAB reporter.
Example 10: Transcript Reporter Assay: Methods of Detecting Target
Transcripts or Genes Using a mecA -luxAB Reporter System
In another example, a method for detecting a target mecA gene is provided using a
mecA-luxAB reporter system. Here, mecA is the target transcript, and luxAB is the reporter
molecule.
1. Construction of the Reporter Construct
A vector comprising a reporter construct encoding luxAB can be constructed via
standard molecular biological techniques by incorporating the reporter construct into a shuttle
vector capable of propagating in both E. coli and S. aureus. The vector can contain an origin
of replication that is functional in E. coli and a selectable marker that is expressed in E. coli
and suitable for allowing the growth of E. coli cells transformed with the vector and grown
under selective conditions. The vector can also contain an origin of replication that is
functional in S. aureus and a selectable marker that is expressed in S. aureus and suitable for
allowing the growth of E. coli cells transformed with the vector and grown under selective
conditions. Propagation of the vector for conducting in vitro manipulations and for
verification of manipulations can be accomplished via a suitable laboratory cloning strain of
E. coli and the final modified vector can then be introduced into S. aureus strains.
The reporter construct can be first introduced into a S. aureus cell for transcribing
the construct and producing the reporter transcript.
2. Construction ofa cis-Repressed Reporter Transcript
Methods are provided for constructing a cis-repressed reporter transcript that can
bind to a mecA-target transcript. The reporter transcript can be constructed via standard
molecular biological techniques. The luxA and luxB genes serve as reporter genes and can be
derived from Vibrio harveyi. The genes lack a transcriptional promoter, and each contains its
own ribosomal binding site (RBS). When both the luxA and luxB genes are translated in a
cell, the luxA and luxB proteins complex to form the active luciferase enzyme (LuxAB). See
Farinha, M.A. and A.M. Kropinski, Construction of broad-host-range plasmid vectors for
easy visible selection and analysis of promoters. J. Bacteriol., 1990. 172(6): p. 3496-3499.
The cis-repressing sequence can be situated upstream of the luxAB genes and
downstream of a promoter and includes a sequence that is complementary to the luxA RBS.
A linker sequence can separate the complementary regions of the cis-repressing sequence and
the luxA sequence. After transcription of the vector, the complementary regions of the cis
repressing sequence and the luxA RBS sequence complex, creating a stem loop that prevents
docking of a ribosome and hence translation.
The stem loop of the reporter transcript is designed to destabilize and form an
open complex when it interacts with a naturally-occurring mecA transcript sequence
(endogenous to the cell). To activate translation of the luxA gene sequence, the natural mecA
transcript serves as a trans-activating RNA that binds to the cis-repressed reporter transcript
and opens the inhibitory stem loop that sequesters the RBS of the luxA gene. Once the RBS
is not sequestered by the cis-repressing sequence, translation of luxA can occur.
Transcription of the reporter construct is accomplished via operatively linking the reporter
sequence to a constitutive promoter, upstream of the cis-repressing sequence.
An example of a target mecA gene sequence is shown in Figure 29. The sequence
is a mecA gene loci DNA sequence (from Staphylococcus aureus subsp. aureus SA40,
complete genome GenBank: CP003604.1; SEQ ID N0:15) and can be used for generating a
reporter construct comprising a reporter sequence and a cis-repressing sequence. The -10
position 2901, the transcription start position 2902, the RBS 2903, the coding region (in grey
904) and the transcription termination sequence 2905 are shown.
Figure 30 shows an exemplary mecA transcript sequence that can be used for
designing a reporter transcript (SEQ ID N0:16), according to an embodiment of the
invention. The RBS 3001 and the coding sequence 3002 are shown for mecA.
Figure 31 is an example of a luxAB gene loci DNA sequence that can be used for
designing a reporter transcript, according to an embodiment of the invention. The luxAB gene
loci DNA sequence was obtained from Vibrio fischeri genes luxA and luxB for luciferase
alpha and beta subunits (GenBank: X06758.1) (SEQ ID NO: 17). The -10 position 3101, the
transcription start position 3102, the RBS for lux A 3103, the luxA coding sequence 3104
(gray shading), the RBS for luxB 3105, and the luxB coding sequence (gray shading) 3106
are shown.
Figure 32 is an example of a luxAB transcript sequence that can be used for
designing a reporter transcript (SEQ ID N0:18). The RBS for lux A 3201, the luxA coding
sequence 3202 (gray shading), the RBS for luxB 3203, and the luxB coding sequence (gray
shading) 3204 are shown.
Figure 33 is an example of a luxAB cis-repressed transcript sequence that can be
used in a reporter transcript (SEQ ID NO: 19). The cis-repressing sequence (dotted line box)
3301, the RBS for lux A 3302, the luxA coding sequence 3303 (gray shading), the RBS for
luxB 3304, and the luxB coding sequence (gray shading) 3305 are shown.
3. Methods for Detecting the Presence or Absence of a mecA Target
Transcript Using the Reporter Transcript
Examples are provided for detecting the presence or absence of a mecA target
transcript in a cell using the reporter transcripts of the invention. Figure 34 shows an
example of a cell comprising a vector 3400 that encodes a reporter transcript 1410, where
there is no endogenous mecA transcript in the cell 3401 (e.g., the cell's genome does not
contain the mecA gene). In this case, the cis-repressing sequence 3420 binds to the RBS 3430
of the luxAB genes. In some embodiments, the cis-repressing sequence 3420 can bind to a
portion of or all of the RBS of the luxA gene, the RBS of the luxB gene, or both. This binding
event blocks and prevents the translation of the luxAB genes, and the reporter molecule (e.g.,
luciferase) is not produced in the cell. Thus, no signal is detected, indicating the absence of
the mecA gene in the cell.
In another example, the cell includes an endogenous mecA transcript (e.g., the
cell's genome contains the mecA gene). Figure 35 shows a vector 3400 introduced into a cell
3401. The vector 3400 encodes the reporter transcript 3410, which includes a cis-repressing
sequence 3420 and a reporter sequence (luxA and luxB genes). When the mecA transcript
3510 present in the cell binds to the cis-repressing sequence 1420, the inhibitory hairpin loop
opens up and the RBS 3430 for the luxA gene is exposed. Translation of the reporter
sequences (luxA and luxB) can occur, resulting in the formation of a luxAB enzyme 3520.
The luxAB enzyme 3520 produces a detectable luminescent signal 3530. In this manner, the
transcript reporter vector 3400 reports the presence of endogenous mecA transcripts 3 510
within a cell 3401.
While the invention has been particularly shown and described with reference to a
preferred embodiment and various alternate embodiments, it will be understood by persons
skilled in the relevant art that various changes in form and details can be made therein
without departing from the spirit and scope of the invention.
All references, issued patents and patent applications cited within the body of the
instant specification are hereby incorporated by reference in their entirety, for all purposes.
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INFORMAL SEQUENCE LISTING
SEQ IDNO: 1
Native Pl pac-site
CCACTAAAAAGCATGATCATTGATCACTCTAATGATCAACATGCAGGTGATCACATTGCGGC
TGAAATAGCGGAAAAACAAAGAGTTAATGCCGTTGTCAGTGCCGCAGTCGAGAATGCGAAGC
GCCAAAATAAGCGCATAAATGATCGTTCAGATGATCATGACGTGATCACCCGC
SEQ IDN0:2
P 1 pac-site with silent mutations, lower case letters signify mutated bases
CCACTAAAAAGCATGATaATaGAcCACTCTAAcGAcCAACATGCAGGgGAgCACATTGCGGC
TGAAATAGCGGAAAAgCAgAGgGTgAATGCCGTTGTCAGTGCCGCAGTCGAGAATGCGAAGC
GCCAAAATAAGCGCATAAAcGAcCGTTCAGAcGAcCATGACGTtATtACCCGC
SEQ IDNO: 3
Pl lytic replicon containing the Cl repressor-controlled P53 promoter, the promoter P53
antisense, the repL genes, and an in-frame deletion of the kilA gene
CACTATAGGGCGAATTGGCGGAAGGCCGTCAAGGCCGCATTTGGGCCCGGCGCGCCGGATCC
GCTAGCTCTAGACTGGCAGGTTTCTGAGCAGATCGTCCAACCCGATCTGGATCGGGTCAGAA
AAATTTGCTCTAATAAATTTCGTTTTCTAAGTGCAAAGAATCACCATTTCGAGCTGGTGATT
GAAGGTTGATGCAAATTTGGAGAAAAAATGCAACAAACATTCAATGCGGATATGAATATATC
AAACCTTCATCAAAATGTCGATCCTTCAACCACTCTGCCCGTTATTTGTGGTGTTGAAATTA
CGACCGACCGCGCTGGCCGTTACAACCTTAATGCTCTACACAGAGCGAGCGGACTCGGTGCC
CATAAAGCGCCAGCTCAATGGCTAAGAACGCTGTCAGCTAAACAGCTCATCGAAGAGCTTGA
AAAAGAAACTATGCAGAATTGCATAGTTTCGTTCACAAGCAATGGAAGCAGGATTTCTTTCA
CGACTCGTATAACCGGCAAAGGTCAGCAGTGGCTGATGAAGCGATTGCTTGATGCTGGTGTG
CTGGTACCTGTCGCGGCAACGCGCTAACAGACGTAGTAAGAACCACCAGCATTGTAATGCTG
GCTAAAGTCACTTTCCTGAGCTGTATAACGATGAGCGATTTTACTTTTTCTGGCTATGAATT
GGCCTGCTTTGTAACACACTCCGGTCTATCCCGTAGCGCCGGGCATATCCTGTCGCAATGTG
CAAATCTCGCGGCAACAACCAGTGAATACTTCATTCACAAGCCTCACCGCCTGATCGCGGCA
GAAACTGGTTATAGCCAATCAACCGTCGTTCGTGCATTCCGTGAAGCTGTAAACAAAGGAAT
TCTGTCTGTAGAGATTGTTATCGGCGATCACCGTGAACGTCGCGCTAACCTGTACCGGTTTA
CACCATCCTTTTTGGCCTTCGCACAACAAGCCAAAAATGCGCTGATAGAAAGCAAATTAAAG
ATCTCTTCAGCGGCAACCAAGGTTAAAGCTGTTCTCGCTAAGACATTGGCTTTATTTAATTT
TTTATCCACACCCCCATGTCAAAATGATACCCCCTCCCCCTGTCAGGATGACGTGGCAATAA
AGAATAAGAAGTCACAAGTTAAAAAAACAAAAAGATCAGTTTCCGGCGGTGCCGGAACAACC
AGCCTCAAAAAATTGACTTCATGGATCGCTAAGGCAAAAGCAAAGGCTGACAATCTGCGGTT
ATCCAAAAAACGCACTCAAAAACATGAGTTCAAGCAGAAAGTAGAGGCGGCTGCGCGGAAAT
ATGCTTACCTGAAGAACAAGCGTTCGCCTGATATTGGCGGGATATCAAACTTCGATAACCTA
CCGCATTGCATGACGGTAAACGAAGCTCTTAATGCGGTTTTAGCCAAAAATAAAGATAACGA
ACAATGGGGTATACCGGCAGGATTCAGAGGGTAATGAATTGCTCTAATTATAACCATGCATA
CTTTCAACACCTCTAGTTTGCCATGAGGCAAACTCATAGGTGTCCTGGTAAGAGGACACTGT
TGCCAAAACTGGACGCCCCATTATTGCAATTAATAAACAACTAACGGACAATTCTACCTAAC
AATAAGTGGCTTAAAAAAACCCGCCCCGGCGGGTTTTTTTATCTAGAGCTAGCGGATCCGGC
GCGCCGGGCCCTTCTGGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTTCCAG
SEQ IDNO: 4
Pblast promoter sequence
CGTCAGGTGGCACTTTTCGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTCTAAATACA
TTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAG
GAAGAGT
SEQ IDNO: 5
S. aureus pT181 plasmid origin or replication copy number variant pT181cop-623 repC
TTTGCGGAAAGAGTTAGTAAGTTAACAGAAGACGAGCCAAACCTAAATGGTTTAGCAGGAAA
CTTAGATAAAAAAATGAATCCAGAATTATATTCAGAACAGGAACAGCAACAAGAGCAACAAA
AGAATCAAAAACGAGATAGAGGTATGCACTTATAGAACATGCATTTATGCCGAGAAAACTTA
TTGGTTGGAATGGGCTATGTGTTAGCTAACTTGTTAGCGAGTTGGTTGGACTTGAATTGGGA
TTAATCCCAAGAAAGTACCGGCTCAACAACCCATAAAGCCCTGTAGGTTCCGNCCAATAAGG
AAATTGGAATAAAGCAATAAAAGGAGTTGAAGAAATGAAATTCAGAGAAGCCTTTGAGAATT
TTATAACAAGTAAGTATGTACTTGGTGTTTTAGTAGTCTTAACTGTTTACCAGATAATACAA
ATGCTTAAATAAAAAAAGACTTGATCTGATTAGACCAAATCTTTTGATAGTGTTATATTAAT
AACAAAATAAAAAGGAGTCGCTCACGCCCTACCAAAGTTTGTGAACGACATCATTCAAAGAA
AAAAACACTGAGTTGTTTTTATAATCTTGTATATTTAGATATTAAACGATATTTAAATATAC
ATCAAGATATATATTTGGGTGAGCGATTACTTAAACGAAATTGAGATTAAGGAGTCGATTTT
TTATGTATAAAAACAATCATGCAAATCATTCAAATCATTTGGAAAATCACGATTTAGACAAT
TTTTCTAAAACCGGCTACTCTAATAGCCGGTTGGACGCACATACTGTGTGCATATCTGATCC
AAAATTAAGTTTTGATGCAATGACGATCGTTGGAAATCTCAACCGAGACAACGCTCAGGCCC
TTTCTAAATTTATGAGTGTAGAGCCCCAAATAAGACTTTGGGATATTCTTCAAACAAAGTTT
AAAGCTAAAGCACTTCAAGAAAAAGTTTATATTGAATATGACAAAGTGAAAGCAGATAGTTG
GGATAGACGTAATATGCGTATTGAATTTAATCCAAACAAACTTACACGAGATGAAATGATTT
GGTTAAAACAAAATATAATAAGCTACATGGAAGATGACGGTTTTACAAGATTAGATTTAGCC
TTTGATTTTGAAGATGATTTGAGTGACTACTATGCAATGTCTGATAAAGCAGTTAAGAAAAC
TATTTTTTATGGTCGTAATGGTAAGCCAGAAACAAAATATTTTGGCGTGAGAGATAGTAATA
GATTTATTAGAATTTATAATAAAAAGCAAGAACGTAAAGATAATGCAGATGCTGAAGTTATG
TCTGAACATTTATGGCGTGTAGAAATCGAACTTAAAAGAGATATGGTGGATTACTGGAATGA
TTGCTTTAGTGATTTACATATCTTGCAACCAGATTGGAAAACTATCCAACGCACTGCGGATA
GAGCAATAGTTTTTATGTTATTGAGTGATGAAGAAGAATGGGGAAAGCTTCACAGAAATTCT
AGAACAAAATATAAGAATTTGATAAAAGAAATTTCGCCAGTCGATTTAACGGACTTAATGAA
ATCGACTTTAAAAGCGAACGAAAAACAATTGCAAAAACAAATCGATTTTTGGCAACATGAAT
TTAAATTTTGGAAATAGTGTACATATTAATATTACTGAACAAAAATGATATATTTAAACTAT
TCTAATTTAGGAGGATTTTTTTATGAAGTGTCTATTTAAAAATTTGGGGAATTTATATGAGG
TGAAAGAATAATTTACCCCTATAAACTTTAGCCACCTCAAGTAAAGAGGTAAAATTGTTTAG
TTTATATAAAAAATTTAAAGGTTTGTTTTATAGCGTTTTATTTTGGCTTTGTATTCTTTCAT
TTTTTAGTGTATTAAATGAAATGGTTTTAAATGTTTCTTTACCTGATATTGCAAATCATTTT
AATACTACTCCTGGAATTACAAACTGGGTAAACACTGCATATATGTTAACTTTTTCGATAGG
AACAGCAGTATATGGAAAATTATCTGATTATATAAATATAAAAAAATTGTTAATTATTGGTA
TTAGTTTGAGCTGTCTTGGTTCATTGATTGCTTTTATTGGGCCCACCTAGGCAAATATGCTC
TTACGTGCTATTATTTAAGTGACTATTTAAAAGGAGTTAATAAATATGCGGCAAGGTATTCT
TAAATAAACTGTCAATTTGATAGCGGGAACAAATAATTAGATGTCCTTTTTTAGGAGGGCTT
AGTTTTTTGTACCCAGTTTAAGAATACCTTTATCATGTGATTCTAAAGTATCCAGAGAATAT
CTGTATGCTTTGTATACCTATGGTTATGCATAAAAATCCCAGTGATAAAAGTATTTATCACT
GGGATTTTTATGCCCTTTTGGGTTTTTGAATGGAGGAAAATCACATGAAAATTATTAATATT
GGAGTTTTAGCTCATGTTGATGCAGGAAAAACTACCTTAACAGAAAGCTTATTATATAACAG
TGGAGCGATTACAGAATTAGGAAGCGTGGACAAAGGTACAACGAGGACGGATAATACGCTTT
TAGAACGTCAGAGAGGAATTACAATTCAGACAGGAATAACCTCTTTTCAGTGGGAAAATACG
AAGGTGAACATCATAGACACGCCAGGACATATGGATTTCTTAGCAGAAGTATATCGTTCATT
ATCAGTTTTAGATGGGGCAATTCTACTGATTTCTGCAAAAGATGGCGTACAAGCACAAACTC
GTATATTATTTCATGCACTTAGGAAAATGGGGATTCCCACAATCTTTTTTATCAATAAGATT
GACCAAAATGGAATTGATTTATCAACGGTTTATCAGGATATTAAAGAGAAACTTTCTGCCGA
AATTGTAATCAAACAGAAGGTAGAACTGTATCCTAATATGTGTGTGACGAACTTTACCGAAT
CTGAACAATGGGATACGGTAATAGAGGGAAACGATAACCTTTTAGAGAAATATATGTCCGGT
AAATCATTAGAAGCATTGGAACTCGAACAAGAGGAAAGCATAAGATTTCAGAATTGTTCTCT
GTTCCCTCTTTATCATGGAAGTGCAAAAAGTAATATAGGGATTGATAACCTTATAGAAGTTA
TTACTAATAAATTTTATTCATCAACACATCGAGGTCCGTCTGAACTTTGCGGAAATGTTTTC
AAAATTGAATATACAAAAAAAAGACAACGTCTTGCATATATACGCCTTTATAGTGGAGTACT
ACATTTACGAGATTCGGTTAGAGTATCAGAAAAAGAAAAAATAAAAGTTACAGAAATGTATA
CTTCAATAAATGGTGAATTATGTAAGATTGATAGAGCTTATTCTGGAGAAATTGTTATTTTG
CAAAATGAGTTTTTGAAGTTAAATAGTGTTCTTGGAGATACAAAACTATTGCCACAGAGAAA
AAAGATTGAAAATCCGCACCCTCTACTACAAACAACTGTTGAACCGAGTAAACCTGAACAGA
GAGAAATGTTGCTTGATGCCCTTTTGGAAATCTCAGATAGTGATCCGCTTCTACGATATTAC
GTGGATTCTACGACACATGAAATTATACTTTCTTTCTTAGGGAAAGTACAAATGGAAGTGAT
TAGTGCACTGTTGCAAGAAAAGTATCATGTGGAGATAGAACTAAAAGAGCCTACAGTCATTT
ATATGGAGAGACCGTTAAAAAATGCAGAATATACCATTCACATCGAAGTGCCGCCAAATCCT
TTCTGGGCTTCCATTGGTTTATCTGTATCACCGCTTCCGTTGGGAAGTGGAATGCAGTATGA
GAGCTCGGTTTCTCTTGGATACTTAAATCAATCATTTCAAAATGCAGTTATGGAAGGGGTAC
GCTATGGTTGCGAACAAGGATTATATGGTTGGAATGTGACGGATTGTAAAATCTGTTTTAAG
TACGGTTTATACTATAGCCCTGTTAGTACTCCAGCAGATTTTCGGATGCTTACTCCTATTGT
ACTGGAGCAAGCCTTTAGAAAAGCTGGAACAGAATTGTTAGAGCCATATCTTAGTTTTAAAG
TTTATGCACCACAGGAATATCTTTCNCGGGCATATAACGATGCTCCCAAATATTGTGCAAAT
ATCGTAAATACTCAACTGAAAAATAATGAGGTCATTATTATTGGAGAAATTCCTGCTCGATG
TATTCAAGATTATCGCAATGATTTAACTTTTTTTACAAATGGGCTTAGTGTTTGTTTAGCAG
AGCTAAAAGGATATCAGGTTACCACTGGCGAACCTGTTTGCCAGACCCGTCGTCTAAATAGT
CGGATAGATAAAGTAAGATATATGTTCAATAAAATAACTTAGTGCGTTTTATGTTGTTATAT
AAATATGGTTTCTTATTAAATAAGATGAAATATTCTTTAATATAGATTTGAATTAAAGTGGA
AAGGAGGAGATTGTTATTATAAACTACAAGTGGATATTGTGTCCTATTTGTGGAAATAAAAC
AAGACTACGAATACGAGTGGATACTATACTTAAAAATTTCCCTTTATACAGCCCCAAATGTA
AGAACGAAACTTTAATTAATGTTCAAAAAATGAATATAATAACAATCAAAGAGCCAGACGCC
AAGACGCAGAGCCGATAATTTGAGAAATGAAACTCTCATCTTATCGGCTCTTTTTGTTTATC
TGAATTTTACTGACTAGCCTTCAATATTTCC
SEQ IDNO: 6
P 1 pacA gene, lower case letters signify deleted pac-site sequence
GTGACCTGGGACGATCACAAGAAGAATTTTGCTCGCCTGGCGCGAGATGGTGGTTACACCAT
CGCACAGTATGCCGCCGAGTTTAATCTTAACCCTAATACCGCACGTCGTTATCTCCGTGCCT
TCAAAGAAGACACCAGGACTACGGACAGCCGCAAGCCAAATAAGCCAGTCAGGAAGccacta
aaaagcatgatcattgatcactctaatgatcaacatgcaggtgatcacattgcggctgaaat
agcggaaaaacaaagagttaatgccgttgtcagtgccgcagtcgagaatgcgaagcgccaaa
ataagcgcataaatgatcgttcagatgatcatgacgtgatcacccgcGCCCACCGGACCTTA
CGTGATCGCCTGGAACGCGACACCCTGGATGATGATGGTGAACGCTTTGAATTCGAAGTTGG
CGATTACCTGATAGATAACGTTGAAGCGCGGAAGGCCGCGCGCGCTATGTTGCGTCGGTCCG
GGGCCGATGTTCTGGAAACCACTCTTCTGGAAAAGTCTCTTTCTCATCTCCTTATGCTGGAG
AACGCCAGGGATACGTGTATTCGCCTGGTGCAGGAAATGCGCGATCAGCAAAAAGACGATGA
TGAAGGTACTCCGCCTGAATACCGTATCGCGAGCATGCTAAACAGCTGTTCCGCGCAGATAA
GCAGCCTGATCAACACCATTTACAGCATCCGGAATAACTATCGAAAAGAAAGCCGGGAGGCG
GAAAAGCACGCTTTATCTATGGGGCAAGCTGGCATTGTTAAGCTGGCATACGAACGAAAGCG
TGAAAATAACTGGTCAGTGCTGGAAGCGGCTGAATTCATCGAGGCGCATGGAGGAAAAGTGC
CGCCCCTGATGCTGGAGCAAATCAAAGCCGATCTGCGTGCTCCTAAGACCAATACCGATGAT
GAGGAAAACCAAACAGCATCTGGCGCTCCATCACTTGAAGATCTGGATAAAATCGCGCGAGA
ACGGGCCGCCAGCCGCCGCGCTGATGCCGCATTGTGGATTGAGCATCGTAGAGAAGAAATTG
CCGATATCGTCGATACAGGTGGTTATGGTGATGTCGATGCGGAAGGCATATCAAACGAAGCA
TGGCTTGAACAGGATCTGGACGAAGACGAGGAGGAAGACGAAGAAGTTACCCGCAAACTGTA
CGGGGATGATGATTAA
SEQ IDNO: 7
Native Pl pacA gene
GTGACCTGGGACGATCACAAGAAGAATTTTGCTCGCCTGGCGCGAGATGGTGGTTACACCAT
CGCACAGTATGCCGCCGAGTTTAATCTTAACCCTAATACCGCACGTCGTTATCTCCGTGCCT
TCAAAGAAGACACCAGGACTACGGACAGCCGCAAGCCAAATAAGCCAGTCAGGAAGCCACTA
AAAAGCATGATCATTGATCACTCTAATGATCAACATGCAGGTGATCACATTGCGGCTGAAAT
AGCGGAAAAACAAAGAGTTAATGCCGTTGTCAGTGCCGCAGTCGAGAATGCGAAGCGCCAAA
ATAAGCGCATAAATGATCGTTCAGATGATCATGACGTGATCACCCGCGCCCACCGGACCTTA
CGTGATCGCCTGGAACGCGACACCCTGGATGATGATGGTGAACGCTTTGAATTCGAAGTTGG
CGATTACCTGATAGATAACGTTGAAGCGCGGAAGGCCGCGCGCGCTATGTTGCGTCGGTCCG
GGGCCGATGTTCTGGAAACCACTCTTCTGGAAAAGTCTCTTTCTCATCTCCTTATGCTGGAG
AACGCCAGGGATACGTGTATTCGCCTGGTGCAGGAAATGCGCGATCAGCAAAAAGACGATGA
TGAAGGTACTCCGCCTGAATACCGTATCGCGAGCATGCTAAACAGCTGTTCCGCGCAGATAA
GCAGCCTGATCAACACCATTTACAGCATCCGGAATAACTATCGAAAAGAAAGCCGGGAGGCG
GAAAAGCACGCTTTATCTATGGGGCAAGCTGGCATTGTTAAGCTGGCATACGAACGAAAGCG
TGAAAATAACTGGTCAGTGCTGGAAGCGGCTGAATTCATCGAGGCGCATGGAGGAAAAGTGC
CGCCCCTGATGCTGGAGCAAATCAAAGCCGATCTGCGTGCTCCTAAGACCAATACCGATGAT
GAGGAAAACCAAACAGCATCTGGCGCTCCATCACTTGAAGATCTGGATAAAATCGCGCGAGA
ACGGGCCGCCAGCCGCCGCGCTGATGCCGCATTGTGGATTGAGCATCGTAGAGAAGAAATTG
CCGATATCGTCGATACAGGTGGTTATGGTGATGTCGATGCGGAAGGCATATCAAACGAAGCA
TGGCTTGAACAGGATCTGGACGAAGACGAGGAGGAAGACGAAGAAGTTACCCGCAAACTGTA
CGGGGATGATGATTAA
SEQ IDN0:8
terS gene, lower case characters signify deleted sequence
ATGAACGAAAAACAAAAGAGATTCGCAGATGAATATATAATGAATGGATGTAATGGTAAAAA
AGCAGCAATTTCAGCAggttatagtaagaaaacagcagagtctttagcaagtcgattgttaa
gaaatgttaatgtttcggaatatattaaagaacgattagaacagatacaagaagagcgttta
atgagcattacagaagctttagcgttatctgcttctattgctagaggagaacctcaagaggc
ttacagtaagaaatatgaccatttaaacgatgaagtggaaaaagaggttacttacacaatca
caccaacttttgaagagcgtcagagatctattgaccacatactaaaagttcatggtgcgtat
atcgacaaaaaagaaattactcagaagaatattgagattaatattAGATCTATTGACCACAT
ACTAAAAGTTCATGGTGCGTATATCGACAAAAAAGAAATTACTCAGAAGAATATTGAGATTA
ATATTGGTGAGTACGATGACGAAAGTTAA
SEQ IDN0:9
Sequence containing native terS gene
AATTGGCAGTAAAGTGGCAGTTTTTGATACCTAAAATGAGATATTATGATAGTGTAGGATAT
TGACTATCTTACTGCGTTTCCCTTATCGCAATTAGGAATAAAGGATCTATGTGGGTTGGCTG
ATTATAGCCAATCCTTTTTTAATTTTAAAAAGCGTATAGCGCGAGAGTTGGTGGTAAATGAA
ATGAACGAAAAACAAAAGAGATTCGCAGATGAATATATAATGAATGGATGTAATGGTAAAAA
AGCAGCAATTTCAGCAGGTTATAGTAAGAAAACAGCAGAGTCTTTAGCAAGTCGATTGTTAA
GAAATGTTAATGTTTCGGAATATATTAAAGAACGATTAGAACAGATACAAGAAGAGCGTTTA
ATGAGCATTACAGAAGCTTTAGCGTTATCTGCTTCTATTGCTAGAGGAGAACCTCAAGAGGC
TTACAGTAAGAAATATGACCATTTAAACGATGAAGTGGAAAAAGAGGTTACTTACACAATCA
CACCAACTTTTGAAGAGCGTCAGAGATCTATTGACCACATACTAAAAGTTCATGGTGCGTAT
ATCGACAAAAAAGAAATTACTCAGAAGAATATTGAGATTAATATTGGTGAGTACGATGACGA
AAGTTAAATTAAACTTTAACAAACCATCTAATGTTTTCAACAG
SEQ ID NO: IO
SaPibov2 integrase gene
TCATAAATATTTAACTATTTCTTTCTGTGTACTAGGGTACAAATGACCGTATCGGTTATATA
CTTCATTACTATCAGCATGGCCTAAACGCTGTGCTATTACCATGATACTTGCGCCATGATTG
ACTAGCATAGACGCATGGCTATGTCTTAACTCATGAATTACAATTCTAGGGAATGTCTGACC
GTCTGGTAGTTGTTCATCTAATACTTTTAATGCAGCGGTAAACCAACGATCTATAGTTGATT
CACTATAAGCTTTGAAGAATGTACCGAATAATACATAATCATCTTTATATACATTGTTTTCT
TTGTACCATTTTAAATATTCTTTGATATCATTCATCATGTGAACAGGTAAGTATATATCACG
TATTGCTGCTTTTGTTTTAGGGGCTGTCACTTCACCGTGATAGTCTGTTTTGTTAATATGTA
TGAAATCATCATCATAGTTAATATCACGCCATGTGAGGGCTCTAATTTCGCCCTTACGTGCA
CCAGAGTAAAACAGTAGCTTAAAGAATAACTTTTGTTGTTGTGTAGCTAAAGCCTCATAGAA
TTGATTGAATTGTTCTAATGTCCAATAGTTCAAACGCTTATTTGATTCTATTTCAAAGTTAC
CTACTAGAGAGGCTACATTTTGCTTTAGATCATGAAACTTCATAGCATGGTTAAGTAACGAT
ACTAAGAACACGTGCATTTTCTTTAGGTACTCTCCAGAGTGTCCCTCTTTTAACTTCGTATT
CTGAAACTTCATAATATCTTGTGTAGTCATATTAAACACGTCCATAGACTTAAAATAGGGTA
GCAAATGGTTGTTTGTATGTGTCTTTAATGCTTTCACACTAGATGACTTACGACGTGCAGAA
TACCACTCTATATACTCATCTACGAGCTTATCAAAGGGCAGTTTGTTTATCTGTCCTACACC
CTCTAACTCGTCCATAATTTCATTACATTTCTTCAATGCCTCTTTACGCTGTTTAAAGCCAC
TCTTTTTTATTTCTTTACGTTGATTAAATTTATCATAGTATTTTATACGAAAATAGTATGTA
CCACGTTTAGCGTCTTTATATATGTTGTGGGATAGGTTTAAGTTGTGTTCTATGGGAATCAC
SEQ IDNO:ll
pGWP 10001 full sequence
CTCGGGCCGTCTCTTGGGCTTGATCGGCCTTCTTGCGCATCTCACGCGCTCCTGCGGCGGCC
TGTAGGGCAGGCTCATACCCCTGCCGAACCGCTTTTGTCAGCCGGTCGGCCACGGCTTCCGG
CGTCTCAACGCGCTTTGAGATTCCCAGCTTTTCGGCCAATCCCTGCGGTGCATAGGCGCGTG
GCTCGACCGCTTGCGGGCTGATGGTGACGTGGCCCACTGGTGGCCGCTCCAGGGCCTCGTAG
AACGCCTGAATGCGCGTGTGACGTGCCTTGCTGCCCTCGATGCCCCGTTGCAGCCCTAGATC
GGCCACAGCGGCCGCAAACGTGGTCTGGTCGCGGGTCATCTGCGCTTTGTTGCCGATGAACT
CCTTGGCCGACAGCCTGCCGTCCTGCGTCAGCGGCACCACGAACGCGGTCATGTGCGGGCTG
GTTTCGTCACGGTGGATGCTGGCCGTCACGATGCGATCCGCCCCGTACTTGTCCGCCAGCCA
CTTGTGCGCCTTCTCGAAGAACGCCGCCTGCTGTTCTTGGCTGGCCGACTTCCACCATTCCG
GGCTGGCCGTCATGACGTACTCGACCGCCAACACAGCGTCCTTGCGCCGCTTCTCTGGCAGC
AACTCGCGCAGTCGGCCCATCGCTTCATCGGTGCTGCTGGCCGCCCAGTGCTCGTTCTCTGG
CGTCCTGCTGGCGTCAGCGTTGGGCGTCTCGCGCTCGCGGTAGGCGTGCTTGAGACTGGCCG
CCACGTTGCCCATTTTCGCCAGCTTCTTGCATCGCATGATCGCGTATGCCGCCATGCCTGCC
CCTCCCTTTTGGTGTCCAACCGGCTCGACGGGGGCAGCGCAAGGCGGTGCCTCCGGCGGGCC
ACTCAATGCTTGAGTATACTCACTAGACTTTGCTTCGCAAAGTCGTGACCGCCTACGGCGGC
TGCGGCGCCCTACGGGCTTGCTCTCCGGGCTTCGCCCTGCGCGGTCGCTGCGCTCCCTTGCC
AGCCCGTGGATATGTGGACGATGGCCGCGAGCGGCCACCGGCTGGCTCGCTTCGCTCGGCCC
GTGGACAACCCTGCTGGACAAGCTGATGGACAGGCTGCGCCTGCCCACGAGCTTGACCACAG
GGATTGCCCACCGGCTACCACTATAGGGCGAATTGGCGGAAGGCCGTCAAGGCCGCATTTGG
GCCCGGCGCGCCGGATCCGCTAGCTCTAGACCTCTAGACCAGCCAGGACAGAAATGCCTCGA
CTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATGAAGGCACGAACC
CAGTGGACATAAGCCTGTTCGGTTCGTAAGCTGTAATGCAAGTAGCGTGCCCGTTCCGGGCC
TTTTGACATGTGACTTTCGTTACCCTCGCGTCAAAAAGAGTTTTTACGAAAGGAAGCATAAG
TGACCTGGGACGATCACAAGAAGAATTTTGCTCGCCTGGCGCGAGATGGTGGTTACACCATC
GCACAGTATGCCGCCGAGTTTAATCTTAACCCTAATACCGCACGTCGTTATCTCCGTGCCTT
CAAAGAAGACACCAGGACTACGGACAGCCGCAAGCCAAATAAGCCAGTCAGGAAGCCACTAA
AAAGCATGATCATTGATCACTCTAATGATCAACATGCAGGTGATCACATTGCGGCTGAAATA
GCGGAAAAACAAAGAGTTAATGCCGTTGTCAGTGCCGCAGTCGAGAATGCGAAGCGCCAAAA
TAAGCGCATAAATGATCGTTCAGATGATCATGACGTGATCACCCGCGCCCACCGGACCTTAC
GTGATCGCCTGGAACGCGACACCCTGGATGATGATGGTGAACGCTTTGAATTCGAAGTTGGC
GATTACCTGATAGATAACGTTGAAGCGCGGAAGGCCGCGCGCGCTATGTTGCGTCGGTCCGG
GGCCGATGTTCTGGAAACCACTCTTCTGGAAAAGTCTCTTTCTCATCTCCTTATGCTGGAGA
ACGCCAGGGATACGTGTATTCGCCTGGTGCAGGAAATGCGCGATCAGCAAAAAGACGATGAT
GAAGGTACTCCGCCTGAATACCGTATCGCGAGCATGCTAAACAGCTGTTCCGCGCAGATAAG
CAGCCTGATCAACACCATTTACAGCATCCGGAATAACTATCGAAAAGAAAGCCGGGAGGCGG
AAAAGCACGCTTTATCTATGGGGCAAGCTGGCATTGTTAAGCTGGCATACGAACGAAAGCGT
GAAAATAACTGGTCAGTGCTGGAAGCGGCTGAATTCATCGAGGCGCATGGAGGAAAAGTGCC
GCCCCTGATGCTGGAGCAAATCAAAGCCGATCTGCGTGCTCCTAAGACCAATACCGATGATG
AGGAAAACCAAACAGCATCTGGCGCTCCATCACTTGAAGATCTGGATAAAATCGCGCGAGAA
CGGGCCGCCAGCCGCCGCGCTGATGCCGCATTGTGGATTGAGCATCGTAGAGAAGAAATTGC
CGATATCGTCGATACAGGTGGTTATGGTGATGTCGATGCGGAAGGCATATCAAACGAAGCAT
GGCTTGAACAGGATCTGGACGAAGACGAGGAGGAAGACGAAGAAGTTACCCGCAAACTGTAC
GGGGATGATGATTAATTAAAAAAACCCGCCCCGGCGGGTTTTTTTATCTAGAGCTAGCGGAT
CCGGCGCGCCGGGCCCTTCTGGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTTCCAGCACT
ATAGGGCGAATTGGCGGAAGGCCGTCAAGGCCGCATTTGGGCCCGGCGCGCCGGATCCGCTA
GCTCTAGACTGGCAGGTTTCTGAGCAGATCGTCCAACCCGATCTGGATCGGGTCAGAAAAAT
TTGCTCTAATAAATTTCGTTTTCTAAGTGCAAAGAATCACCATTTCGAGCTGGTGATTGAAG
GTTGATGCAAATTTGGAGAAAAAATGCAACAAACATTCAATGCGGATATGAATATATCAAAC
CTTCATCAAAATGTCGATCCTTCAACCACTCTGCCCGTTATTTGTGGTGTTGAAATTACGAC
CGACCGCGCTGGCCGTTACAACCTTAATGCTCTACACAGAGCGAGCGGACTCGGTGCCCATA
AAGCGCCAGCTCAATGGCTAAGAACGCTGTCAGCTAAACAGCTCATCGAAGAGCTTGAAAAA
GAAACTATGCAGAATTGCATAGTTTCGTTCACAAGCAATGGAAGCAGGATTTCTTTCACGAC
TCGTATAACCGGCAAAGGTCAGCAGTGGCTGATGAAGCGATTGCTTGATGCTGGTGTGCTGG
TACCTGTCGCGGCAACGCGCTAACAGACGTAGTAAGAACCACCAGCATTGTAATGCTGGCTA
AAGTCACTTTCCTGAGCTGTATAACGATGAGCGATTTTACTTTTTCTGGCTATGAATTGGCC
TGCTTTGTAACACACTCCGGTCTATCCCGTAGCGCCGGGCATATCCTGTCGCAATGTGCAAA
TCTCGCGGCAACAACCAGTGAATACTTCATTCACAAGCCTCACCGCCTGATCGCGGCAGAAA
CTGGTTATAGCCAATCAACCGTCGTTCGTGCATTCCGTGAAGCTGTAAACAAAGGAATTCTG
TCTGTAGAGATTGTTATCGGCGATCACCGTGAACGTCGCGCTAACCTGTACCGGTTTACACC
ATCCTTTTTGGCCTTCGCACAACAAGCCAAAAATGCGCTGATAGAAAGCAAATTAAAGATCT
CTTCAGCGGCAACCAAGGTTAAAGCTGTTCTCGCTAAGACATTGGCTTTATTTAATTTTTTA
TCCACACCCCCATGTCAAAATGATACCCCCTCCCCCTGTCAGGATGACGTGGCAATAAAGAA
TAAGAAGTCACAAGTTAAAAAAACAAAAAGATCAGTTTCCGGCGGTGCCGGAACAACCAGCC
TCAAAAAATTGACTTCATGGATCGCTAAGGCAAAAGCAAAGGCTGACAATCTGCGGTTATCC
AAAAAACGCACTCAAAAACATGAGTTCAAGCAGAAAGTAGAGGCGGCTGCGCGGAAATATGC
TTACCTGAAGAACAAGCGTTCGCCTGATATTGGCGGGATATCAAACTTCGATAACCTACCGC
ATTGCATGACGGTAAACGAAGCTCTTAATGCGGTTTTAGCCAAAAATAAAGATAACGAACAA
TGGGGTATACCGGCAGGATTCAGAGGGTAATGAATTGCTCTAATTATAACCATGCATACTTT
CAACACCTCTAGTTTGCCATGAGGCAAACTCATAGGTGTCCTGGTAAGAGGACACTGTTGCC
AAAACTGGACGCCCCATTATTGCAATTAATAAACAACTAACGGACAATTCTACCTAACAATA
AGTGGCTTAAAAAAACCCGCCCCGGCGGGTTTTTTTATCTAGAGCTAGCGGATCCGGCGCGC
CGGGCCCTTCTGGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTTCCAGCCAGCCTTCGACC
ACATACCCACCGGCTCCAACTGCGCGGCCTGCGGCCTTGCCCCATCAATTTTTTTAATTTTC
TCTGGGGAAAAGCCTCCGGCCTGCGGCCTGCGCGCTTCGCTTGCCGGTTGGACACCAAGTGG
AAGGCGGGTCAAGGCTCGCGCAGCGACCGCGCAGCGGCTTGGCCTTGACGCGCCTGGAACGA
CCCAAGCCTATGCGAGTGGGGGCAGTCGAAGGCGAAGCCCGCCCGCCTGCCCCCCGAGACCT
GCAGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGC
CTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGT
AGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGA
AGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCC
CGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAA
CTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTT
GAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGA
TCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTC
GTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATG
GCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCA
AAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATAC
GCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTG
CCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTT
TTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGAT
GGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCAT
TGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAAT
CGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATC
AGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCA
TAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTT
TTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCCCCCCT
GCAGGTCCCGAGCCTCACGGCGGCGAGTGCGGGGGTTCCAAGGGGGCAGCGCCACCTTGGGC
AAGGCCGAAGGCCGCGCAGTCGATCAACAAGCCCCGGAGGGGCCACTTTTTGCCGGAGGGGG
AGCCGCGCCGAAGGCGTGGGGGAACCCCGCAGGGGTGCCCTTCTTTGGGCACCAAAGAACTA
GATATAGGGCGAAATGCGAAAGACTTAAAAATCAACAACTTAAAAAAGGGGGGTACGCAACA
GCTCATTGCGGCACCCCCCGCAATAGCTCATTGCGTAGGTTAAAGAAAATCTGTAATTGACT
GCCACTTTTACGCAACGCATAATTGTTGTCGCGCTGCCGAAAAGTTGCAGCTGATTGCGCAT
GGTGCCGCAACCGTGCGGCACCCCTACCGCATGGAGATAAGCATGGCCACGCAGTCCAGAGA
AATCGGCATTCAAGCCAAGAACAAGCCCGGTCACTGGGTGCAAACGGAACGCAAAGCGCATG
AGGCGTGGGCCGGGCTTATTGCGAGGAAACCCACGGCGGCAATGCTGCTGCATCACCTCGTG
GCGCAGATGGGCCACCAGAACGCCGTGGTGGTCAGCCAGAAGACACTTTCCAAGCTCATCGG
ACGTTCTTTGCGGACGGTCCAATACGCAGTCAAGGACTTGGTGGCCGAGCGCTGGATCTCCG
TCGTGAAGCTCAACGGCCCCGGCACCGTGTCGGCCTACGTGGTCAATGACCGCGTGGCGTGG
GGCCAGCCCCGCGACCAGTTGCGCCTGTCGGTGTTCAGTGCCGCCGTGGTGGTTGATCACGA
CGACCAGGACGAATCGCTGTTGGGGCATGGCGACCTGCGCCGCATCCCGACCCTGTATCCGG
GCGAGCAGCAACTACCGACCGGCCCCGGCGAGGAGCCGCCCAGCCAGCCCGGCATTCCGGGC
ATGGAACCAGACCTGCCAGCCTTGACCGAAACGGAGGAATGGGAACGGCGCGGGCAGCAGCG
CCTGCCGATGCCCGATGAGCCGTGTTTTCTGGACGATGGCGAGCCGTTGGAGCCGCCGACAC
GGGTCACGCTGCCGCGCCGGTAGCACTTGGGTTGCGCAGCAACCCGTAAGTGCGCTGTTCCA
GACTATCGGCTGTAGCCGCCTCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCG
GTGCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCA
CCGTTTTTATCAGGCTCTGGGAGGCAGAATAAATGATCATATCGTCAATTATTACCTCCACG
GGGAGAGCCTGAGCAAACTGGCCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGT
AGTCAATAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAAC
CGACGACCGGGTCGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATACTTATTCA
GGCGTAGCACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTG
CCACTCATCGCACTCGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTT
TATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTT
CAATAATATTGAAAAAGGAAGAGTATGAAGTTTGGAAATATTTGTTTTTCGTATCAACCACC
AGGTGAAACTCATAAGCTAAGTAATGGATCGCTTTGTTCGGCTTGGTATCGCCTCAGAAGAG
TAGGGTTTGATACATATTGGACCTTAGAACATCATTTTACAGAGTTTGGTCTTACGGGAAAT
TTATTTGTTGCTGCGGCTAACCTGTTAGGAAGAACTAAAACATTAAATGTTGGCACTATGGG
GGTTGTTATTCCGACAGCACACCCAGTTCGACAGTTAGAAGACGTTTTATTATTAGATCAAA
TGTCGAAAGGTCGTTTTAATTTTGGAACCGTTCGAGGGCTATACCATAAAGATTTTCGAGTA
TTTGGTGTTGATATGGAAGAGTCTCGAGCAATTACTCAAAATTTCTACCAGATGATAATGGA
AAGCTTACAGACAGGAACCATTAGCTCTGATAGTGATTACATTCAATTTCCTAAGGTTGATG
TATATCCCAAAGTGTACTCAAAAAATGTACCAACCTGTATGACTGCTGAGTCCGCAAGTACG
ACAGAATGGCTAGCAATACAAGGGCTACCAATGGTTCTTAGTTGGATTATTGGTACTAATGA
AAAAAAAGCACAGATGGAACTCTATAATGAAATTGCGACAGAATATGGTCATGATATATCTA
AAATAGATCATTGTATGACTTATATTTGTTCTGTTGATGATGATGCACAAAAGGCGCAAGAT
GTTTGTCGGGAGTTTCTGAAAAATTGGTATGACTCATATGTAAATGCGACCAATATCTTTAA
TGATAGCAATCAAACTCGTGGTTATGATTATCATAAAGGTCAATGGCGTGATTTTGTTTTAC
AAGGACATACAAACACCAATCGACGTGTTGATTATAGCAATGGTATTAACCCTGTAGGCACT
CCTGAGCAGTGTATTGAAATCATTCAACGTGATATTGATGCAACGGGTATTACAAACATTAC
ATGCGGATTTGAAGCTAATGGAACTGAAGATGAAATAATTGCTTCCATGCGACGCTTTATGA
CACAAGTCGCTCCTTTCTTAAAAGAACCTAAATAAATTACTTATTTGATACTAGAGATAATA
AGGAACAAGTTATGAAATTTGGATTATTTTTTCTAAACTTTCAGAAAGATGGAATAACATCT
GANGAAACGTTGGATAATATGGTAAAGACTGTCACGTTAATTGATTCAACTAAATATCATTT
TAATACTGCCTTTGTTAATGAACATCACTTTTCAAAAAATGGTATTGTTGGAGCACCTATTA
CCGCAGCTGGTTTTTTATTAGGGTTAACAAATAAATTACATATTGGTTCATTAAATCAAGTA
ATTACCACCCATCACCCTGTACGTGTAGCAGAAGAAGCCAGTTTATTAGATCAAATGTCAGA
GGGACGCTTCATTCTTGGTTTTAGTGACTGCGAAAGTGATTTCGAAATGGAATTTTTTAGAC
GTCATATCTCATCAAGGCAACAACAATTTGAAGCATGCTATGAAATAATTAATGACGCATTA
ACTACAGGTTATTGTCATCCCCAAAACGACTTTTATGATTTTCCAAAGGTTTCAATTAATCC
ACACTGTTACAGTGAGAATGGACCTAAGCAATATGTATCCGCTACATCAAAAGAAGTCGTCA
TGTGGGCAGCGAAAAAGGCACTGCCTTTAACATTTAAGTGGGAGGATAATTTAGAAACCAAA
GAACGCTATGCAATTCTATATAATAAAACAGCACAACAATATGGTATTGATATTTCGGATGT
TGATCATCAATTAACTGTAATTGCGAACTTAAATGCTGATAGAAGTACGGCTCAAGAAGAAG
TGAGAGAATACTTAAAAGACTATATCACTGAAACTTACCCTCAAATGGACAGAGATGAAAAA
ATTAACTGCATTATTGAAGAGAATGCAGTTGGGTCTCATGATGACTATTATGAATCGACAAA
ATTAGCAGTGGAAAAAACAGGGTCTAAAAATATTTTATTATCCTTTGAATCAATGTCCGATA
TTAAAGATGTAAAAGATATTATTGATATGTTGAACCAAAAAATCGAAATGAATTTACCATAA
AGTAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAGACGGCATGA
TGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGT
GAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCA
CCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGG
TTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTG
GTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGT
GAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAATTCCGGATGAGCA
TTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTAC
GGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTG
ACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCA
GTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATAC
GCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGT
CTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATT
TATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGAAGACGAAAGGGCCTCGTGATAC
GCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTT
CGGGGAAATGTGCGCGCCCGCGTTCCTGCTGGCGCTGGGCCTGTTTCTGGCGCTGGACTTCC
CGCTGTTCCGTCAGCAGCTTTTCGCCCACGGCCTTGATGATCGCGGCGGCCTTGGCCTGCAT
ATCCCGATTCAACGGCCCCAGGGCGTCCAGAACGGGCTTCAGGCGCTCCCGAAGGT
SEQ IDN0:12
S. aureus P clpB Promoter Sequence
GTCTAGTTAATGTGTAACGTAACATTAGCTAGATTTTTTTATTCAAAAAAATATTTACAAAT
ATTAGGAAATTTAAGTGTAAAAGAGTTGATAAATGATTATATTGGGACTATAATATAATTAA
GGTC
SEQ IDN0:13
RN10616 genomic sequence loci showing the cp80a terS deletion and complementation.
terS=Bracketed Text, Deletion=Underlined, Complement=Bold
ATTAGACAACAAACAAGTCATTGAAAATTCCGACTTATTATTCAAAAAGAAATTTGATAGCG
CAGATATACAAGCTAGGTTAAAAGTAGGCGATAAGGTAGAAGTTAAAACAATCGGTTATAGA
ATACACTTTTTAAATTTATATCCGGTCTTATACGAAGTAAAGAAGGTAGATAAACAATGATT
AAACAAATACTAAGACTATTATTCTTACTAGCAATGTATGAGTTAGGTAAGTATGTAACTGA
GCAAGTATATATTATGATGACGGCTAATGATGATGTAGAGGTGCCGAGTGACTTCGCGAAGT
TGAGCGATCAGTCAGATTTGATGAGGGCGGAGGTGACGGAGTAGATGATGTGGTTAGTCATA
GCAATTATATTACTAGTCATCTTATTGTTTGGTGTGATGTTGCAAGCTGAACAGTTAAAAGG
CGATGTGAAAGTTAAAGAGCGGGAGATAGAGATATTAAGAAGTAGATTGAGACATTTTGAAG
ATTAAAAATATTTGTATGGAGGGTATTCATGACTAAAAAGAAATATGGATTAAAATTATCAA
CAGTTCGAAAGTTAGAAGATGAGTTGTGTGATTATCCTAATTATCATAAGCAACTCGAAGAT
TTAAGAAGTGAAATAATGACACCATGGATTCCAACAGATACAAATATAGGCGGGGAGTTTGT
ACCGTCTAATACATCGAAAACAGAAATGGCAGTAACTAATTATCTTTGTAGTATACGAAGAG
GTAAAATCCTTGAGTTTAAGAGCGCTATTGAACGTATAATCAACACATCAAGTAGGAAAGAA
CGCGAATTCATTCAAGAGTATTATTTTAATAAAAAGGAATTAGTGAAAGTTTGTGATGACAT
ACACATTTCTGATAGAACTGCTCATAGAATCAAAAGGAAAATCATATCTAGATTGGCGGAAG
AGTTAGGGGAAGAGTGAAATTGGCAGTAAAGTGGCAGTTTTTGATACCTAAAATGAGATATT
ATGATAGTGTAGGATATTGACTATCTTACTGCGTTTCCCTTATCGCAATTAGGAATAAAGGA
TCTATGTGGGTTGGCTGATTATAGCCAATCCTTTTTTAATTTTAAAAAGCGTATAGCGCGAG
AGTTGGTGGTAAATGAA
[[ATGAACGAAAAACAAAAGAGATTCGCAGATGAATATATAATGAATGGATGTAATGGTAAA
AAAGCAGCAATTTCAGCAGGTTATAGTAAGAAAACAGCAGAGTCTTTAGCAAGTCGATTGTT
AAGAAATGTTAATGTTTCGGAATATATTAAAGAACGATTAGAACAGATACAAGAAGAGCGTT
TAATGAGCATTACAGAAGCTTTAGCGTTATCTGCTTCTATTGCTAGAGGAGAACCTCAAGAG
GCTTACAGTAAGAAATATGACCATTTAAACGATGAAGTGGAAAAAGAGGTTACTTACACAAT
CACACCAACTTTTGAAGAGCGTCAGAGATCTATTGACCACATACTAAAAGTTCATGGTGCGT
ATATCGACAAAAAAGAAATTACTCAGAAGAATATTGAGATTAATATTGGTGAGTACGATGAC
GAAAGTTAA] ]
ATTAAACTTTAACAAACCATCTAATGTTTTCAACAGAAACATATTCGAAATACTAACCAATT
ACGATAACTTCACTGAAGTACATTACGGTGGAGGTTCGAGTGGTAAGTCTCACGGCGTTATA
CAAAAAGTTGTACTTAAAGCATTGCAAGACTGGAAATATCCTAGGCGTATACTATGGCTTAG
AAAAGTCCAATCAACAATTAAAGATAGTTTATTCGAAGATGTCAAAGATTGTTTGATAAACT
TCGGTATTTGGGACATGTGCCTTTGGAATAAGACTGATAACAAAGTTGAATTGCCAAACGGC
GCAGTTTTTTTGTTTAAAGGATTAGATAACCCAGAGAAAATAAAGTCGATAAAAGGCATATC
AGACATAGTCATGGAAGAAGCGTCTGAATTCACACTAAATGATTACACGCAATTAACGTTGC
GTTTGAGGGAGCGTAAACACGTGAATAAGCAAATATTTTTGATGTTTAACCCAGTATCTAAA
CTGAATTGGGTTTATAAGTATTTCTTTGAACATGGTGAACCAATGGAAAATGTCATGATTAG
ACAATCTAGTTATCGAGATAATAAGTTTCTTGATGAAATGACACGACAAAACTTAGAGTTGT
TAGCAAATCGTAATCCAGCATATTACAAAATTTATGCGTTAGGTGAATTTTCTACACTAGAC
AAATTGGTTTTCCCTAAGTATGAAAAACGTTTAATAAATAAAGATGAGTTAAGACATTTACC
TTCTTATTTTGGATTGGACTTTGGCTACGTTAATGATCCTAGTGCTTTTATACATTCTAAAA
TAGATGTAAAGAAAAAGAAGTTATACATCATTGAAGAGTATGTTAAACAAGGTATGCTGAAT
GATGAAATAGCTAATGTCATAAAGCAACTTGGTTATGCTAAAGAAGAAATTACAGCAGATAG
TGCAGAACAAAAAAGTATAGCTGAATTAAGGAATCTAGGGCTTAAAAGGATTTTACCAACCA
AAAAAGGGAAGGGCTCGGTTGTACAAGGGTTACAATTCTTAATGCAATTTGAAATCATTGTT
GATGAACGTTGTTTCAAGACTATTGAAGAGTTTGACAACTACACATGGCAAAAGGACAAAGA
TACAGGTGAATATACCAATGAACCAGTAGATACATACAATCATTGTATCGATTCGTTGCGTT
ATTCAGTGGAACGATTC
SEQ IDN0:14
pGW80AOOO 1 Full Sequence
GGCGCCATGGTTAAGGGCCCTTTGCGGAAAGAGTTAGTAAGTTAACAGAAGACGAACCAAAA
CTAAATGGTTTAGCAGGAAACTTAGATAAAAAAATGAATCCAGAATTATATTCAGAACAGGA
ACAGCAACAAGAACAACAAAAGAATCAAAAACGAGATAGAGGTATGCACTTATAGAACATGC
ATTTATGCCGAGAAAACTTATTGGTTGGAATGGGCTATGTGTTAGCTAACTTGTTAGCGAGT
TGGTTGGACTTGAATTGGGATTAATCCCAAGAAAGTACCAACTCAACAACACATAAAGCCCT
GTAGGTTCCGACCAATAAGGAAATTGGAATAAAGCAATAAAAGGAGTTGAAGAAATGAAATT
CAGAGAAGCCTTTGAGAATTTTATAACAAGTAAGTATGTACTTGGTGTTTTAGTAGTCTTAA
CTGTTTACCAGATAATACAAATGCTTAAATAAAAAAAGACTTGATCTGATTAGACCAAATCT
TTTGATAGTGTTATATTAATAACAAAATAAAAAGGAGTCGCTCACGCCCTACCAAAGTTTGT
GAACGACATCATTCAAAGAAAAAAACACTGAGTTGTTTTTATAATCTTGTATATTTAGATAT
TAAACGATATTTAAATATACATCAAGATATATATTTGGGTGAGCGATTACTTAAACGAAATT
GAGATTAAGGAGTCGATTTTTTATGTATAAAAACAATCATGCAAATCATTCAAATCATTTGG
AAAATCACGATTTAGACAATTTTTCTAAAACCGGCTACTCTAATAGCCGGTTGGACGCACAT
ACTGTGTGCATATCTGATCCAAAATTAAGTTTTGATGCAATGACGATCGTTGGAAATCTCAA
CCGAGACAACGCTCAGGCCCTTTCTAAATTTATGAGTGTAGAGCCCCAAATAAGACTTTGGG
ATATTCTTCAAACAAAGTTTAAAGCTAAAGCACTTCAAGAAAAAGTTTATATTGAATATGAC
AAAGTGAAAGCAGATAGTTGGGATAGACGTAATATGCGTATTGAATTTAATCCAAACAAACT
TACACGAGATGAAATGATTTGGTTAAAACAAAATATAATAAGCTACATGGAAGATGACGGTT
TTACAAGATTAGATTTAGCCTTTGATTTTGAAGATGATTTGAGTGACTACTATGCAATGTCT
GATAAAGCAGTTAAGAAAACTATTTTTTATGGTCGTAATGGTAAGCCAGAAACAAAATATTT
TGGCGTGAGAGATAGTAATAGATTTATTAGAATTTATAATAAAAAGCAAGAACGTAAAGATA
ATGCAGATGCTGAAGTTATGTCTGAACATTTATGGCGTGTAGAAATCGAACTTAAAAGAGAT
ATGGTGGATTACTGGAATGATTGCTTTAGTGATTTACATATCTTGCAACCAGATTGGAAAAC
TATCCAACGCACTGCGGATAGAGCAATAGTTTTTATGTTATTGAGTGATGAAGAAGAATGGG
GAAAGCTTCACAGAAATTCTAGAACAAAATATAAGAATTTGATAAAAGAAATTTCGCCAGTC
GATTTAACGGACTTAATGAAATCGACTTTAAAAGCGAACGAAAAACAATTGCAAAAACAAAT
CGATTTTTGGCAACATGAATTTAAATTTTGGAAATAGTGTACATATTAATATTACTGAACAA
AAATGATATATTTAAACTATTCTAATTTAGGAGGATTTTTTTATGAAGTGTCTATTTAAAAA
TTTGGGGAATTTATATGAGGTGAAAGAATAATTTACCCCTATAAACTTTAGCCACCTCAAGT
AAAGAGGTAAAATTGTTTAGTTTATATAAAAAATTTAAAGGTTTGTTTTATAGCGTTTTATT
TTGGCTTTGTATTCTTTCATTTTTTAGTGTATTAAATGAAATGGTTTTAAATGTTTCTTTAC
CTGATATTGCAAATCATTTTAATACTACTCCTGGAATTACAAACTGGGTAAACACTGCATAT
ATGTTAACTTTTTCGATAGGAACAGCAGTATATGGAAAATTATCTGATTATATAAATATAAA
AAAATTGTTAATTATTGGTATTAGTTTGAGCTGTCTTGGTTCATTGATTGCTTTTATTGGGC
CCACCTAGGCAAATATGCTCTTACGTGCTATTATTTAAGTGACTATTTAAAAGGAGTTAATA
AATATGCGGCAAGGTATTCTTAAATAAACTGTCAATTTGATAGCGGGAACAAATAATTAGAT
GTCCTTTTTTAGGAGGGCTTAGTTTTTTGTACCCAGTTTAAGAATACCTTTATCATGTGATT
CTAAAGTATCCAGAGAATATCTGTATGCTTTGTATACCTATGGTTATGCATAAAAATCCCAG
TGATAAAAGTATTTATCACTGGGATTTTTATGCCCTTTTGGGTTTTTGAATGGAGGAAAATC
ACATGAAAATTATTAATATTGGAGTTTTAGCTCATGTTGATGCAGGAAAAACTACCTTAACA
GAAAGCTTATTATATAACAGTGGAGCGATTACAGAATTAGGAAGCGTGGACAAAGGTACAAC
GAGGACGGATAATACGCTTTTAGAACGTCAGAGAGGAATTACAATTCAGACAGGAATAACCT
CTTTTCAGTGGGAAAATACGAAGGTGAACATCATAGACACGCCAGGACATATGGATTTCTTA
GCAGAAGTATATCGTTCATTATCAGTTTTAGATGGGGCAATTCTACTGATTTCTGCAAAAGA
TGGCGTACAAGCACAAACTCGTATATTATTTCATGCACTTAGGAAAATGGGGATTCCCACAA
TCTTTTTTATCAATAAGATTGACCAAAATGGAATTGATTTATCAACGGTTTATCAGGATATT
AAAGAGAAACTTTCTGCCGAAATTGTAATCAAACAGAAGGTAGAACTGTATCCTAATATGTG
TGTGACGAACTTTACCGAATCTGAACAATGGGATACGGTAATAGAGGGAAACGATAACCTTT
TAGAGAAATATATGTCCGGTAAATCATTAGAAGCATTGGAACTCGAACAAGAGGAAAGCATA
AGATTTCAGAATTGTTCTCTGTTCCCTCTTTATCATGGAAGTGCAAAAAGTAATATAGGGAT
TGATAACCTTATAGAAGTTATTACTAATAAATTTTATTCATCAACACATCGAGGTCCGTCTG
AACTTTGCGGAAATGTTTTCAAAATTGAATATACAAAAAAAAGACAACGTCTTGCATATATA
CGCCTTTATAGTGGAGTACTACATTTACGAGATTCGGTTAGAGTATCAGAAAAAGAAAAAAT
AAAAGTTACAGAAATGTATACTTCAATAAATGGTGAATTATGTAAGATTGATAGAGCTTATT
CTGGAGAAATTGTTATTTTGCAAAATGAGTTTTTGAAGTTAAATAGTGTTCTTGGAGATACA
AAACTATTGCCACAGAGAAAAAAGATTGAAAATCCGCACCCTCTACTACAAACAACTGTTGA
ACCGAGTAAACCTGAACAGAGAGAAATGTTGCTTGATGCCCTTTTGGAAATCTCAGATAGTG
ATCCGCTTCTACGATATTACGTGGATTCTACGACACATGAAATTATACTTTCTTTCTTAGGG
AAAGTACAAATGGAAGTGATTAGTGCACTGTTGCAAGAAAAGTATCATGTGGAGATAGAACT
AAAAGAGCCTACAGTCATTTATATGGAGAGACCGTTAAAAAATGCAGAATATACCATTCACA
TCGAAGTGCCGCCAAATCCTTTCTGGGCTTCCATTGGTTTATCTGTATCGCCGCTTCCGTTG
GGAAGTGGAATGCAGTATGAGAGCTCGGTTTCTCTTGGATACTTAAATCAATCATTTCAAAA
TGCAGTTATGGAAGGGGTACGCTATGGTTGCGAACAAGGATTATATGGTTGGAATGTGACGG
ATTGTAAAATCTGTTTTAAGTACGGTTTATACTATAGCCCTGTTAGTACTCCAGCAGATTTT
CGGATGCTTACTCCTATTGTACTGGAGCAAGCCTTTAGAAAAGCTGGAACAGAATTGTTAGA
GCCATATCTTAGTTTTAAAGTTTATGCACCACAGGAATATCTTTCACGGGCATATAACGATG
CTCCCAAATATTGTGCAAATATCGTAAATACTCAACTGAAAAATAATGAGGTCATTATTATT
GGAGAAATTCCTGCTCGATGTATTCAAGATTATCGCAATGATTTAACTTTTTTTACAAATGG
GCTTAGTGTTTGTTTAGCAGAGCTAAAAGGATATCAGGTTACCACTGGCGAACCTGTTTGCC
AGACCCGTCGTCTAAATAGTCGGATAGATAAAGTAAGATATATGTTCAATAAAATAACTTAG
TGCGTTTTATGTTGTTATATAAATATGGTTTCTTATTAAATAAGATGAAATATTCTTTAATA
TAGATTTGAATTAAAGTGGAAAGGAGGAGATTGTTATTATAAACTACAAGTGGATATTGTGT
CCTAGTTGTGGAAATAAAACAAGACTACGAATACGAGTGGATACTATACTTAAAAATTTCCC
TTTATACAGCCCCAAATGTAAGAACGAAACTTTAATTAATGTTCAAAAAATGAATATAATAA
CAATCAAAGAGCCAGACGCCAAGACGCAGAGCCGATAATTTGAGAAATGAAACTCTCATCTT
ATCGGCTCTTTTTGTTTATCTGAATTTTACTGACTAGCCTTCAATATTTCCGCGGCCAGCTT
ACTATGCCATTATTAAGCTTGTAATATCGGAGGGTTTATTAATTGGCAGTAAAGTGGCAGTT
TTTGATACCTTAAATGAGATATTATGATAGTGTAGGATATTGACTATCGTACTGCGTTTCCC
TACCGCAAATTAGGAATAAAGGATCTATGTGGGTTGGCTGATTATAGCCAATCCTTTTTTAA
TTTTAAAAAGCGTATAGCGCGAGAGTTGGTGGTAAATGAAATGAACGAAAAACAAAAGAGAT
TCGCAGATGAATATATAATGAATGGATGTAATGGTAAAAAAGCAGCAATTACAGTAGGTTAT
AGTAAGAAAACAGCAGAGTCTTTAGCAAGTCGATTGTTAAGAAATGTTAATGTTTCGGAATA
TATTAAAGAACGATTAGAACAGGTACAAGAAGAGCGTTTAATGAGTATTACAGAAGCTTTAG
CGTTATCTGCTTCTATTGCTAGAGGAGAACCTCAAGAGGCTTACAGTAAGAAATATGACCAT
TTAAACGATGAAGTGGAAAAAGAGGTTACTTACACAATCACACCAACTTTTGAAGAGCGTCA
GAGATCTATTGACCACATACTAAAAGTACATGGTGCGTATATCGATAAAAAAGAAATTACTC
AGAAGAATATTGAGATTAATATTGGTGAGTACGATGACGAAAGTTAAATTGAACTTTAACAA
ACCGTCTAATGTTTTCAATAGCCGCGGGGGCCCAACACACCAACTTTTGAAGAGCGTCAGAG
ATCTATTGACCACATACTAAAAGTACATGGTGCGTATATCGATAAAAAAGAAATTACTCAGA
AGAATATTGAGATTAATATTGGTGAGTACGATGACGAAAGTTAAATTAAACTTTAACAAACC
GTCTAATGTTTTCAATAGCCGCGGGGGCCCAACGAGCGGCCGCATAGTTAAGCCAGCCCCGA
CACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAG
ACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAAC
GCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATG
GTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATT
TTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAAT
AATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTT
GCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGA
AGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTG
AGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGC
GCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCA
GAATGACTTGGTTGAGTACTCACCGGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAA
GAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACA
ACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCG
CCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA
TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCT
TCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTC
GGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCG
GTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACG
GGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGAT
TAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTC
ATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCT
TAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTG
AGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGG
TGGTTTTTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA
GCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTC
TGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCG
ATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCG
GGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACCTGA
GATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGG
TATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGC
CTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGAT
GCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTG
GCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAA
CCGTATTACCGCCTTTGAGTGAGCTGGCGGGTCTAGTTAATGTGTAACGTAACATTAGCTAG
ATTTTTTTATTCAAAAAAATATTTACAAATATTAGGAAATTTAAGTGTAAAAGAGTTGATAA
ATGATTATATTGGGACTATAATATAATTAAGGTCGATTGAATTCGTTAACTAATTAATCACC
AAAAAGGAATAGAGTATGAAGTTTGGAAATATTTGTTTTTCGTATCAACCACCAGGTGAAAC
TCATAAGCAAGTAATGGATCGCTTTGTTCGGCTTGGTATCGCCTCAGAAGAGGTAGGGTTTG
ATACATATTGGACCTTAGAACATCATTTTACAGAGTTTGGTCTTACGGGAAATTTATTTGTT
GCTGCGGCTAACCTGTTAGGAAGAACTAAAACATTAAATGTTGGCACTATGGGGGTTGTTAT
TCCGACAGCACACCCAGTTCGACAGTTAGAAGACGTTTTATTATTAGATCAAATGTCGAAAG
GTCGTTTTAATTTTGGAACCGTTCGAGGGCTATACCATAAAGATTTTCGAGTATTTGGTGTT
GATATGGAAGAGTCTCGAGCAATTACTCAAAATTTCTACCAGATGATAATGGAAAGCTTACA
GACAGGAACCATTAGCTCTGATAGTGATTACATTCAATTTCCTAAGGTTGATGTATATCCCA
AAGTGTACTCAAAAAATGTACCAACCTGTATGACTGCTGAGTCCGCAAGTACGACAGAATGG
CTAGCAATACAAGGGCTACCAATGGTTCTTAGTTGGATTATTGGTACTAATGAAAAAAAAGC
ACAGATGGAACTCTATAATGAAATTGCGACAGAATATGGTCATGATATATCTAAAATAGATC
ATTGTATGACTTATATTTGTTCTGTTGATGATGATGCACAAAAGGCGCAAGATGTTTGTCGG
GAGTTTCTGAAAAATTGGTATGACTCATATGTAAATGCGACCAATATCTTTAATGATAGCAA
TCAAACTCGTGGTTATGATTATCATAAAGGTCAATGGCGTGATTTTGTTTTACAAGGACATA
CAAACACCAATCGACGTGTTGATTATAGCAATGGTATTAACCCCGTAGGCACTCCTGAGCAG
TGTATTGAAATCATTCAACGTGATATTGATGCAACGGGTATTACAAACATTACATGCGGATT
TGAAGCTAATGGAACTGAAGATGAAATAATTGCTTCCATGCGACGCTTTATGACACAAGTCG
CTCCTTTCTTAAAAGAACCTAAATAAATTACTTATTTGATACTAGAGATAATAAGGAACAAG
TTATGAAATTTGGATTATTTTTTCTAAACTTTCAGAAAGATGGAATAACATCTGAAGAAACG
TTGGATAATATGGTAAAGACTGTCACGTTAATTGATTCAACTAAATATCATTTTAATACTGC
CTTTGTTAATGAACATCACTTTTCAAAAAATGGTATTGTTGGAGCACCTATTACCGCAGCTG
GTTTTTTATTAGGGTTAACAAATAAATTACATATTGGTTCATTAAATCAAGTAATTACCACC
CATCACCCTGTACGTGTAGCAGAAGAAGCCAGTTTATTAGATCAAATGTCAGAGGGACGCTT
CATTCTTGGTTTTAGTGACTGCGAAAGTGATTTCGAAATGGAATTTTTTAGACGTCATATCT
CATCAAGGCAACAACAATTTGAAGCATGCTATGAAATAATTAATGACGCATTAACTACAGGT
TATTGCCATCCCCAAAACGACTTTTATGATTTTCCAAAGGTTTCAATTAATCCACACTGTTA
CAGTGAGAATGGACCTAAGCAATATGTATCCGCTACATCAAAAGAAGTCGTCATGTGGGCAG
CGAAAAAGGCACTGCCTTTAACGTTTAAGTGGGAGGATAATTTAGAAACCAAAGAACGCTAT
GCAATTCTATATAATAAAACAGCACAACAATATGGTATTGATATTTCGGATGTTGATCATCA
ATTAACTGTAATTGCGAACTTAAATGCTGATAGAAGTACGGCTCAAGAAGAAGTGAGAGAAT
ACTTAAAAGACTATATCACTGAAACTTACCCTCAAATGGACAGAGATGAAAAAATTAACTGC
ATTATTGAAGAGAATGCAGTTGGGTCTCATGATGACTATTATGAATCGACAAAATTAGCAGT
GGAAAAAACAGGGTCTAAAAATATTTTATTATCCTTTGAATCAATGTCCGATATTAAAGATG
TAAAAGATATTATTGATATGTTGAACCAAAAAATCGAAATGAATTTACCATAATAAAATTAA
AGGCAATTTCTATATTAGATTGCCTTTTTGGCGCGCCTATTCTAATGCATAATAAATACTGA
TAACATCTTATATTTTGTATTATATTTTGTATTATCGTTGACATGTATAATTTTGATATCAA
AAACTGATTTTCCCTCTATTATTTTCGAGATTTATTTTCTTAATTCTCTTTAACAAACTAGA
AATATTGTATATACAAAAAATTATAAATAATAGATGAATAGTTTAATTATAGGTGTTCATCA
ATCGAAAAAGCAACGTATCTTATTTAAAGTGCGTTGCTTTTTTCTCATTTATAAGGTTAAAT
AATTCTCATATATCAAGCAAAGTGACA
SEQ ID N0:15 (mecA gene loci DNA Sequence (from Staphylococcus aureus subsp. aureus
SA40, complete genome GenBank: CP003604.1))
TATACTACAAATGTAGTCTTATATAAGGAGGATATTGATGAAAAAGATAAAAATTGTTCCAC
TTATTTTAATAGTTGTAGTTGTCGGGTTTGGTATATATTTTTATGCTTCAAAAGATAAAGAA
ATTAATAATACTATTGATGCAATTGAAGATAAAAATTTCAAACAAGTTTATAAAGATAGCAG
TTATATTTCTAAAAGCGATAATGGTGAAGTAGAAATGACTGAACGTCCGATAAAAATATATA
ATAGTTTAGGCGTTAAAGATATAAACATTCAGGATCGTAAAATAAAAAAAGTATCTAAAAAT
AAAAAACGAGTAGATGCTCAATATAAAATTAAAACAAACTACGGTAACATTGATCGCAACGT
TCAATTTAATTTTGTTAAAGAAGATGGTATGTGGAAGTTAGATTGGGATCATAGCGTCATTA
TTCCAGGAATGCAGAAAGACCAAAGCATACATATTGAAAATTTAAAATCAGAACGTGGTAAA
ATTTTAGACCGAAACAATGTGGAATTGGCCAATACAGGAACAGCATATGAGATAGGCATCGT
TCCAAAGAATGTATCTAAAAAAGATTATAAAGCAATCGCTAAAGAACTAAGTATTTCTGAAG
ACTATATCAAACAACAAATGGATCAAAATTGGGTACAAGATGATACCTTCGTTCCACTTAAA
ACCGTTAAAAAAATGGATGAATATTTAAGTGATTTCGCAAAAAAATTTCATCTTACAACTAA
TGAAACAGAAAGTCGTAACTATCCTCTAGAAAAAGCGACTTCACATCTATTAGGTTATGTTG
GTCCCATTAACTCTGAAGAATTAAAACAAAAAGAATATAAAGGCTATAAAGATGATGCAGTT
ATTGGTAAAAAGGGACTCGAAAAACTTTACGATAAAAAGCTCCAACATGAAGATGGCTATCG
TGTCACAATCGTTGACGATAATAGCAATACAATCGCACATACATTAATAGAGAAAAAGAAAA
AAGATGGCAAAGATATTCAACTAACTATTGATGCTAAAGTTCAAAAGAGTATTTATAACAAC
ATGAAAAATGATTATGGCTCAGGTACTGCTATCCACCCTCAAACAGGTGAATTATTAGCACT
TGTAAGCACACCTTCATATGACGTCTATCCATTTATGTATGGCATGAGTAACGAAGAATATA
ATAAATTAACCGAAGATAAAAAAGAACCTCTGCTCAACAAGTTCCAGATTACAACTTCACCA
GGTTCAACTCAAAAAATATTAACAGCAATGATTGGGTTAAATAACAAAACATTAGACGATAA
AACAAGTTATAAAATCGATGGTAAAGGTTGGCAAAAAGATAAATCTTGGGGTGGTTACAACG
TTACAAGATATGAAGTGGTAAATGGTAATATCGACTTAAAACAAGCAATAGAATCATCAGAT
AACATTTTCTTTGCTAGAGTAGCACTCGAATTAGGCAGTAAGAAATTTGAAAAAGGCATGAA
AAAACTAGGTGTTGGTGAAGATATACCAAGTGATTATCCATTTTATAATGCTCAAATTTCAA
ACAAAAATTTAGATAATGAAATATTATTAGCTGATTCAGGTTACGGACAAGGTGAAATACTG
ATTAACCCAGTACAGATCCTTTCAATCTATAGCGCATTAGAAAATAATGGCAATATTAACGC
ACCTCACTTATTAAAAGACACGAAAAACAAAGTTTGGAAGAAAAATATTATTTCCAAAGAAA
ATATCAATCTATTAACTGATGGTATGCAACAAGTCGTAAATAAAACACATAAAGAAGATATT
TATAGATCTTATGCAAACTTAATTGGCAAATCCGGTACTGCAGAACTCAAAATGAAACAAGG
AGAAACTGGCAGACAAATTGGGTGGTTTATATCATATGATAAAGATAATCCAAACATGATGA
TGGCTATTAATGTTAAAGATGTACAAGATAAAGGAATGGCTAGCTACAATGCCAAAATCTCA
GGTAAAGTGTATGATGAGCTATATGAGAACGGTAATAAAAAATACGATATAGATGAATAACA
AAACAGTGAAGCAATCCGTAACGATGGTTGCTTCACTGTTTT
SEQ ID N0:16 (mecA transcript sequence)
UAGUCUUAUAUAAGGAGGAUAUUGAUGAAAAAGAUAAAAAUUGUUCCACUUAUUUUAAUAGU
UGUAGUUGUCGGGUUUGGUAUAUAUUUUUAUGCUUCAAAAGAUAAAGAAAUUAAUAAUACUA
UUGAUGCAAUUGAAGAUAAAAAUUUCAAACAAGUUUAUAAAGAUAGCAGUUAUAUUUCUAAA
AGCGAUAAUGGUGAAGUAGAAAUGACUGAACGUCCGAUAAAAAUAUAUAAUAGUUUAGGCGU
UAAAGAUAUAAACAUUCAGGAUCGUAAAAUAAAAAAAGUAUCUAAAAAUAAAAAACGAGUAG
AUGCUCAAUAUAAAAUUAAAACAAACUACGGUAACAUUGAUCGCAACGUUCAAUUUAAUUUU
GUUAAAGAAGAUGGUAUGUGGAAGUUAGAUUGGGAUCAUAGCGUCAUUAUUCCAGGAAUGCA
GAAAGACCAAAGCAUACAUAUUGAAAAUUUAAAAUCAGAACGUGGUAAAAUUUUAGACCGAA
ACAAUGUGGAAUUGGCCAAUACAGGAACAGCAUAUGAGAUAGGCAUCGUUCCAAAGAAUGUA
UCUAAAAAAGAUUAUAAAGCAAUCGCUAAAGAACUAAGUAUUUCUGAAGACUAUAUCAAACA
ACAAAUGGAUCAAAAUUGGGUACAAGAUGAUACCUUCGUUCCACUUAAAACCGUUAAAAAAA
UGGAUGAAUAUUUAAGUGAUUUCGCAAAAAAAUUUCAUCUUACAACUAAUGAAACAGAAAGU
CGUAACUAUCCUCUAGAAAAAGCGACUUCACAUCUAUUAGGUUAUGUUGGUCCCAUUAACUC
UGAAGAAUUAAAACAAAAAGAAUAUAAAGGCUAUAAAGAUGAUGCAGUUAUUGGUAAAAAGG
GACUCGAAAAACUUUACGAUAAAAAGCUCCAACAUGAAGAUGGCUAUCGUGUCACAAUCGUU
GACGAUAAUAGCAAUACAAUCGCACAUACAUUAAUAGAGAAAAAGAAAAAAGAUGGCAAAGA
UAUUCAACUAACUAUUGAUGCUAAAGUUCAAAAGAGUAUUUAUAACAACAUGAAAAAUGAUU
AUGGCUCAGGUACUGCUAUCCACCCUCAAACAGGUGAAUUAUUAGCACUUGUAAGCACACCU
UCAUAUGACGUCUAUCCAUUUAUGUAUGGCAUGAGUAACGAAGAAUAUAAUAAAUUAACCGA
AGAUAAAAAAGAACCUCUGCUCAACAAGUUCCAGAUUACAACUUCACCAGGUUCAACUCAAA
AAAUAUUAACAGCAAUGAUUGGGUUAAAUAACAAAACAUUAGACGAUAAAACAAGUUAUAAA
AUCGAUGGUAAAGGUUGGCAAAAAGAUAAAUCUUGGGGUGGUUACAACGUUACAAGAUAUGA
AGUGGUAAAUGGUAAUAUCGACUUAAAACAAGCAAUAGAAUCAUCAGAUAACAUUUUCUUUG
CUAGAGUAGCACUCGAAUUAGGCAGUAAGAAAUUUGAAAAAGGCAUGAAAAAACUAGGUGUU
GGUGAAGAUAUACCAAGUGAUUAUCCAUUUUAUAAUGCUCAAAUUUCAAACAAAAAUUUAGA
UAAUGAAAUAUUAUUAGCUGAUUCAGGUUACGGACAAGGUGAAAUACUGAUUAACCCAGUAC
AGAUCCUUUCAAUCUAUAGCGCAUUAGAAAAUAAUGGCAAUAUUAACGCACCUCACUUAUUA
AAAGACACGAAAAACAAAGUUUGGAAGAAAAAUAUUAUUUCCAAAGAAAAUAUCAAUCUAUU
AACUGAUGGUAUGCAACAAGUCGUAAAUAAAACACAUAAAGAAGAUAUUUAUAGAUCUUAUG
CAAACUUAAUUGGCAAAUCCGGUACUGCAGAACUCAAAAUGAAACAAGGAGAAACUGGCAGA
CAAAUUGGGUGGUUUAUAUCAUAUGAUAAAGAUAAUCCAAACAUGAUGAUGGCUAUUAAUGU
UAAAGAUGUACAAGAUAAAGGAAUGGCUAGCUACAAUGCCAAAAUCUCAGGUAAAGUGUAUG
AUGAGCUAUAUGAGAACGGUAAUAAAAAAUACGAUAUAGAUGAAUAACAAAACAGUGAAGCA
AUCCGUAACGAUGGUUGCUUCACUGUUUU
SEQ ID NO: 17 (luxAB gene loci DNA sequence (from Vibrio fischeri genes luxA and luxB
for luciferase alpha and beta subunits - GenBank: X06758.1))
GGCTTAAATAAACAGAATCACCAAAAAGGAATAGAGTATGAAGTTTGGAAATATTTGTTTTT
CGTATCAACCACCAGGTGAAACTCATAAGCTAAGTAATGGATCGCTTTGTTCGGCTTGGTAT
CGCCTCAGAAGAGTAGGGTTTGATACATATTGGACCTTAGAACATCATTTTACAGAGTTTGG
TCTTACGGGAAATTTATTTGTTGCTGCGGCTAACCTGTTAGGAAGAACTAAAACATTAAATG
TTGGCACTATGGGGGTTGTTATTCCGACAGCACACCCAGTTCGACAGTTAGAAGACGTTTTA
TTATTAGATCAAATGTCGAAAGGTCGTTTTAATTTTGGAACCGTTCGAGGGCTATACCATAA
AGATTTTCGAGTATTTGGTGTTGATATGGAAGAGTCTCGAGCAATTACTCAAAATTTCTACC
AGATGATAATGGAAAGCTTACAGACAGGAACCATTAGCTCTGATAGTGATTACATTCAATTT
CCTAAGGTTGATGTATATCCCAAAGTGTACTCAAAAAATGTACCAACCTGTATGACTGCTGA
GTCCGCAAGTACGACAGAATGGCTAGCAATACAAGGGCTACCAATGGTTCTTAGTTGGATTA
TTGGTACTAATGAAAAAAAAGCACAGATGGAACTCTATAATGAAATTGCGACAGAATATGGT
CATGATATATCTAAAATAGATCATTGTATGACTTATATTTGTTCTGTTGATGATGATGCACA
AAAGGCGCAAGATGTTTGTCGGGAGTTTCTGAAAAATTGGTATGACTCATATGTAAATGCGA
CCAATATCTTTAATGATAGCAATCAAACTCGTGGTTATGATTATCATAAAGGTCAATGGCGT
GATTTTGTTTTACAAGGACATACAAACACCAATCGACGTGTTGATTATAGCAATGGTATTAA
CCCTGTAGGCACTCCTGAGCAGTGTATTGAAATCATTCAACGTGATATTGATGCAACGGGTA
TTACAAACATTACATGCGGATTTGAAGCTAATGGAACTGAAGATGAAATAATTGCTTCCATG
CGACGCTTTATGACACAAGTCGCTCCTTTCTTAAAAGAACCTAAATAAATTACTTATTTGAT
ACTAGAGATAATAAGGAACAAGTTATGAAATTTGGATTATTTTTTCTAAACTTTCAGAAAGA
TGGAATAACATCTGANGAAACGTTGGATAATATGGTAAAGACTGTCACGTTAATTGATTCAA
CTAAATATCATTTTAATACTGCCTTTGTTAATGAACATCACTTTTCAAAAAATGGTATTGTT
GGAGCACCTATTACCGCAGCTGGTTTTTTATTAGGGTTAACAAATAAATTACATATTGGTTC
ATTAAATCAAGTAATTACCACCCATCACCCTGTACGTGTAGCAGAAGAAGCCAGTTTATTAG
ATCAAATGTCAGAGGGACGCTTCATTCTTGGTTTTAGTGACTGCGAAAGTGATTTCGAAATG
GAATTTTTTAGACGTCATATCTCATCAAGGCAACAACAATTTGAAGCATGCTATGAAATAAT
TAATGACGCATTAACTACAGGTTATTGTCATCCCCAAAACGACTTTTATGATTTTCCAAAGG
TTTCAATTAATCCACACTGTTACAGTGAGAATGGACCTAAGCAATATGTATCCGCTACATCA
AAAGAAGTCGTCATGTGGGCAGCGAAAAAGGCACTGCCTTTAACATTTAAGTGGGAGGATAA
TTTAGAAACCAAAGAACGCTATGCAATTCTATATAATAAAACAGCACAACAATATGGTATTG
ATATTTCGGATGTTGATCATCAATTAACTGTAATTGCGAACTTAAATGCTGATAGAAGTACG
GCTCAAGAAGAAGTGAGAGAATACTTAAAAGACTATATCACTGAAACTTACCCTCAAATGGA
CAGAGATGAAAAAATTAACTGCATTATTGAAGAGAATGCAGTTGGGTCTCATGATGACTATT
ATGAATCGACAAAATTAGCAGTGGAAAAAACAGGGTCTAAAAATATTTTATTATCCTTTGAA
TCAATGTCCGATATTAAAGATGTAAAAGATATTATTGATATGTTGAACCAAAAAATCGAAAT
GAATTTACCATAATAAAATTAAAGGCAATTTCTATATTAGATTGCCTTTTTAAATTTC
SEQ ID NO: 18 (luxAB Transcript Sequence)
AAUCACCAAAAAGGAAUAGAGUAUGAAGUUUGGAAAUAUUUGUUUUUCGUAUCAACCACCAG
GUGAAACUCAUAAGCUAAGUAAUGGAUCGCUUUGUUCGGCUUGGUAUCGCCUCAGAAGAGUA
GGGUUUGAUACAUAUUGGACCUUAGAACAUCAUUUUACAGAGUUUGGUCUUACGGGAAAUUU
AUUUGUUGCUGCGGCUAACCUGUUAGGAAGAACUAAAACAUUAAAUGUUGGCACUAUGGGGG
UUGUUAUUCCGACAGCACACCCAGUUCGACAGUUAGAAGACGUUUUAUUAUUAGAUCAAAUG
UCGAAAGGUCGUUUUAAUUUUGGAACCGUUCGAGGGCUAUACCAUAAAGAUUUUCGAGUAUU
UGGUGUUGAUAUGGAAGAGUCUCGAGCAAUUACUCAAAAUUUCUACCAGAUGAUAAUGGAAA
GCUUACAGACAGGAACCAUUAGCUCUGAUAGUGAUUACAUUCAAUUUCCUAAGGUUGAUGUA
UAUCCCAAAGUGUACUCAAAAAAUGUACCAACCUGUAUGACUGCUGAGUCCGCAAGUACGAC
AGAAUGGCUAGCAAUACAAGGGCUACCAAUGGUUCUUAGUUGGAUUAUUGGUACUAAUGAAA
AAAAAGCACAGAUGGAACUCUAUAAUGAAAUUGCGACAGAAUAUGGUCAUGAUAUAUCUAAA
AUAGAUCAUUGUAUGACUUAUAUUUGUUCUGUUGAUGAUGAUGCACAAAAGGCGCAAGAUGU
UUGUCGGGAGUUUCUGAAAAAUUGGUAUGACUCAUAUGUAAAUGCGACCAAUAUCUUUAAUG
AUAGCAAUCAAACUCGUGGUUAUGAUUAUCAUAAAGGUCAAUGGCGUGAUUUUGUUUUACAA
GGACAUACAAACACCAAUCGACGUGUUGAUUAUAGCAAUGGUAUUAACCCUGUAGGCACUCC
UGAGCAGUGUAUUGAAAUCAUUCAACGUGAUAUUGAUGCAACGGGUAUUACAAACAUUACAU
GCGGAUUUGAAGCUAAUGGAACUGAAGAUGAAAUAAUUGCUUCCAUGCGACGCUUUAUGACA
CAAGUCGCUCCUUUCUUAAAAGAACCUAAAUAAAUUACUUAUUUGAUACUAGAGAUAAUAAG
GAACAAGUUAUGAAAUUUGGAUUAUUUUUUCUAAACUUUCAGAAAGAUGGAAUAACAUCUGA
AGAAACGUUGGAUAAUAUGGUAAAGACUGUCACGUUAAUUGAUUCAACUAAAUAUCAUUUUA
AUACUGCCUUUGUUAAUGAACAUCACUUUUCAAAAAAUGGUAUUGUUGGAGCACCUAUUACC
GCAGCUGGUUUUUUAUUAGGGUUAACAAAUAAAUUACAUAUUGGUUCAUUAAAUCAAGUAAU
UACCACCCAUCACCCUGUACGUGUAGCAGAAGAAGCCAGUUUAUUAGAUCAAAUGUCAGAGG
GACGCUUCAUUCUUGGUUUUAGUGACUGCGAAAGUGAUUUCGAAAUGGAAUUUUUUAGACGU
CAUAUCUCAUCAAGGCAACAACAAUUUGAAGCAUGCUAUGAAAUAAUUAAUGACGCAUUAAC
UACAGGUUAUUGUCAUCCCCAAAACGACUUUUAUGAUUUUCCAAAGGUUUCAAUUAAUCCAC
ACUGUUACAGUGAGAAUGGACCUAAGCAAUAUGUAUCCGCUACAUCAAAAGAAGUCGUCAUG
UGGGCAGCGAAAAAGGCACUGCCUUUAACAUUUAAGUGGGAGGAUAAUUUAGAAACCAAAGA
ACGCUAUGCAAUUCUAUAUAAUAAAACAGCACAACAAUAUGGUAUUGAUAUUUCGGAUGUUG
AUCAUCAAUUAACUGUAAUUGCGAACUUAAAUGCUGAUAGAAGUACGGCUCAAGAAGAAGUG
AGAGAAUACUUAAAAGACUAUAUCACUGAAACUUACCCUCAAAUGGACAGAGAUGAAAAAAU
UAACUGCAUUAUUGAAGAGAAUGCAGUUGGGUCUCAUGAUGACUAUUAUGAAUCGACAAAAU
UAGCAGUGGAAAAAACAGGGUCUAAAAAUAUUUUAUUAUCCUUUGAAUCAAUGUCCGAUAUU
AAAGAUGUAAAAGAUAUUAUUGAUAUGUUGAACCAAAAAAUCGAAAUGAAUUUACCAUAAUA
AAAUUAAAGGCAAUUUCUAUAUUAGAUUGCCUUUU
SEQ ID NO: 19 (cis-repressed luxAB transcript sequence)
AAAGGCAUGAAAAAACUUGGUAUCUUCACCAACACCUAGCUUUUUGAAGGAAUUGAGUAUGA
AGUUUGGAAAUAUUGGUUGUUCGUAUCAACCACCAGGUGAAACUCAUAAGCUAAAGGCAUGA
AAAAACUAGGUGAUCUUCACCAACACCUAGUUUUUUCAAGGAAUUGAGUAUGAAGUUUGGAA
AUAUUUGUUUUUCGUAUCAACCACCAGGUGAAACUCAUAAGCUAAGUAAUGGAUCGCUUUGU
UCGGCUUGGUAUCGCCUCAGAAGAGUAGGGUUUGAUACAUAUUGGACCUUAGAACAUCAUUU
UACAGAGUUUGGUCUUACGGGAAAUUUAUUUGUUGCUGCGGCUAACCUGUUAGGAAGAACUA
AAACAUUAAAUGUUGGCACUAUGGGGGUUGUUAUUCCGACAGCACACCCAGUUCGACAGUUA
GAAGACGUUUUAUUAUUAGAUCAAAUGUCGAAAGGUCGUUUUAAUUUUGGAACCGUUCGAGG
GCUAUACCAUAAAGAUUUUCGAGUAUUUGGUGUUGAUAUGGAAGAGUCUCGAGCAAUUACUC
AAAAUUUCUACCAGAUGAUAAUGGAAAGCUUACAGACAGGAACCAUUAGCUCUGAUAGUGAU
UACAUUCAAUUUCCUAAGGUUGAUGUAUAUCCCAAAGUGUACUCAAAAAAUGUACCAACCUG
UAUGACUGCUGAGUCCGCAAGUACGACAGAAUGGCUAGCAAUACAAGGGCUACCAAUGGUUC
UUAGUUGGAUUAUUGGUACUAAUGAAAAAAAAGCACAGAUGGAACUCUAUAAUGAAAUUGCG
ACAGAAUAUGGUCAUGAUAUAUCUAAAAUAGAUCAUUGUAUGACUUAUAUUUGUUCUGUUGA
UGAUGAUGCACAAAAGGCGCAAGAUGUUUGUCGGGAGUUUCUGAAAAAUUGGUAUGACUCAU
AUGUAAAUGCGACCAAUAUCUUUAAUGAUAGCAAUCAAACUCGUGGUUAUGAUUAUCAUAAA
GGUCAAUGGCGUGAUUUUGUUUUACAAGGACAUACAAACACCAAUCGACGUGUUGAUUAUAG
CAAUGGUAUUAACCCUGUAGGCACUCCUGAGCAGUGUAUUGAAAUCAUUCAACGUGAUAUUG
AUGCAACGGGUAUUACAAACAUUACAUGCGGAUUUGAAGCUAAUGGAACUGAAGAUGAAAUA
AUUGCUUCCAUGCGACGCUUUAUGACACAAGUCGCUCCUUUCUUAAAAGAACCUAAAUAAAU
UACUUAUUUGAUACUAGAGAUAAUAAGGAACAAGUUAUGAAAUUUGGAUUAUUUUUUCUAAA
CUUUCAGAAAGAUGGAAUAACAUCUGAAGAAACGUUGGAUAAUAUGGUAAAGACUGUCACGU
UAAUUGAUUCAACUAAAUAUCAUUUUAAUACUGCCUUUGUUAAUGAACAUCACUUUUCAAAA
AAUGGUAUUGUUGGAGCACCUAUUACCGCAGCUGGUUUUUUAUUAGGGUUAACAAAUAAAUU
ACAUAUUGGUUCAUUAAAUCAAGUAAUUACCACCCAUCACCCUGUACGUGUAGCAGAAGAAG
CCAGUUUAUUAGAUCAAAUGUCAGAGGGACGCUUCAUUCUUGGUUUUAGUGACUGCGAAAGU
GAUUUCGAAAUGGAAUUUUUUAGACGUCAUAUCUCAUCAAGGCAACAACAAUUUGAAGCAUG
CUAUGAAAUAAUUAAUGACGCAUUAACUACAGGUUAUUGUCAUCCCCAAAACGACUUUUAUG
AUUUUCCAAAGGUUUCAAUUAAUCCACACUGUUACAGUGAGAAUGGACCUAAGCAAUAUGUA
UCCGCUACAUCAAAAGAAGUCGUCAUGUGGGCAGCGAAAAAGGCACUGCCUUUAACAUUUAA
GUGGGAGGAUAAUUUAGAAACCAAAGAACGCUAUGCAAUUCUAUAUAAUAAAACAGCACAAC
AAUAUGGUAUUGAUAUUUCGGAUGUUGAUCAUCAAUUAACUGUAAUUGCGAACUUAAAUGCU
GAUAGAAGUACGGCUCAAGAAGAAGUGAGAGAAUACUUAAAAGACUAUAUCACUGAAACUUA
CCCUCAAAUGGACAGAGAUGAAAAAAUUAACUGCAUUAUUGAAGAGAAUGCAGUUGGGUCUC
AUGAUGACUAUUAUGAAUCGACAAAAUUAGCAGUGGAAAAAACAGGGUCUAAAAAUAUUUUA
UUAUCCUUUGAAUCAAUGUCCGAUAUUAAAGAUGUAAAAGAUAUUAUUGAUAUGUUGAACCA
AAAAAUCGAAAUGAAUUUACCAUAAUAAAAUUAAAGGCAAUUUCUAUAUUAGAUUGCCUUUU
Claims (27)
1. A bacterial cell packaging system for packaging a reporter nucleic acid molecule into a non-replicative transduction particle, said bacterial cell packaging system comprising a host bacterial cell comprising: a lysogenized bacteriophage genome comprising a first bacteriophage gene comprising a deletion of a first packaging initiation site sequence of said first bacteriophage gene that prevents packaging of a bacteriophage nucleic acid molecule into said non-replicative transduction particle; and a plasmid comprising a reporter nucleic acid molecule comprising a reporter gene and a second bacteriophage gene comprising a second packaging initiation site sequence that facilitates the packaging of a replica of said plasmid comprising said reporter nucleic acid molecule into said non-replicative transduction particle, wherein said second bacteriophage gene encodes a protein, wherein said replica of said plasmid comprising a reporter nucleic acid molecule forms a replicon for packaging into said non-replicative transduction particle, wherein the first and second packaging initiation site sequence is a pac-site sequence.
2. A bacterial cell packaging system for packaging a reporter nucleic acid molecule into a non-replicative transduction particle, said bacterial cell packaging system comprising a host bacterial cell comprising: a lysogenized bacteriophage genome comprising a deletion of a first bacteriophage gene comprising a first packaging initiation site sequence, wherein deletion of said first bacteriophage gene prevents packaging of a bacteriophage nucleic acid molecule into said non-replicative transduction particle; and a plasmid comprising a reporter gene and a second bacteriophage gene, wherein said second bacteriophage gene encodes a second packaging initiation site sequence and facilitates the packaging of a replica of said plasmid comprising said reporter nucleic acid molecule into said non-replicative transduction particle, wherein said second bacteriophage gene is capable of expressing a protein that is encoded by said gene, wherein said replica of said plasmid comprising a reporter nucleic acid molecule forms a replicon amenable to packaging into said non-replicative transduction particle, wherein the first and second packaging initiation site sequence is a pac-site sequence.
3. A bacterial cell packaging system for packaging a reporter nucleic acid molecule into a non-replicative transduction particle, said bacterial cell packaging system comprising a host bacterial cell comprising: a lysogenized bacteriophage genome comprising a first bacteriophage packaging initiation site sequence within a first bacteriophage gene, wherein said first bacteriophage packaging initiation site sequence comprises a silent mutation that prevents cleavage of the packaging initiation sequence, but does not disrupt the expression of the first bacteriophage gene product that encompasses the first packaging initiation site sequence and prevents packaging of a bacteriophage nucleic acid molecule into said non-replicative transduction particle; and a plasmid comprising a reporter nucleic acid molecule comprising a reporter gene and comprising a second bacteriophage packaging initiation site sequence, wherein said second bacteriophage packaging initiation site sequence lacks said mutation and facilitates the packaging of a replica of said plasmid comprising said reporter nucleic acid molecule into said non-replicative transduction particle, wherein said replica of said plasmid comprising said reporter nucleic acid molecule forms a replicon for packaging into said non-replicative transduction particle, wherein the first and second packaging initiation site sequence is a pac-site sequence.
4. The bacterial cell packaging system of any one of claims 1 to 3, wherein said reporter nucleic acid molecule is operatively linked to a promoter.
5. The bacterial cell packaging system of any one of claims 1 to 3, wherein said reporter nucleic acid molecule comprises an origin of replication.
6. The bacterial cell packaging system of any one of claims 1 to 3, wherein said replicon comprises a concatamer amenable to packaging into said non-replicative transduction particle.
7. The bacterial cell packaging system of claim 1 or claim 2, wherein said second bacteriophage gene comprises the sequence set forth in SEQ ID NO:9.
8. The bacterial cell packaging system of claim 1, wherein said first bacteriophage gene comprises the sequence of SEQ ID NO:8.
9. The bacterial cell packaging system of claim 1 or claim 2, wherein said first and said second bacteriophage genes each comprises a small terminase (terS) gene comprising said packaging initiation site sequence.
10. The bacterial cell packaging system of claim 9, wherein said terS gene is a S. aureus bacteriophage φ11 or φ80α terS gene.
11. The bacterial cell packaging system of claim 3, wherein said first and said second bacteriophage packaging initiation site sequences each comprise a packaging initiation site sequence from a small terminase gene, or said first and said second bacteriophage packaging initiation site sequences each comprise a pac-site sequence from a small terminase (terS) gene of an S. aureus bacteriophage φ11 or φ80α.
12. The bacterial cell packaging system of any one of claims 1 to 3, wherein said replicon is derived from a S. aureus pT181 plasmid origin of replication.
13. The bacterial cell packaging system of any one of claims 1 to 3, wherein said replicon comprises the sequence set forth in SEQ ID NO:5.
14. The bacterial cell packaging system of claim 1 or claim 2, wherein said second bacteriophage gene is operatively linked to a promoter.
15. The bacterial cell packaging system of claim 14, wherein said promoter is an inducible promoter or a constitutive promoter.
16. The bacterial cell packaging system of any one of claims 1 to 4, wherein said bacteriophage comprises a S. aureus bacteriophage φ80α or a bacteriophage φ11.
17. The bacterial cell packaging system of any one of claims 1 to 3, wherein said bacterial cell comprises: an Escherichia coli (E. coli) cell; a Gram-negative cell; or a Gram-positive cell.
18. The bacterial cell packaging system of any one of claims 1 to 4, wherein said bacterial cell comprises an S. aureus cell.
19. The bacterial cell packaging system of any one of claims 1 to 4, wherein said reporter nucleic acid molecule comprises a reporter gene.
20. The bacterial cell packaging system of claim 19, wherein said reporter gene encodes a detectable and/or a selectable marker.
21. The bacterial cell packaging system of claim 20, wherein said reporter gene is selected from the group consisting of genes encoding enzymes mediating luminescence reactions (luxA, luxB, luxAB, luc, ruc, nluc), genes encoding enzymes mediating colorimetric reactions (lacZ, HRP), genes encoding fluorescent proteins (GFP, eGFP, YFP, RFP, CFP, BFP, mCherry, near-infrared fluorescent proteins), nucleic acid molecules encoding affinity peptides (His-tag, 3X-FLAG), and genes encoding selectable markers (ampC, tet(M), CAT, erm).
22. A method for packaging a reporter nucleic acid molecule into a non-replicative transduction particle, comprising: providing conditions to said bacterial cell packaging system of any one of claims 1 to 18 that induce a lytic phase of said bacteriophage to produce non-replicative transduction particles packaged with said plasmid comprising said reporter nucleic acid molecule; and isolating said non-replicative transduction particle comprising a replica of said plasmid comprising said reporter nucleic acid molecule.
23. The method of claim 22, wherein said non-replicative transduction particle does not contain a replicated bacteriophage genome.
24. A composition comprising a non-replicative transduction particle comprising a replica of said plasmid comprising said reporter nucleic acid molecule produced by the method of claim 22 or 23.
25. A method for detecting a presence or an absence of a bacterial cell in a sample, comprising: introducing into a sample a non-replicative transduction particle produced by the method of claim 22 or 23 comprising a reporter gene encoding a reporter molecule and lacking a bacteriophage genome under conditions such that said non-replicative transduction particle can transduce said bacterial cell and wherein said reporter gene can be expressed in said bacterial cell; providing conditions for activation of said reporter molecule; and detecting for a presence or an absence of a reporter signal transmitted from said expressed reporter molecule, wherein a presence of said reporter signal correctly indicates said presence of said bacterial cell.
26. The method of claim 20, wherein said bacterial cell is a Methicillin Resistant Staphylococcus aureus (MRSA) cell or a Methicillin Sensitive Staphylococcus aureus (MSSA) cell.
27. The method of claim 25 or 26, further comprising: providing an antibiotic to said sample at a pre-determined concentration and detecting a presence or absence of said reporter signal to determine whether said bacterial cell is resistant or sensitive to said antibiotic; or providing varying pre-determined concentrations antibiotic to said sample and detecting the amount of said reporter signal to determine the minimum inhibitory concentration of said bacterial cell to said antibiotic.
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201361779177P | 2013-03-13 | 2013-03-13 | |
US61/779,177 | 2013-03-13 | ||
US201361897040P | 2013-10-29 | 2013-10-29 | |
US61/897,040 | 2013-10-29 | ||
US201461939126P | 2014-02-12 | 2014-02-12 | |
US61/939,126 | 2014-02-12 | ||
PCT/US2014/026536 WO2014160418A2 (en) | 2013-03-13 | 2014-03-13 | Non-replicative transduction particles and transduction particle-based reporter systems |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ712344A NZ712344A (en) | 2021-03-26 |
NZ712344B2 true NZ712344B2 (en) | 2021-06-29 |
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