CN109517774A - It is a kind of from the pseudomonas aeruginosa of main light emission and its construction method and application - Google Patents
It is a kind of from the pseudomonas aeruginosa of main light emission and its construction method and application Download PDFInfo
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Abstract
The invention discloses a kind of for converting the transferring plasmid of pseudomonas aeruginosa, its according to clockwise successively contain promoter ori, ampicillin resistance AmpR, transposon sequence, bond site oriT, wherein, transposon sequence, which contains, can make base sequence and resistant gene of the pseudomonas aeruginosa from main light emission, and the both ends of resistant gene have difR and difL sequence；A kind of pseudomonas aeruginosa from main light emission of non-resistant label is also disclosed, gene of the bacterium from main light emission can be made by containing in the genome of the pseudomonas aeruginosa, and not have resistance screening gene in the genome of pseudomonas aeruginosa.In the present invention by transferring plasmid pUC18T-mini-Tn7T-lux-dif-Apr and helper plasmid participate in building from main light emission pseudomonas aeruginosa, not needing to add any substrate can shine；Also, photogenic colony just can be seen by naked eyes in a dark environment；There is no resistant gene, physiological status, drug susceptibility are more applicable for operate in large scale closer in wild type；Luminous intensity is high, stability is strong.
Description
Technical field
It is especially a kind of from the pseudomonas aeruginosa of main light emission and its building side the present invention relates to gene engineering technology field
Method and application.
Background technique
Pseudomonas aeruginosa, also referred to as Pseudomonas aeruginosa are a kind of Gram-negative bacterias, are widespread in nature, and are somebody's turn to do
Bacterium is stronger to extraneous environmental resistance, at humidity can long term survival, it is insensitive to ultraviolet light, damp and hot 55 DEG C, 1h could be killed
It goes out；The bacterium is a kind of conditioned pathogen, could only be caused a disease under certain specified conditions.Prolonged application hormone, immunosuppressor, change
Treatment, radiotherapy etc. cause patient immune function low, can cause serious or even lethal infection；In addition, postoperative patient
Also the infection of this bacterium is easily suffered from, therefore, pseudomonas aeruginosa becomes in hospital, especially the important pathogen of intensive care unit infection
One of bacterium.
Transposons (Transposon, Tn) is also known as transposable element or skip over, it can be from a part of inhereditary material
Another part is jumped to, so as to cause hereditary variation.Tn7 transposons is a kind of fixed point insert type transposons, is fixedly inserted into thin
After the glmS gene of bacterium.GlmS gene is related with the synthesis of cell wall, is a kind of indispensable gene.Tn7 transposons has because of it
It is inserted into after chromosome on the no any influence of expression of bacterial gene, the growth conditions for not influencing bacterium, insertion point is clear, takes
The features such as with big genetic fragment, has been used as a kind of genetic manipulation tool application in various bacteria.
Conventional genetic operation requires to carry resistant maker gene, for screening positive transformants result.Utilizing swivel base subsystem
Purpose base sequence is integrated into bacterial genomes by system also needs resistance screening to mark, but since the bacterial strain contains resistant base
Cause may make troubles to subsequent genetic manipulation and application, and such as there may be cross tolerances in drug screening/evaluation.
The sequence recombination system for being commonly used for excision resistance marker at present includes the Cre/loxP system for deriving from bacteriophage P1, swivel base
The TnpR/res system of sub- γ δ, the Flp/FRT system of saccharomyces cerevisiae.But these systems require that resolvase gene will be contained
Plasmid be transferred to host strain, inducing expression and after eliminating resistant gene also needs to remove the plasmid, in practical application very not
Just.Xer-cise specific recombination systems mainly work in bacterial chromosome reproduction process, and Xer resolvase acts on
The dif sequence of chromosome separation stage identification duplication end during cell division, catalysis chromosome dimer dissociation, thus shape
At chromosome monosomy.All there is Xer-cise system in the bacteriums such as Escherichia coli, comma bacillus.However Xer-cise system is for cutting
Except resistant gene is just to be reported recent years, have been used for the genetic manipulation of Mycobacterium at present, not yet sees verdigris vacation
Monad.Compared with system described above, the advantage of Xer-cise sequence-specific recombination system is, is compiled using bacterial strain itself
The Xer recombinase of code does not need artificial expression foreign protein to realize the dissociation of resistant gene, eliminates many unnecessary fiber crops
It is tired.
Summary of the invention
Based on the above issues, a kind of nonreactive is provided it is an object of the invention to overcome above-mentioned the deficiencies in the prior art place
The pseudomonas aeruginosa of property selection markers and its construction method.
To achieve the above object, the technical solution that the present invention takes includes the following aspects:
In the first aspect, the present invention provides a kind of for converting the transferring plasmid of pseudomonas aeruginosa, the transfer
Plasmid according to clockwise successively contain promoter ori, ampicillin resistance AmpR, transposon sequence, bond site oriT,
In, the transposon sequence, which contains, can make base sequence and resistant gene of the pseudomonas aeruginosa from main light emission, and described is anti-
Property gene both ends have difR and difL sequence.It should be noted that DifR and DifL sequence is selected from Xer-cise specificity
The excision of resistant gene can be realized using the resolvase of the endogenous expression of host strain for recombination system, the system.
Preferably, described to make base sequence LuxCDABE gene order of the pseudomonas aeruginosa from main light emission.Due to
The base sequence of LuxCDABE is the common knowledge of the technical field of the invention, not listed here to save space
The specific base sequence of LuxCDABE.
Preferably, the resistant gene is apramycin resistance gene or/and gentamicin resistance gene.
Preferably, the transposon sequence successively contains inverted repeats Tn7R, resistant gene, promoter by clockwise
Promoter, LuxCDABE gene and inverted repeats Tn7F.Wherein, the base sequence of Tn7R is preferably such as SEQ ID NO.1
Shown, the base sequence of Tn7L is preferably as shown in SEQ ID NO.2.
In the second aspect, the present invention provides a kind of for converting the building side of the transferring plasmid of pseudomonas aeruginosa
Method, comprising the following steps:
1) it after starting plasmids pUC18T-mini-Tn7T-lux-Tp is digested with restriction enzyme Xba I and BamH I, returns
Receive the function fragment A of about 11kb；
2) the Apr gene after Xba I and BamH I digestion PCR amplification recycles function fragment B；
3) function fragment A and function fragment B are attached, obtain the plasmid pUC18 T-mini-Tn7T- of about 11.8kb
Lux-dif-Apr to get.
In the third aspect, the present invention provides a kind of pseudomonas aeruginosa from main light emission, the pseudomonas aeruginosas
Genome in containing gene of the bacterium from main light emission can be made, and there is no resistance screening base in the genome of pseudomonas aeruginosa
Cause.
Preferably, described to make gene LuxCDABE gene of the bacterium from main light emission.
In the fourth aspect, the present invention provides a kind of construction methods of pseudomonas aeruginosa from main light emission, including such as
Lower step:
(1) transferring plasmid described in first aspect is provided；
(2) above-mentioned transferring plasmid and helper plasmid containing transposase gene are transferred to pseudomonas aeruginosa simultaneously, are applied to
The LB plate of Apr resistance, obtains the pseudomonas aeruginosa from main light emission.It is wherein transferred to the preferred electricity of mode and turns or engage transfer.It answers
When explanation, which is applicable not only to the building of pseudomonas aeruginosa, applies also for building with Xer-cise sequence
The bacterium of column specific recombination systems, such as Escherichia coli, comma bacillus；The construction method is applicable not only to the structure of photogen
It builds, is also applied for expressing other foreign proteins；Helper plasmid preferred pTNS3 or pTNS2；The gene of the encoding transposase is
TnsABCD, base sequence is as shown in SEQ IDNO.3.
Preferably, the construction method further includes step (3): by step (2) obtain from main light emission pseudomonas aeruginosa
Secondary culture filters out the photogen of resistant gene loss, obtain non-resistant selection markers from main light emission pseudomonas aeruginosa.
At the 5th aspect, the present invention provides the pseudomonas aeruginosas in terms of third in screening resisting pseudomonas aeruginosa
Application in the drug of infection.
In conclusion the invention has the benefit that
1) present invention is applied to verdigris vacation as specific recombination site for the first time by Tn7 transposon system in conjunction with dif sequence
In monad, this method is that one-step method constructs autonomous photogen, it is only necessary to dif sequence are connected to resistant gene both ends, utilize place
Resistance excision can be realized in the resolvase of the main endogenous expression of bacterium；Compared to other specific recombination systems such as TnpR/res, Flp/
FRT system, this method are simple and efficient to handle without artificial expression external source resolvase；
2) building is participated in by transferring plasmid pUC18T-mini-Tn7T-lux-dif-Apr and helper plasmid in the present invention
From main light emission pseudomonas aeruginosa, not needing to add any substrate can shine；Also, just can in a dark environment by naked eyes
See photogenic colony；
3) of the invention false single from main light emission verdigris relative to existing resistant label from main light emission mycobacteria
Born of the same parents bacterium does not have a resistant gene, and physiological status, drug susceptibility are closer in wild type, so being more applicable for grasping on a large scale
Make；
4) that the present invention is prepared is high from main light emission pseudomonas aeruginosa luminous intensity, stability is strong, CFU and RLU it
Between there are corresponding relationships, it is possible to use RLU (relative light unit) to replace CFU (Colony Forming Unit) raw as analysis bacterium
The foundation of long situation；The microorganism growth, activity, distribution in the environment etc. can need to only be carried out by the detection to light
Real time on-line monitoring, this be all other biological reporter gene it is incomparable；
5) method from main light emission pseudomonas aeruginosa of present invention building non-resistant selection markers is also applied for other bands
There is a bacterium of Xer-cise recombination system, greatly easy genetic manipulation is photobacteria using providing convenience.
Detailed description of the invention
Fig. 1 is pUC18T-mini-Tn7T-lux-Tp plasmid map；
Fig. 2 is pUC18T-mini-Tn7T-lux-dif-Apr plasmid map；
Fig. 3 is the building flow diagram of plasmid pUC18 T-mini-Tn7T-lux-dif-Apr；
Fig. 4 is helper plasmid pTNS3 map；
Fig. 5 is the aspect graph of photogen；
Fig. 6 is that the photogen obtained after passage is applied to the result containing Apr resistant panel and antibiotic-free plate respectively
Figure；Wherein 2,18,20,22, No. 24 are cloned on non-resistant plate (plate on the left side) and grow, Apr plate (the right it is flat
Plate) on do not grow；
Fig. 7 is the electrophoresis result figure that PCR screens non-resistant A1PA；Wherein swimming lane M:2kb plus；Swimming lane 1-2: nonreactive
AlPA；Swimming lane 3-4: resistant AlPA；Swimming lane 5: plasmid pUC18 T-mini-Tn7T-lux-dif-Apr；
Fig. 8 is the growth curve of AlPA in the present invention, and the time is abscissa, and logRLU is ordinate；
Fig. 9 is the growth curve of AlPA in the present invention, wherein the time is abscissa, OD600For ordinate, PA is that verdigris is false
Monad, ALPA are selectable marker-free prepared by embodiment 2 from main light emission pseudomonas aeruginosa.
Specific embodiment
The invention discloses a kind of construction methods from main light emission pseudomonas aeruginosa of selectable marker-free, utilize
Luciferase gene LuxCDABE is integrated into the genome of pseudomonas aeruginosa by Tn7 Transposon System, realizes luciferase
The expression of efficient stable in pseudomonas aeruginosa；There is no in genetic engineering transformation process for constructed pseudomonas aeruginosa
Resistance screening label, without add substrate can be realized it is luminous.
In some embodiments, this method uses two kinds of plasmids: helper plasmid pTNS3 and transferring plasmid pUC18T- altogether
mini-Tn7T-lux-dif-Apr；Transferring plasmid contains using inverted repeat series Tn7L and Tn7R as the transposon sequence of flank,
Transposon sequence contains apramycin resistance gene Apr, luciferase gene LuxCDABE and the repetition in the same direction positioned at the both ends Apr
Sequence D ifR and DifL, DifR and DifL sequence is the recognition site of Xer-cise specific recombination systems, without importing expression
The excision of resistant gene can be realized in the base sequence of external source resolvase；Helper plasmid includes the gene of encoding transposase
TnsABCD；In some embodiments, construction method cotransformation helper plasmid (pTNS3 or pTNS2) of the invention and transfer matter
Grain pUC18T-mini-Tn7T-lux-dif-Apr；In some embodiments, the gene of inverted repeats and encoding transposase
Selected from Tn7 Transposon System.
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention is described further.Molecular biology experiment technology employed in following embodiments includes that PCR amplification, plasmid mention
It takes, plasmid conversion, DNA fragmentation connection, digestion, gel electrophoresis etc., all using conventional method, for details, reference can be made to " molecular clonings
Experiment guide " (third edition, Sambrook J, Russe11 DW, Janssen K, Argentine J. Huang Peitang etc. are translated, and 2002,
Beijing: Science Press).
PCR reaction TransTaq-T DNA Polymerase, dNTP and related reagent used are equal in following embodiment
It buys from Beijing Quan Shijin biotechnology；Antibiotic ammonia benzyl, apramycin are purchased from Mei Lun Bioisystech Co., Ltd；Escherichia coli
Competence DH5a is bought in Guangzhou Dongsheng Biotechnology Co., Ltd；DNA connection reaction is all made of Takara treasured biotech firm
T4DNA connection kit；Extraction of plasmid DNA kit, DNA QIAquick Gel Extraction Kit are purchased from Magen (Mei Ji) biotech firm；Biorad
Electric converter (Biorad GenePulser Xcel1) and electric revolving cup are purchased from Biorad company.
The building of 1 transferring plasmid pUC18T-mini-Tn7T-lux-dif-Apr of embodiment
(1) transferring plasmid pUC18T-mini-Tn7T-lux-dif-Apr is constructed according to flow chart 3.As shown in Fig. 2, plasmid
PUC18T-mini-Tn7T-lux-dif-Apr successively contains replication origin (ori) in the direction of the clock, ammonia benzyl resistance base
Cause (AmpR), apramycin resistance gene (Apr), luciferase gene (LuxCDABE), positioned at the in the same direction of AprR gene both ends
Repetitive sequence DifR and DifL.The function of each element is as follows:
LuxCDABE: i.e. luciferase gene, be shine needed for enzyme gene, the expression of the gene allow host strain from
Main light emission.
Apramycin resistance gene (AprR): apramycin resistance gene (Apr) is a kind of selected marker, is obtained for screening
Obtain purpose bacterial strain；Apramycin resistance gene (Apr) can be such that host strain obtains after pseudomonas aeruginosa and expression in escherichia coli
The resistance to apramycin (Apr) is obtained, can be grown in the culture medium containing Apr；(Apramycin is abbreviated as apramycin
It Apr) is a kind of common resistance screening drug, the Apr concentration for pseudomonas aeruginosa is 100 μ g/mL, is used for Escherichia coli
Apr concentration be 50 μ g/mL.
Direct repeat dif: the sequence can be identified by a kind of resolvase of host strain itself, and efficiently be cut off, because
And apramycin resistance gene can be removed in subsequent operation.
Ampicillin resistance gene (AmpR): being a kind of selection markers, obtains purpose bacterial strain for screening；In large intestine bar
After being expressed in bacterium, host strain can be made to obtain the resistance to ampicillin (Amp), can be grown in the culture medium containing Amp.
Ori: it is responsible for the element that plasmid replicates in Escherichia coli.
(2) the specific construction method of transferring plasmid pUC18T-mini-Tn7T-lux-apr-dif is as follows:
S1, as shown in Figure 1, starting plasmids pUC18T-mini-Tn7T-lux-Tp (by Virginia medical care university Joanna
The laboratory B.Goldberg give, and plasmid map is shown in Fig. 1) with after restriction enzyme Xba I and BamH I digestion, recycling is about
The function fragment A of 11kb；
S2, with plasmid pMABH1 (preservation of this laboratory) be template, with primer Dif-apr-F:5 '-CGGGATCCATGGG ATTCGCATAATGTATATTATGTTAAATCACCACCGACTATTTG-3 ' (SEQ ID NO.4) and primer DIF-APR-R:
5’-TGCTCTAGAAGCTTATTTAACATAATATACATTATGCGAATCAGCTCAGCCAATCGAC-3’(SEQ ID NO.5)
(wherein underscore part is dif sequence) amplifies dif-Apr；
S3, with Xba I and BamH I digestion, obtain the function fragment B of 954bp, which is that both ends have and come from verdigris
The apramycin Apr genetic fragment of pseudomonad dif sequence；
S4, after mixing function fragment A and function fragment B, ligase is added and is attached, obtains recombinant plasmid
PUC18T-mini-Tn7T-lux-dif-Apr converts E. coli competent DH5 α, is screened using the LB solid plate containing Amp
Positive colony out, picking monoclonal are cultivated into LB liquid medium, extract plasmid pUC18 T-mini-Tn7T-lux-dif-
Apr；Identification plasmid pUC18 T-mini-Tn7T-lux-dif-Apr is digested with Hind III and Nco I, correct plasmid is through enzyme
Glue is run after cutting the result is that respectively there is a band at 10931bp and 934bp；The correct plasmid of digestion is sent into sequencing again, to select
There is no the plasmids of mutation for dif segment, to carry out subsequent experimental.
A kind of construction method from main light emission pseudomonas aeruginosa of the non-resistant selection markers of embodiment 2
1. electrotransformation
(1) P. aeruginosa bacterium competence is prepared, it will bacterium (pseudomonas aeruginosa, Pseudomonas be turned
Aeruginosa it) is inoculated in 6mL LB culture medium, 37 DEG C, 200rpm shaken cultivation, overnight, is dispensed into the EP pipe of 4 1.5mL
In, room temperature, 12000g is centrifuged 2min, collects thallus.It is suspended again with the 300mM sucrose solution of 1mL, washing thalline, room temperature,
12000g, is centrifuged 2min, and evacuation raffinate is repeated 2 times.With the 300mM sucrose solution resuspended bacterium solution of 200 μ L and mix merging.It will
Competent cell is distributed into 100uL/ pipe, puts -80 DEG C of refrigerators, saves backup and (preferably prepare new competence every time).
(2) the competence pseudomonas aeruginosa of 100 μ L is transferred in the electric revolving cup of 2mm, the transferring plasmid of 50ng is added
Helper plasmid pTNS3 (the pTNS3 map ginseng of pUC18T-mini-Tn7T-lux-dif-Apr (being made by embodiment 1) and 50ng
See Fig. 4).Soft pressure-vaccum is placed 10 minutes after mixing well on ice, and the moisture on revolving cup electric to the greatest extent is wiped before addition electroporation.
(3) electrotransformation is carried out with Biorad electric converter, with 25 μ F, 200 Ω, 2.5kV impulse wave carries out electrotransformation.This mistake
Cheng Ruo competent cell is washed unclean or plasmid saliferous is more, it may occur that explosion.
(4) it is rapidly added 1mL LB culture medium, in 37 DEG C after mixing, vibrates (200rpm) 1h, sufficiently bringing back to life bacterium makes it
Resistance expression.
(5) 100 μ L bacterium solutions are taken to be coated on the LB culture medium containing 100 μ g/mL apramycin resistances, remaining bacterium solution exists
It is centrifuged 2min under 16000g, is resuspended after discarding supernatant liquid with 200 μ L LB culture mediums, bacterium solution is coated on again containing 100g/mL
On the LB culture medium of Apr resistance.In 37 DEG C of cultures until generating obvious bacterium colony.
2. detecting whether that swivel base occurs
1) single bacterium grown on picking Apr plate is fallen in the 1.5mL centrifuge tube containing 20 μ L sterile waters, is mixed, will be centrifuged
Pipe is put into luminometer device (GLOMAX2020 of Pu Luomaige company), detects luminous value size.Rule of thumb, general to shine
Value 100 it is below regard as not shining, luminous value is 105Or higher very strong, the form of photogen of regarding as shining
As shown in Figure 5.
2) it using photogen obtained above as template, uses
Primer PglmS-down: 5 '-GCACATCGGCGACGTGCTCTC-3 ' (SEQ ID NO.6)
And PTn7R: 5 '-CACAGCATAACTGGACTGATTTC-3 ' (SEQ ID NO.7)
PCR amplification is carried out, if having band at the place 272bp or so, proves that swivel base occurs for the single colonie, is labeled as
AlPA。
3. secondary culture AlPA
1) it takes AlPA to be inoculated with 5mL into the LB liquid medium without Apr with 1:10000, is placed in 37 DEG C, 200rpm is permanent
Warm shaking table culture；When carrying out first generation passage, strain, 750+250 μ L of μ L bacterium solution, 80% glycerol are saved.It is typically maintained in sweet
Oily 20-30%, is stored in -80 DEG C.
2) as bacterium solution OD600When reaching 0.7, then the bacterium solution is inoculated with 50mL with 1:10000 and is trained to the LB liquid without Apr
It supports in base, is placed in 37 DEG C, 200rpm constant-temperature table culture.
3) after repeating step 2) and reaching 4 times or so, it will be laid on the LB plate of non-resistant after bacterium solution dilution, is placed in 37
DEG C constant incubator culture, is observed afterwards for 24 hours.
4. preliminary screening resistance loses AlPA
If the bacterium of same bacterium colony can grow on non-resistant plate and not grow on the LB plate containing 100 μ g/mL Apr
Get up, then tentatively concludes there is no Apr gene in the bacterium.
Specific screening step is as follows: the single colonie that picking previous step secondary culture obtains synchronizes the LB for being inoculated into non-resistant
Plate and LB plate containing 100 μ g/mL Apr.Then, plate is placed in 37 DEG C of constant incubator cultures to observe afterwards for 24 hours, is tied
Fruit is as shown in Figure 6.
The non-resistant AlPA of 5.PCR verifying screening
According to plasmid pUC18 T-mini-Tn7T-lux-dif-Apr nucleic acid sequence information, software primer is utilized
For primier 5.0 in dif-Apr-dif sequence both ends design primer, the specific primer of final design is as follows:
Delapr-F:5 '-CACCACCGACTATTTG-3 ' (SEQ ID NO.8) and
delapr-R:5’-AGCTCAGCCAATCGAC-3’(SEQ ID NO.9)。
Colony PCR amplification is carried out with the primer again and whether electrophoresis verifying dif-Apr-dif segment loses, as a result such as Fig. 7
It is shown.Meanwhile taken from the AlPA that has lost of identification resistance 700 μ L bacterium solutions (i.e. non-resistant selection markers from main light emission verdigris
Pseudomonad) plus 300 μ L, 80% glycerol, after mixing, it is stored in -80 DEG C.
The stability of photoluminescence of the verifying of embodiment 3 non-resistant AlPA
1) non-resistant AlPA made from secondary culture embodiment 2
A. it takes AlPA with 1:10000 inoculation 5mL into LB liquid medium, is placed in 37 DEG C, the training of 200rpm constant-temperature table
It supports；
B. as bacterium solution OD600When reaching 0.7, then by the bacterium solution with 1:10000 inoculation 5mL into LB liquid medium, juxtaposition
In 37 DEG C, 200rpm constant-temperature table culture；
C. after repeating step b for several times, it will be laid on the LB plate of non-resistant after bacterium solution dilution, is placed in 37 DEG C of constant temperature trainings
Case culture is supported, is observed afterwards for 24 hours.
2) AlPA proportion is counted
After bacterium colony is grown on plate, 200 single colonies of random picking simultaneously detect RLUs with luminometer.If tested
The RLUs for surveying single colonie is 5 times or more of PA single colonie RLUs, then determines the single colonie for photogenic colony.AlPA proportion is
The clump count of the number of photogen/total.The results show that ratio shared by photogen is 100%, because bacterium can be made from main light emission
Gene (LuxCDABE) be integrated into bacterium (AlPA) genome, it is sufficiently stable, will not lose, and other contain by plasmid
There is the bacterium of bioluminescence gene, in the case where not adding resistance, easily lose, it is very unstable.
3) prepare bacteria to be tested
1) it obtains AlPA single colonie: AlPA being inoculated in non-resistant LB plate culture in a manner of streak inoculation, to 37 DEG C
Single colonie can be obtained in culture 15h；
2) it is inoculated with single colonie culture: picking from the plate single colonie, be inoculated into the LB culture medium of 5mL, 37 DEG C, 200rpm is permanent
Warm shaking table culture；
3) OD to Liquid Culture AlPA600In 0.5-0.7,5mL LB culture medium is inoculated into the ratio of 1:10000,
37 DEG C, 200rpm constant-temperature table culture.
4) OD in different time periods is measured600And it RLUs and notes down
It is denoted as 0h to be inoculated with the moment, when starting measurement, 200 μ L bacterium solutions is just taken from culture medium at interval of 1h, detects RLUs
And OD600And it notes down；Until the measurement of RLUs (relative light unit) is continued until that bacterium solution RLUs starts reduction.
5) growth curve is drawn
Pass through analytical procedure 4) the data obtained, using the time as abscissa, logRLU/mL is ordinate, draw growth curve:
T-logRLU/mL, as shown in Figure 8；
Using the time as abscissa, OD600For ordinate, growth curve: T-OD is drawn600, as shown in Figure 9.According to Fig. 8 and figure
Result shown in 9 it is found that non-resistant selection markers of the invention the stable luminescence from main light emission pseudomonas aeruginosa (AlPA)
The time of growth was up to 10 hours.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
SEQUENCE LISTING
<110>Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health
<120>a kind of from the pseudomonas aeruginosa of main light emission and its construction method and application
<130> 2018
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 199
<212> DNA
<213>artificial sequence
<400> 1
tgtgggcgga caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt 60
cttaaactag acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat 120
actggacttt tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc 180
gtattaaaga ggggcgtgg 199
<210> 2
<211> 166
<212> DNA
<213>artificial sequence
<400> 2
aaccagataa gtgaaatcta gttccaaact attttgtcat ttttaatttt cgtattagct 60
tacgacgcta cacccagttc ccatctattt tgtcactctt ccctaaataa tccttaaaaa 120
ctccatttcc acccctccca gttcccaact attttgtccg cccaca 166
<210> 3
<211> 6110
<212> DNA
<213> E.coli
<400> 3
atggctaaag caaactcttc attttctgaa gtgcaaattg cccgtcgtat taaagagggg 60
cgtggccaag ggcatggtaa agactatatt ccatggctaa cagtacaaga agttccttct 120
tcaggtcgtt cccaccgtat ttattctcat aagacgggac gagtccatca tttgctatct 180
gacttagagc ttgctgtttt tctcagtctt gagtgggaga gcagcgtgct agatatacgc 240
gagcagttcc ccttattacc tagtgatacc aggcagattg caatagatag tggtattaag 300
catcctgtta ttcgtggtgt agatcaggtt atgtctactg attttttagt ggactgcaaa 360
gatggtcctt ttgagcagtt tgctattcaa gtcaaacctg cagcagcctt acaagacgag 420
cgtaccttag aaaaactaga actagagcgt cgctattggc agcaaaagca aattccttgg 480
ttcattttta ctgataaaga aataaatccc gtagtaaaag aaaatattga atggctttat 540
tcagtgaaaa cagaagaagt ttctgcggag cttttagcac aactatcccc attggcccat 600
atcctgcaag aaaaaggaga tgaaaacatt atcaatgtct gtaagcaggt tgatattgct 660
tatgatttgg agttaggcaa aacattgagt gagatacgag ccttaaccgc aaatggtttt 720
attaagttca atatttataa gtctttcagg gcaaataagt gtgcagatct ctgtattagc 780
caagtagtga atatggagga gttgcgctat gtggcaaatt aatgaggttg tgctatttga 840
taatgatccg tatcgcattt tggctataga ggatggccaa gttgtctgga tgcaaataag 900
cgctgataaa ggagttccac aagctagggc tgagttgttg ctaatgcagt atttagatga 960
aggccgctta gttagaactg atgaccctta tgtacatctt gatttagaag agccgtctgt 1020
agattctgtc agcttccaga agcgcgagga ggattatcga aaaattcttc ctattattaa 1080
tagtaaggat cgtttcgacc ctaaagtcag aagcgaactc gttgagcatg tggtccaaga 1140
acataaggtt actaaggcta cagtttataa gttgttacgc cgttactggc agcgtggtca 1200
aacgcctaat gcattaattc ctgactacaa aaacagcggt gcaccagggg aaagacgttc 1260
agcgacagga acagcaaaga ttggccgagc cagagaatat ggtaagggtg aaggaaccaa 1320
ggtaacgccc gagattgaac gcctttttag gttgaccata gaaaagcacc tgttaaatca 1380
aaaaggtaca aagaccaccg ttgcctatag acgatttgtg gacttgtttg ctcagtattt 1440
tcctcgcatt ccccaagagg attacccaac actacgtcag tttcgttatt tttatgatcg 1500
agaataccct aaagctcagc gcttaaagtc tagagttaaa gcaggggtat ataaaaaaga 1560
cgtacgaccc ttaagtagta cagccacttc tcaggcgtta ggccctggga gtcgttatga 1620
gattgatgcc acgattgctg atatttattt agtggatcat catgatcgcc aaaaaatcat 1680
aggaagacca acgctttaca ttgtgattga tgtgtttagt cggatgatca cgggctttta 1740
tatcggcttt gaaaatccgt cttatgtggt ggcgatgcag gcttttgtaa atgcttgctc 1800
tgacaaaacg gccatttgtg cccagcatga tattgagatt agtagctcag actggccgtg 1860
tgtaggtttg ccagatgtgt tgctagcgga ccgtggcgaa ttaatgagtc atcaggtcga 1920
agccttagtt tctagtttta atgtgcgagt ggaaagtgct ccacctagac gtggcgatgc 1980
taaaggcata gtggaaagca cttttagaac actacaagcc gagtttaagt cctttgcacc 2040
tggcattgta gagggcagtc ggatcaaaag ccatggtgaa acagactata ggttagatgc 2100
atctctgtcg gtatttgagt tcacacaaat tattttgcgt acgatcttat tcagaaataa 2160
ccatctggtg atggataaat acgatcgaga tgctgatttt cctacagatt taccgtctat 2220
tcctgtccag ctatggcaat ggggtatgca gcatcgtaca ggtagtttaa gggctgtgga 2280
gcaagagcag ttgcgagtag cgttactgcc tcgccgaaag gtctctattt cttcatttgg 2340
cgttaatttg tggggtttgt attactcggg gtcagagatt ctgcgtgagg gttggttgca 2400
gcggagcact gatatagcta gacctcaaca tttagaagcg gcttatgacc cagtgctggt 2460
tgatacgatt tatttgtttc cgcaagttgg cagccgtgta ttttggcgct gtaatctgac 2520
ggaacgtagt cggcagttta aaggtctctc attttgggag gtttgggata tacaagcaca 2580
agaaaaacac aataaagcca atgcgaagca ggatgagtta actaaacgca gggagcttga 2640
ggcgtttatt cagcaaacca ttcagaaagc gaataagtta acgcccagta ctactgagcc 2700
caaatcaaca cgcattaagc agattaaaac taataaaaaa gaagccgtga cctcggagcg 2760
taaaaaacgt gcggagcatt tgaagccaag ctcttcaggt gatgaggcta aagttattcc 2820
tttcaacgca gtggaagcgg atgatcaaga agattacagc ctacccacat acgtgcctga 2880
attatttcag gatccaccag aaaaggatga gtcatgagtg ctacccggat tcaagcagtt 2940
tatcgtgata cgggggtaga ggcttatcgt gataatcctt ttatcgaggc cttaccacca 3000
ttacaagagt cagtgaatag tgctgcatca ctgaaatcct ctttacagct tacttcctct 3060
gacttgcaaa agtcccgtgt tatcagagct cataccattt gtcgtattcc agatgactat 3120
tttcagccat taggtacgca tttgctacta agtgagcgta tttcggtcat gattcgaggt 3180
ggctacgtag gcagaaatcc taaaacagga gatttacaaa agcatttaca aaatggttat 3240
gagcgtgttc aaacgggaga gttggagaca tttcgctttg aggaggcacg atctacggca 3300
caaagcttat tgttaattgg ttgttctggt agtgggaaga cgacctctct tcatcgtatt 3360
ctagccacgt atcctcaggt gatttaccat cgtgaactca atgtagagca ggtggtgtat 3420
ttgaaaatag actgctcgca taatggttcg ctaaaagaaa tctgcttgaa ttttttcaga 3480
gcgttggatc gagccttggg ctcgaactat gagcgtcgtt atggcttaaa acgtcatggt 3540
atagaaacca tgttggcttt gatgtcgcaa atagccaatg cacatgcttt agggttgttg 3600
gttattgatg aaattcagca tttaagccgc tctcgttcgg gtggatctca agagatgctg 3660
aacttttttg tgacgatggt gaatattatt ggcgtaccag tgatgttgat tggtacccct 3720
aaagcacgag agatttttga ggctgatttg cggtctgcac gtagaggggc agggtttgga 3780
gctatattct gggatcctat acaacaaacg caacgtggaa agcccaatca agagtggatc 3840
gcttttacgg ataatctttg gcaattacag cttttacaac gcaaagatgc gctgttatcg 3900
gatgaggtcc gtgatgtgtg gtatgagcta agccaaggag tgatggacat tgtagtaaaa 3960
ctttttgtac tcgctcagct ccgtgcgcta gctttaggca atgagcgtat taccgctggt 4020
ttattgcggc aagtgtatca agatgagtta aagcctgtgc accccatgct agaggcatta 4080
cgctcgggta tcccagaacg cattgctcgt tattctgatc tagtcgttcc cgagattgat 4140
aaacggttaa tccaacttca gctagatatc gcagcgatac aagaacaaac accagaagaa 4200
aaagcccttc aagagttaga taccgaagat cagcgtcatt tatatctgat gctgaaagag 4260
gattacgatt caagcctgtt aattcccact attaaaaaag cgtttagcca gaatccaacg 4320
atgacaagac aaaagttact gcctcttgtt ttgcagtggt tgatggaagg cgaaacggta 4380
gtgtcagaac tagaaaagcc ctccaagagt aaaaaggttt cggctataaa ggtagtcaag 4440
cccagcgact gggatagctt gcctgatacg gatttacgtt atatctattc acaacgccaa 4500
cctgaaaaaa ccatgcatga acggttaaaa gggaaagggg taatagtgga tatggcgagc 4560
ttatttaaac aagcaggtta gccatgagaa actttcctgt tccgtactcg aatgagctga 4620
tttatagcac tattgcacgg gcaggcgttt atcaagggat tgttagtcct aagcagctgt 4680
tggatgaggt gtatggcaac cgcaaggtgg tcgctacctt aggtctgccc tcgcatttag 4740
gtgtgatagc aagacatcta catcaaacag gacgttacgc tgttcagcag cttatttatg 4800
agcatacctt attcccttta tatgctccgt ttgtaggcaa ggagcgccga gacgaagcta 4860
ttcggttaat ggagtaccaa gcgcaaggtg cggtgcattt aatgctagga gtcgctgctt 4920
ctagagttaa gagcgataac cgctttagat actgccctga ttgcgttgct cttcagctaa 4980
ataggtatgg ggaagccttt tggcaacgag attggtattt gcccgctttg ccatattgtc 5040
caaaacacgg tgctttagtc ttctttgata gagctgtaga tgatcaccga catcaatttt 5100
gggctttggg tcatactgag ctgctttcag actaccccaa agactcccta tctcaattaa 5160
cagcactagc tgcttatata gcccctctgt tagatgctcc acgagcgcaa gagctttccc 5220
caagccttga gcagtggacg ctgttttatc agcgcttagc gcaggatcta gggctaacca 5280
aaagcaagca cattcgtcat gacttggtgg cggagagagt gaggcagact tttagtgatg 5340
aggcactaga gaaactggat ttaaagttgg cagagaacaa ggacacgtgt tggctgaaaa 5400
gtatattccg taagcataga aaagccttta gttatttaca gcatagtatt gtgtggcaag 5460
ccttattgcc aaaactaacg gttatagaag cgctacagca ggcaagtgct cttactgagc 5520
actctataac gacaagacct gttagccagt ctgtgcaacc taactctgaa gatttatctg 5580
ttaagcataa agactggcag caactagtgc ataaatacca aggaattaag gcggcaagac 5640
agtctttaga gggtggggtg ctatacgctt ggctttaccg acatgacagg gattggctag 5700
ttcactggaa tcaacagcat caacaagagc gtctggcacc cgcccctaga gttgattgga 5760
accaaagaga tcgaattgct gtacgacaac tattaagaat cataaagcgt ctagatagta 5820
gccttgatca cccaagagcg acatcgagct ggctgttaaa gcaaactcct aacggaacct 5880
ctcttgcaaa aaatctacag aaactgcctt tggtagcgct ttgcttaaag cgttactcag 5940
agagtgtgga agattatcaa attagacgga ttagccaagc ttttattaag cttaaacagg 6000
aagatgttga gcttaggcgc tggcgattat taagaagtgc aacgttatct aaagagcgga 6060
taactgagga agcacaaaga ttcttggaaa tggtttatgg ggaagagtga 6110
<210> 4
<211> 56
<212> DNA
<213>artificial sequence
<400> 4
cgggatccat gggattcgca taatgtatat tatgttaaat caccaccgac tatttg 56
<210> 5
<211> 58
<212> DNA
<213>artificial sequence
<400> 5
tgctctagaa gcttatttaa cataatatac attatgcgaa tcagctcagc caatcgac 58
<210> 6
<211> 21
<212> DNA
<213>artificial sequence
<400> 6
gcacatcggc gacgtgctct c 21
<210> 7
<211> 23
<212> DNA
<213>artificial sequence
<400> 7
cacagcataa ctggactgat ttc 23
<210> 8
<211> 16
<212> DNA
<213>artificial sequence
<400> 8
caccaccgac tatttg 16
<210> 9
<211> 16
<212> DNA
<213>artificial sequence
<400> 9
agctcagcca atcgac 16
Claims (10)
1. a kind of for converting the transferring plasmid of pseudomonas aeruginosa, which is characterized in that the transferring plasmid according to clockwise according to
It is secondary to contain promoter ori, ampicillin resistance AmpR, transposon sequence, bond site oriT, wherein the transposon sequence
Containing can make gene and resistant gene of the pseudomonas aeruginosa from main light emission, the both ends of the resistant gene with difR and
DifL sequence.
2. transferring plasmid according to claim 1, which is characterized in that described to make base of the pseudomonas aeruginosa from main light emission
Because of LuxCDABE gene.
3. transferring plasmid according to claim 1, which is characterized in that the resistant gene is apramycin resistance gene
Or/and gentamicin resistance gene.
4. transferring plasmid according to claim 1, which is characterized in that the transposon sequence is by clockwise successively containing anti-
To repetitive sequence Tn7R, resistant gene, promoter promoter, LuxCDABE gene and inverted repeats Tn7L.
5. a kind of for converting the construction method of the transferring plasmid of pseudomonas aeruginosa, which comprises the following steps:
1) after starting plasmids pUC18T-mini-Tn7T-lux-Tp is digested with restriction enzyme Xba I and BamH I, recycling is about
The function fragment A of 11kb；
2) the Apr gene after Xba I and BamH I digestion PCR amplification recycles function fragment B；
3) function fragment A and function fragment B are attached, obtain the plasmid pUC18 T-mini-Tn7T-lux- of about 11.8kb
Dif-Apr to get.
6. a kind of pseudomonas aeruginosa from main light emission, which is characterized in that contain energy in the genome of the pseudomonas aeruginosa
Make gene of the bacterium from main light emission, and there is no resistance screening gene in the genome of pseudomonas aeruginosa.
7. pseudomonas aeruginosa according to claim 6, which is characterized in that described to make gene of the bacterium from main light emission
LuxCDABE gene.
8. a kind of construction method of the pseudomonas aeruginosa from main light emission, which comprises the steps of:
(1) the described in any item transferring plasmids of Claims 1 to 4 are provided；
(2) above-mentioned transferring plasmid and helper plasmid containing transposase gene are transferred to pseudomonas aeruginosa simultaneously, it is anti-is applied to Apr
The LB plate of property, obtains the pseudomonas aeruginosa from main light emission.
9. the construction method of pseudomonas aeruginosa according to claim 8, which is characterized in that further include step (3): by step
(2) obtain from main light emission pseudomonas aeruginosa secondary culture, filter out the photogen of resistant gene loss, obtain non-resistant sieve
Choosing label from main light emission pseudomonas aeruginosa.
10. application of the pseudomonas aeruginosa described in claim 6 or 7 in the drug of screening resisting pseudomonas aeruginosa infection.
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| CN112342229A (en) * | 2020-11-10 | 2021-02-09 | 中国科学院广州生物医药与健康研究院 | Construction method and application of resistance-marker-free self-luminescent klebsiella pneumoniae |
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| CN111662950A (en) * | 2020-06-30 | 2020-09-15 | 陕西省微生物研究所 | Application of recombinant luminous pseudomonas aeruginosa PAO1-lux in bacterial adhesion detection of medical material |
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Application publication date: 20190326 |