CN108064300A - A kind of arsenite inhibiting factor reporter plasmid and its construction method and application - Google Patents
A kind of arsenite inhibiting factor reporter plasmid and its construction method and application Download PDFInfo
- Publication number
- CN108064300A CN108064300A CN201780001826.1A CN201780001826A CN108064300A CN 108064300 A CN108064300 A CN 108064300A CN 201780001826 A CN201780001826 A CN 201780001826A CN 108064300 A CN108064300 A CN 108064300A
- Authority
- CN
- China
- Prior art keywords
- arsenite
- inhibiting factor
- plhpars9
- pllpars9
- plasmid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Plant Pathology (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Disclose a kind of arsenite inhibiting factor reporter plasmid and its construction method and application.Arsenite inhibiting factor reporter plasmid is pLHPars9 and pLLPars9, and nucleotide sequence is respectively such as SEQ ID NO:1 and SEQ ID NO:Shown in 2.Arsenite inhibiting factor reporter plasmid of the present invention is all very sensitive to arsenite, detection range is 0.04~50 μM, wide dynamic detection range is presented, therefore, arsenite inhibiting factor reporter plasmid of the present invention can be used as biosensor, for the detection of arsenite.In addition, arsenite inhibiting factor reporter plasmid pLHPars9 of the present invention is except can detect arsenite, it may also be used for detection antimonite.
Description
Technical field
The invention belongs to genetic engineering and technical field of molecular biology, it is more particularly related to a kind of arsenious acid
Salt inhibiting factor reporter plasmid and its construction method and application.
Background technology
Arsenic is widely distributed in as naturally occurring element in entire environment, it exists with inorganic or organic form, inorganic
Form has high toxicity.The guidelines for drinking water quality that the World Health Organization works out is 10 μ g/L, but in some countries, underground water is subject to arsenic
Concentration is higher than the pollution of permissible level, this can constitute a threat to citizen's health.Exposed To Arsenic can draw from drinking water and food for a long time
A variety of diseases are played, including cancer, angiocardiopathy, neurotoxicity and diabetes.Further Exposed To Arsenic in order to prevent, quick, warp
It helps effective local analysis technology and necessitates to monitor the arsenic in supplying water.
Detection method based on bacterium is a kind of when being subject to arsenic pollution, monitors the emerging technology of arsenic inducible gene expression.
With tradition based on fixed equipment, be not suitable for the method detected on the spot compared with, based on the detection method of bacterium for arsenic just
Ground detection is relatively stable and cheap.Importantly, this can measure the bioavilability of arsenic, to illustrate contact and dosage
Between difference.The report that the key component of assay method based on bacterium is made of promoter/operon and reporter gene
Gene.The synthetic biology occurred recently promotes the arsenic based on bacterium by redesigning the bacterium operon of naturally occurring
The development of biosensor, to realize the response more sensitive to the arsenic in environment.
Arsenic reactivity operon in Escherichia coli can adjust that (ArsR belongs to by arsenite inhibiting factor (ArsR)
Smt/ArsR families), family's element can be used as transcription inhibitory factor, when there is no during arsenic compound, block RNA polymerase entrance
It is combined afterwards with promoter/operon sequence of target gene.After arsenic, and isolation of promoter, and then activated gene is expressed.
Plasmid R773 and escherichia coli chromosome ArsR is homodimer, is each had positioned at its DNA binding domain initial position
Cys32-Val-Cys-Asp-Leu-Cys arsenic binding sequences.ArsR in Acidithiobacillus ferrooxidans strain GF does not have in the position
Binding sequence, but its cysteine residues is located at 95,96 and 102.On ArsR albumen and its combination operon and to it
The more information being adjusted will be helpful to rewrite biosensor basic molecular composition, including inhibiting factor, promoter and
DNA binding sites.
The reporter gene that people have developed various structures carrys out the induction of gene expression.Ideally, it is good
Reporter gene should show hypersensitivity and specific, low endogenous background and the wide dynamic range of reaction.In addition, based on report
The detection method of gene should be easy to use, relatively cheap and safe.Autofluorescence reporter gene green fluorescent protein (GFP) is one
The preferable reporter gene of kind Noninvasive.Both, without additional substrate, reality can be provided without cell cracking in analytic process
When detect.But do not include enlarging function due to detecting, sensibility is relatively low, so as to limit its application.Enzyme reports base
Cause for example, luciferase and beta galactosidase have hypersensitivity, thus is widely applied.Beta galactosidase report
The problem of accusing gene is, naturally occurring in bacterium to cause high background.In the research reported, using firefly
Luciferase rather than bacterial luciferase are as reporter gene, because the quantum yield of firefly luciferase is close to 90%,
And bacterial luciferase is only proximate to 5-10%.Assay method based on luciferase quickly, conveniently, and has extensive linear
Dynamic range.In addition, its sensibility and short-half-life become the ideal chose for measuring interim gene expression.
The content of the invention
It is an object of the invention to：Overcome deficiency present in existing arsenic detection, arsenite inhibiting factor report is provided
Accuse gene plasmid pLHPars9 and pLLPars9 and its construction method and application.
To achieve the above object of the invention, the present invention provides a kind of arsenite inhibiting factor reporter plasmids
PLHPars9 and pLLPars9, wherein, the nucleotide sequence such as SEQ ID NO of pLHPars9:Shown in 1, the nucleosides of pLLPars9
Acid sequence such as SEQ ID NO:Shown in 2.Arsenite inhibiting factor reporter plasmid of the present invention carries ArsR- luciferases
Common original paper, and added in before R773ArsR operons (Fig. 1) respectively from Escherichia coli and Acidithiobacillus ferrooxidans strain GF
(A.ferrooxidans) the two basic change sequence of chromosome.And the metal specificity of the two is different：PLLPars9 is to arsenious acid
Salt has specificity, and pLHPars9 has specificity to arsenite and antimonite.PLHPars9 and pLLPars9 it
Between unique difference be the copy number of plasmid and the corresponding ratio of ArsR promoter/operon sequences in connection.
The construction method of arsenite inhibiting factor reporter plasmid pLHPars9 and pLLPars9 of the present invention are included such as
Lower step：
(1) high copy number plasmid from pGFPuv, containing pUC replication orgins is used, with Fluc
GFPuv genes at gene substitution high copy number plasmid XbaI and EcoRI sites, and digested using HindIII and PvuII
To eliminate lac promoter sequences；
(2) segment of the promoter region containing 773 arsenite inhibiting factor operons of R is synthesized, nucleotide sequence is such as
SEQ ID NO:Shown in 3, and the luciferase genes of upstream at HindIII and XbaI are cloned, obtain pLHPars4；
(3) by the segment of the promoter region containing 773 arsenite inhibiting factor operons of R step (3) Suo Shu together with volume
The segment of the 1st~102, the amino acid of code arsenite inhibiting factor clones to obtain pLHPars5 together；
(4) it is respectively synthesized containing arsenite inhibiting factor binding sequence (EC-BS) in escherichia coli chromosome, such as SEQ
ID NO:Shown in 4 and segment (the AF- containing arsenite inhibiting factor binding sequence in Acidithiobacillus ferrooxidans strain GF
BS), such as SEQ ID NO:It shown in 5, and is inserted respectively between two sites of HindIII and PvuII of pLHPars5, difference gram
Grand acquisition pLHPars7 and pLHPars10；
(5) it is respectively synthesized Escherichia coli/Acidithiobacillus ferrooxidans strain GF chromosome arsenite inhibiting factor binding sequence
(EC-BS/AF-BS), arsenite inhibiting factor binding sequence (EC-BS) and two parts of acidophilus oxygen in two parts of escherichia coli chromosomes
Change arsenite inhibiting factor binding sequence (AF-BS) in ferrous Thiobacillus chromosome, and clone respectively into pLHPars5, point
PLHPars9, pLHPars11 and pLHPars12 are not obtained；
(6) amplification contains EC-BS/AF-BS, arsenite inhibiting factor operon and arsenite inhibiting factor amino acid
The coding region of the 1st~102 is then inserted into pACYC184 matter together with the segment in the luciferase gene downstream of pLHPars9
Between XbaI the and HindIII sites of grain, pLLPars9 is obtained；
Wherein, the pACYC184 plasmids are to be obtained at New England Biolabs (Cat#E4152S), and are contained
Low copy number p15A replication orgins.
Since arsenite inhibiting factor reporter plasmid pLHPars9 and pLLPars9 of the present invention have arsenious acid
Salt specificity, therefore, available for detecting arsenite.In addition, arsenite inhibiting factor reporter plasmid pLHPars9 is also
There is specificity to antimonite, therefore, it may also be used for detection antimonite.
In order to realize foregoing invention purpose, the present invention also provides a kind of arsenite biosensors, are by this hair
Bright arsenite inhibiting factor reporter plasmid pLHPars9 and/or pLLPars9 conversion bacillus coli DH 5 alpha obtains, the life
Object sensor is made of high copy number plasmid or low-copy-number plasmid, very sensitive to arsenite.
In addition, the present invention also provides a kind of antimonite biosensor, be by arsenite of the present invention inhibit because
Sub- reporter plasmid pLHPars9 conversions bacillus coli DH 5 alpha obtains, very sensitive to antimonite.
Compared with the prior art, the present invention has the advantages that：
(1) it is a discovery of the invention that the sensibility of the biosensor based on arsenic reporter gene is depended in the case of no arsenic
The maximum suppression of reporter gene and its highest inductivity in the presence of arsenic.It is present invention demonstrates that high by providing
Copy number plasmid can realize the high level expression of luciferase gene, but this method can cause low-level to induce, this show with
Corresponding promoter/operon sequence is compared, and the amount of endogenous ArsR albumen is too low.Most of promoter/operons are without ArsR
Albumen is for combining, so as to cause any inhibiting.In order to can be adjusted, the present invention be firstly introduced into a kind of Escherichia coli
The expression of ArsR inhibiting factors-Luciferase fusion albumen, expressed fusion protein can apply its promoter/operon anti-
Feedback inhibits.Secondly, two ArsR binding sequences are introduced, one comes from Escherichia coli, another comes from acidophilus ferrous oxide sulphur
Bacillus chromosome.ArsR albumen and preferable ArsR binding sequences using height expression can be carried out high by high copy number plasmid
Horizontal luciferase expression, so as to the detection of arsenite.
(2) secondly, although the expression of luciferase gene is relatively low, the present invention substitutes high copy with low-copy-number plasmid
Number plasmid can still show good inductivity.
(3) Monitoring lower-cut of biosensor of the present invention is 0.04 μM of arsenite (~5 μ g/L), with text so far
The Monitoring lower-cut for offering the optimal biosensor of middle report is the same.In addition, by being based on high copy number plasmid and being copied based on low
The biosensor of shellfish number plasmid is compared, and it is different, Asia to the reaction of arsenite and metal specificity to find it
Arsenate inhibiting factor reporter plasmid pLHPars9 has specificity to arsenite and antimonite.
Description of the drawings
With reference to the accompanying drawings and detailed description, the present invention and advantageous effect are described in detail.
Fig. 1 is that the arsenide of the reporter plasmid of all components of the present invention induces schematic diagram；Wherein, reporter plasmid
After being transformed into bacillus coli DH 5 alpha, with and without 10 μM of sodium arsenites handle respectively 2 it is small when, according to processed cell and without
The control cell of processing is compared, and is measured uciferase activity and is calculated fold induction.
Fig. 2 is that the EMSA of PLHPars11, pLHPars12 and pLHPars9 Reporter gene vector of the present invention compares.A is represented
With 10 μM of arsenites handle e.colidh5αcells 2 it is small when, prepare cell lysate respectively with being copied containing 2 copy AF-BS, 2
The probe mixing of shellfish EC-BS, the 1 EC-BS and AF-EC biotin labelings copied, carry out EMSA analyses, control is without arsenious acid
Salt treatment e.colidh5αcell lysate is mixed with correspondent probe.B represents to handle bacillus coli DH 5 with 10 μM of arsenites
When α cells 2 are small, prepare cell lysate and mix progress EMSA analyses, control with the probe of the EC-BS biotin labelings of 1 copy
It is to handle e.colidh5αcell lysate without arsenite to mix with 1 copy EC-BS probes.
Fig. 3 is the comparison of pLHPars9 and pLLPars9 transformed cells of the present invention.With range of concentrations for 0,0.1,1,10 to
When 100 μM of arsenite processing pLHPars9 and pLLPars9 transformed cells 2 and 4 are small.It collects cell and carries out luciferase survey
It is fixed.
Fig. 4~8 are the dynamic range and detectable limit of pLHPars9 and pLLPars9 transformed cells of the present invention.With 0,50,
100th, 200,400,600 and 800 μM of arsenites (Fig. 4), 0,0.2,0.4,0.6,0.8 and 1.0 μM of arsenite (Fig. 5~6)
PLHPars9 and pLLPars9 transformed cells are handled with 0,0.02,0.04,0.06,0.08 and 0.1 μM of arsenite (Fig. 7~8).
Handle 1 it is small when after, collect cell carry out luciferase assay.
Fig. 9~10 are specific for the metal of pLHPars9 and pLLPars9 transformed cells of the present invention.Respectively with or without 1 μM
K+、Na+、Mg2+、Zn2+、Ca2+、Hg2+、Sb3+、Mn2+、Ni2+、Cr3+、Co2+、Cd2+、Cr2+、Cu2+、As5+And As3+To pLHPars9
With pLLPars9 transformed cells processing 1 it is small when, and collect cell carry out luciferase assay.
Specific embodiment
In order to become apparent from the purpose of the present invention, technical solution and advantageous effects, with reference to embodiments, to this
Invention is further elaborated.It should be appreciated that the embodiment described in this specification is just for the sake of this hair of explanation
It is bright, be not intended to limit the present invention, parameter, ratio of embodiment etc. can adaptation to local conditions make a choice and substance had no to result
It influences.
Embodiment 1
1. plasmid construction
From a series of high copy number plasmids of pGFPuv (Clontech), contain pUC replication orgins (ColE1).Make
Substitute the GFPuv genes at XbaI and EcoRI sites with Fluc gene, and using HindIII and PvuII into
Row digests to eliminate lac promoter sequences.The segment of the 91bp promoter regions containing R 773ArsR operons is synthesized, and is cloned
The luciferase genes of upstream at HindIII and XbaI obtain pLHPars4 carriers.Synthesize the segment containing 91bp promoter regions
Together with the segment of 1-102, the amino acid of coding ArsR, clone obtains pLHPars5.It is respectively synthesized and is dyed containing Escherichia coli
ArsR binding sequences or the segment containing binding sequence in Acidithiobacillus ferrooxidans strain GF (AF-BS) in body (EC-BS), and respectively
It is inserted between two sites of HindIII and PvuII of pLHPars5, clone obtains pLHPars7 and pLHPars10 respectively.This
Outside, the segment of EC-BS/AF-BS, two parts of EC-BS and two part of AF-BS are respectively synthesized, and are cloned respectively into pLHPars5, respectively
Obtain pLHPars9, pLHPars11 and pLHPars12.It is obtained at New England Biolabs (Cat#E4152S)
PACYC184 plasmids contain low copy number p15A replication orgins.Amplification contains EC-BS/AF-BS, ArsR operon and ArsR amino
The coding region of acid 1-102 is then inserted into pACYC184 together with the segment in the luciferase gene downstream of pLHPars9
XbaI and HindIII sites between, obtain pLLPars9.
The nucleotide sequence of gained pLHPars9 such as SEQ ID NO:Shown in 1, the nucleotide sequence such as SEQ of pLLPars9
ID NO:Shown in 2.
In above-mentioned steps, the nucleotides sequence of the segment of the promoter region containing 773 arsenite inhibiting factor operons of R
Row such as SEQ ID NO:Shown in 3, contain arsenite inhibiting factor binding sequence such as SEQ in escherichia coli chromosome (EC-BS)
ID NO:Shown in 4, such as SEQ ID NO of the segment containing binding sequence in Acidithiobacillus ferrooxidans strain GF (AF-BS):Shown in 5,
The nucleotide sequence of the coding region of 1-102, ArsR amino acid such as SEQ ID NO:Shown in 6, luciferase gene sequence is such as
SEQ ID NO:Shown in 7.
2. processing and measure
Plasmid is converted into bacillus coli DH 5 alpha competent cell.Picking single bacterium colony is aggressively shaken inoculation 12- at 37 DEG C
16 it is small when.According to OD values, culture is diluted overnight with the culture medium containing antibiotic, high copy number plasmid is about pressed 1:500 is dilute
It releases, low-copy-number plasmid presses 1:10 dilutions.Diluted cell is further cultured in 37 DEG C of incubators 3-4 it is small when, until O.D. values
Reach 0.6.The cell liquid of 400 μ L is taken, is handled with the sodium arsenite (Sigma) of various concentration.By 50 μ L luciferase substrates and
The DH5 α cells of 20 μ L and the mixing of 5 μ L cell pyrolysis liquids, the machine (Veritas) detected with luciferase measure,
PLHPars9 shows high-caliber luciferase gene expression.
3. electrophoretic mobility measures (EMSA)
1mL overnight cultures are centrifuged 1 minute to prepare cell lysate with 10,000rpm.Sediment is suspended from again
300 μ L lysis buffers (10mM Tris-HCl, pH8.0,0.1M NaCl, 1mM EDTA and 0.5% [w/v] Triton X-
100) in, 30 are cultivated at room temperature with the 25 freshly prepd lysozyme solns of μ L (10mg/mL, pH8.0 in 10mM Tris-HCl)
Minute.After the completion of centrifugation, gel shift measure is carried out using supernatant.1-3 μ g cell lysates, 5 times of 2 μ L are combined into buffering
Liquid and 1 μ L poly- (I-C) mixing, are incubated 5 minutes on ice.1 μ L biotinylated probes are added in mixture, are cultivated at 22 DEG C
30 minutes；Using 6.5% non-denaturing polyacrylamide gel, by each reaction mixture in 0.5 times of TBE at 4 DEG C of 100V
Separation about 50 to 60 minutes.After gel is transferred on NB films, 15mL Block buffers are added at room temperature and continue 20 minutes
It is closed, then with Streptavidin-HRP and by biotin on luminol (Pierce) enhanced chemiluminescence substrate detection trace
The probe of mark.Image is obtained using imager.
Experimental example 1 realizes high-caliber luciferase gene expression using high copy number plasmid
It is with 10 μM of sodium arsenites that transformed cells processing 2 is small in order to assess the arsenite inductivity of luciferase gene
When；As shown in Figure 1, the induction of the arsenite mediation of apparent uciferase activity is not observed.Escherichia coli chromosome
ArsR can be used as trans regulatory factor, for being combined with escherichia coli chromosome and plasmid R773 operons.Therefore, no induction
A simplicity of explanation be, with ArsR combine promoter/operon sequence compared with, free ArsR occupies the majority, and reason is to provide
There is high copy number plasmid in the cell of high level expression luciferase.Arsenite is added, it can be from promoter/operon
Except ArsR, compared with non-binding form, do not make uciferase activity that significant change occur, this is because the startup combined with ArsR
The quantity of son/operon is very limited.
The coexpression of experimental example 2ArsR reduces the basal expression of reporter gene
By the feedback control of reporter gene expression, usually dropped using the coexpression of external source ArsR and luciferase gene
The foundation level of low Poison element enzymatic activity.ArsR can carry out the expression of polycistron formula by reporter gene or be combined into fusion protein
To express.Missing functional analysis is carried out to the ArsR by being co-expressed with beta-lactamase and shows to adjust activity without big
The C-terminal from amino acid 93 to 117 of enterobacteria ArsR；In order to introduce the feedback regulation to luciferase gene expression,
A short ArsR is constructed in the 1-102 amino acid of luciferase N-terminal, to obtain Reporter gene vector pLHPars5
(Fig. 1).This with ArsR 1-102 amino acids short ArsR under R773ArsR promoters/operon with fluorescein
Enzyme is expressed as fusion protein together.With 10 μM of sodium arsenites come handle converted pLHPars5 cell 2 it is small when.Luciferase is surveyed
Surely 1.5 times of inductivities (Fig. 1) of uciferase activity are shown.The inductivity of uciferase activity is not as expected.
Experimental example 3 adds ArsR binding sequences before Ars operons
Common methods in mammlian system are to pass through the additional cis-acting elements of the gene insertion before promoter
It copies preferably to measure the inductivity of reporter gene.It is additional big by being inserted into one between ArsR genes and reporter gene
Enterobacteria ArsR binding sequences copy, and people are reported using this method to reduce in arsenite biosensor building process
The basic background of gene activity.It is slightly lower with being shown compared with the control reporter gene for being not added with additional ArsR binding sequences
Background.In the present invention, an Escherichia coli ArsR combination sequence is built before R773ArsR promoters/operon of pLHPars5
(EC-BS) or Acidithiobacillus ferrooxidans strain GF ArsR binding sequences (AF-BS) are arranged, respectively obtains Reporter gene vector pLHPars7
With pLHPars10 (Fig. 1).With 10 μM of arsenites handle DH5 α transformed cells 2 it is small when show that 2 times of uciferase activity lure
The property led (Fig. 1).Compared with being not added with the pLHPars5 of additional ArsR binding sequences, the pLHPars7 in the reaction with arsenite
It is not improved with pLHPars10.Then, the EC-BS of 2 copies is added respectively before promoter/operon of pLHPars5
Or each 1 copies of AF-BS and EC-BS and AF-BS, respectively obtain pLHPars11, pLHPars12 and pLHPars9 (Fig. 1).Band
There are the Reporter gene vector pLHPars11 of 2 copy EC-BS and the pLHPars12 with 2 copy AF-BS to be induced in arsenite
In do not show effect (Fig. 1).It is worth noting that, the Reporter gene vector with 1 copy EC-BS and 1 copy AF-BS
PLHPars9, can handled with 10 μM of arsenites 2 it is small when after show 5 times of luciferase fold induction (Fig. 1), better than 2
The EC-BS or AF-BS of copy.These are the result shows that the two binding sequences together can be complimentary to one another so that it is better than big
Enterobacteria ArsR individually adds the EC-BS or AF-BS of 2 copies in combining.
Differences of the experimental example 4ArsR in its binding sequence
When interacting for Direct Test and Escherichia coli ArsR, whether individual EC-BS and AF-BS copy different from 2
The EC-BS or AF-BS of shellfish, employ EMSA, and cell lysate is prepared from bacillus coli DH 5 alpha-pLHPars5, with and without 10 μ
When M arsenites processing 2 is small.The ArsR protein for replacing purifying using cell lysate carries out EMSA, to measure natural phase
Interaction.According to the sequence of EC-BS and AF-BS, chemical synthesis 5' ends carry the probe of biotin labeling.Synthesis is with 2 copies
EC-BS, 2 copy AF-BS and AF-BS and EC-BS it is each 1 copy probe, to carry out EMSA analyses.Fig. 2A results are shown
Show, the combination of AF-BS-EC-BS is better than the AF-BS of the copies of EC-BS and 2 of 2 copies, consistent with induction data.This shows ArsR
AF-BS and EC-BS that can be spatially with 1 copy be combined, better than individually being combined with EC-BS or AF-BS.It is looked for by prioritization scheme
To better ArsR binding sequences, and several key positions in ArsR binding sequences are determined.The EC-BS and AF-BS of 1 copy
The nucleotide of junction containing EC-BS and AF-BS helps preferably to be interacted with Escherichia coli ArsR.
In addition, using the probe of the binding sequence with 2 copies, then band is clearly shifted there are two meetings in EMSA results,
This shows there is two kinds, and ArsR dimers and the tetramer coexist or the DNA probe of two kinds of forms；There are one a kind of tools
ArsR dimers, there are two ArsR dimers for another kind tool.In former possibility, dimer and the tetramer coexist, and another
Dimer is only existed in kind possibility.If dimer and the tetramer coexist, even if using a DNA binding sequence row as probe
Both should also detect.As shown in Figure 2 B, a displacement band is only observed, the relatively low strap being equivalent in Fig. 2A, this shows only to deposit
It is coexisted in ArsR dimers rather than with the tetramer.Therefore, two displacement bands with probe are respectively by a dimer of ArsR
It is formed with two dimers.
Addition arsenite can induce ArsR and be dissociated from binding sequence, consistent with above-mentioned conclusion, inhibiting factor need with
Isolation of promoter.Different from MerR families inhibiting factor, without dissociation, but metal combines can induce conformation from undesirable
Promoter is changed into stronger promoter.
Experimental example 5 is compared the biosensor with high copy number plasmid and low-copy-number plasmid
In order to check whether inductivity is different, introduces from plasmid when promoter/operon copy number is relatively low
The low copy number p15A replication orgins of pACYC184, instead of high copy number pUC replication orgins, obtain pLLPars9.With 10 μM
When arsenite processing pLLPars9 transformed cells 2 are small, compared with untreated control cell, which show>10 times lure
The property led, better than pLHPars9 (Fig. 1).It is dense with wider range in order to more specifically be compared pLHPars9 and pLLPars9
The arsenite processing transformed cells of degree, with 0,0.1,1,10 to 100 μM of arsenite handle 2 and 4 it is small when.As expected,
The foundation level of luciferase is far below pLHPars9, while the also significant lower (figure of maximum horizontal in pLLPars9 transformed cells
3).Using high copy number plasmid, interior when 2 is small 0.1 to 1 μM of change dramatically can occur for inductivity, and 4 it is small when interior arsenite
Change in the range more slowly.Using 100 μM of arsenites, the 4 interior uciferase activities for observing pLHPars9 when small
Decline, but do not observed when 2 is small.Induction pattern with low-copy-number plasmid is different from the processed high copy of arsenite
Number plasmid.In addition, under conditions of there is low-copy-number plasmid, do not observe that luciferase activity declines.It is these results indicate that high
The arsenite inductivity of luciferase is different between copy number and low-copy-number plasmid.
The dynamic range and detectable limit of the DH5 α cells of experimental example 6pLHPars9 and pLLPars9 conversion
Application of the biosensor in the field is estimated can be completed in a short time analysis.In some researchs, with arsenic chemical combination
When the shortest time of object processing is 1 small.With the arsenite of various concentration scope (0,50,100,200,400,600 to 800 μM)
Processing with conversion have pLHPars9 or pLLPars9 cell 1 it is small when.Luciferase assay show pLHPars9 and
PLLPars9 biosensors reach peak value in 50 μM and 100 μM of arsenite concentration respectively, and activity begins to decline (figure afterwards
4).In addition, with 0,0.2,0.4,0.8 to 1.0 μM of arsenite handle two kinds of cells 1 it is small when, all show linear response (Fig. 5
~6).In order to study detectable limit, with 0,0.02,0.04,0.06,0.08 and 0.16 μM of arsenite handle cell 1 it is small when.If
Specifying can cause the detectable limit of the arsenite substantially induced concentration to add 2 times of standard deviations (SD) for background, then
PLHPars9 and pLLPars9 biosensors can show 0.04 μM of arseniteDetectable limit (Fig. 7~
8), less than the 10 μ g/L World Health Organization (WHO) guides.Accordingly, it is shown that the two can be employed as arsenite biosensor.
Experimental example 7pLHPars9 and pLLPars9 biosensor are to the difference of the selective reaction of metal
The specificity of both biosensors is tested, and compared with the metal different with 17 kinds.In general, such as
Shown in Fig. 9~10, biosensor pLLPars9 can show more specificity than pLHPars9.In surveyed metal,
PLLPars9 is only induced be subject to arsenite, though slightly inhibited be subject to Hg (II), using arsenate and during other metals not
Observe that it has apparent induction.Biosensor pLHPars9 shows have very big difference with pLLPars9.Except with
Arsenite As (III) reactions are outer, also have reaction to antimonite Sb (III), also have slight reaction to arsenate As (V).
Difference between pLLPars9 and pLHPars9 is the copy number of its promoter/operon and its ratio corresponding to ArsR albumen
Example.Therefore, the metal specificity of ArsR can be by ArsR albumen, the concentration of ArsR combinations promoter/operon or ArsR to it
It is adjusted with reference to the ratio of promoter/operon.
According to the disclosure and teachings of the above specification, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula carries out appropriate change and modification.Therefore, the invention is not limited in specific embodiment disclosed and described above, to this
Some modifications and changes of invention should also be as falling into the scope of the claims of the present invention.In addition, although this specification
In used some specific terms, but these terms are merely for convenience of description, do not limit the present invention in any way.
Sequence table
<110>Guangdong Microbes Inst；Holy Gneuss Co., Ltd
<120>A kind of arsenite inhibiting factor reporter plasmid and its construction method and application
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2686
<212> DNA
<213> pLHPars9 (artificial sequence)
<400> 1
atggaaancg ccaagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 60
tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 120
gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 180
agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg 240
cagctgacac attcgttaag tcatatatgt ttttgacttt ttatccacga atatttcttg 300
cagtattgaa agctttgtga ttaatcatat gcgtttttgg ttatgtgttg tttgacttaa 360
tatcagagcc gagagatact tgttttctac aaaggagagg gaaatgttgc aactaacacc 420
acttcagtta tttaaaaacc tgtccgatga aacccgtttg ggtatcgtgt tgttgctcag 480
ggagatggga gagttgtgcg tgtgtgatct ttgcatggca ctggatcaat cacagcccaa 540
aatatcccgt catctggcga tgctacggga aagtggaatc cttctggatc gtaaacaggg 600
aaaatgggtt cactaccgct tatcaccgca tattccttca tgggctgccc agattattga 660
gcaggcctgg ttaagccaac aggacgacgt tcaggtcatc gcacgcaagc ctctagagga 720
tccccgggta ccggtagaaa aaatggaaga cgccaaaaac ataaagaaag gcccggcgcc 780
attctatccg ctggaagatg gaaccgctgg agagcaactg cataaggcta tgaagagata 840
cgccctggtt cctggaacaa ttgcttttac agatgcacat atcgaggtgg acatcactta 900
cgctgagtac ttcgaaatgt ccgttcggtt ggcagaagct atgaaacgat atgggctgaa 960
tacaaatcac agaatcgtcg tatgcagtga aaactctctt caattcttta tgccggtgtt 1020
gggcgcgtta tttatcggag ttgcagttgc gcccgcgaac gacatttata atgaacgtga 1080
attgctcaac agtatgggca tttcgcagcc taccgtggtg ttcgtttcca aaaaggggtt 1140
gcaaaaaatt ttgaacgtgc aaaaaaagct cccaatcatc caaaaaatta ttatcatgga 1200
ttctaaaacg gattaccagg gatttcagtc gatgtacacg ttcgtcacat ctcatctacc 1260
tcccggtttt aatgaatacg attttgtgcc agagtccttc gatagggaca agacaattgc 1320
actgatcatg aactcctctg gatctactgg tctgcctaaa ggtgtcgctc tgcctcatag 1380
aactgcctgc gtgagattct cgcatgccag agatcctatt tttggcaatc aaatcattcc 1440
ggatactgcg attttaagtg ttgttccatt ccatcacggt tttggaatgt ttactacact 1500
cggatatttg atatgtggat ttcgagtcgt cttaatgtat agatttgaag aagagctgtt 1560
tctgaggagc cttcaggatt acaagattca aagtgcgctg ctggtgccaa ccctattctc 1620
cttcttcgcc aaaagcactc tgattgacaa atacgattta tctaatttac acgaaattgc 1680
ttctggtggc gctcccctct ctaaggaagt cggggaagcg gttgccaaga ggttccatct 1740
gccaggtatc aggcaaggat atgggctcac tgagactaca tcagctattc tgattacacc 1800
cgagggggat gataaaccgg gcgcggtcgg taaagttgtt ccattttttg aagcgaaggt 1860
tgtggatctg gataccggga aaacgctggg cgttaatcaa agaggcgaac tgtgtgtgag 1920
aggtcctatg attatgtccg gttatgtaaa caatccggaa gcgaccaacg ccttgattga 1980
caaggatgga tggctacatt ctggagacat agcttactgg gacgaagacg aacacttctt 2040
catcgttgac cgcctgaagt ctctgattaa gtacaaaggc tatcaggtgg ctcccgctga 2100
attggaatcc atcttgctcc aacaccccaa catcttcgac gcaggtgtcg caggtcttcc 2160
cgacgatgac gccggtgaac ttcccgccgc cgttgttgtt ttggagcacg gaaagacgat 2220
gacggaaaaa gagatcgtgg attacgtcgc cagtcaagta acaaccgcga aaaagttgcg 2280
cggaggagtt gtgtttgtgg acgaagtacc gaaaggtctt accggaaaac tcgacgcaag 2340
aaaaatcaga gagatcctca taaaggccaa gaagggcgga aagatcgccg tgtaactggc 2400
ttcagttaac tgctccggta gcagtaaggc tgtctgcatc taaaaaattt gcctgaacat 2460
atatgtttta tcaaatgcga ggtatttaag atgaaaacgt taatggtatt tgacccggcg 2520
atgtgttgca gcaccggcgt ctgcggtaca gatgttgatc aggctctggt cgatttttct 2580
acagatgtgc aatggctcaa acaatgcggt gtacaaattg agcgtttcaa tcttgcgcaa 2640
caaccgatga gctttgtaca gaacgagaag gtcaaagcgt ttattg 2686
<210> 2
<211> 5654
<212> DNA
<213> pLLPars9 (artificial sequence)
<400> 2
gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt 60
gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt 120
ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga 180
tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga 240
aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt 300
ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc 360
ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat 420
ttattcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt 480
gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt tcagctactg 540
acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgctagcgg agtgtatact 600
ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa 660
aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc 720
actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc 780
ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa 840
agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc 900
agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc 960
tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc 1020
gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac 1080
tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt 1140
gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt 1200
agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg 1260
tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt 1320
cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc 1380
aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga acacattcgt 1440
taagtcatat atgtttttga ctttttatcc acgaatattt cttgcagtat tgaaagcttt 1500
gtgattaatc atatgcgttt ttggttatgt gttgtttgac ttaatatcag agccgagaga 1560
tacttgtttt ctacaaagga gagggaaatg ttgcaactaa caccacttca gttatttaaa 1620
aacctgtccg atgaaacccg tttgggtatc gtgttgttgc tcagggagat gggagagttg 1680
tgcgtgtgtg atctttgcat ggcactggat caatcacagc ccaaaatatc ccgtcatctg 1740
gcgatgctac gggaaagtgg aatccttctg gatcgtaaac agggaaaatg ggttcactac 1800
cgcttatcac cgcatattcc ttcatgggct gcccagatta ttgagcaggc ctggttaagc 1860
caacaggacg acgttcaggt catcgcacgc aagccggatc ctggaagacg ccaaaaacat 1920
aaagaaaggc ccggcgccat tctatccgct ggaagatgga accgctggag agcaactgca 1980
taaggctatg aagagatacg ccctggttcc tggaacaatt gcttttacag atgcacatat 2040
cgaggtggac atcacttacg ctgagtactt cgaaatgtcc gttcggttgg cagaagctat 2100
gaaacgatat gggctgaata caaatcacag aatcgtcgta tgcagtgaaa actctcttca 2160
attctttatg ccggtgttgg gcgcgttatt tatcggagtt gcagttgcgc ccgcgaacga 2220
catttataat gaacgtgaat tgctcaacag tatgggcatt tcgcagccta ccgtggtgtt 2280
cgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa aaaaagctcc caatcatcca 2340
aaaaattatt atcatggatt ctaaaacgga ttaccaggga tttcagtcga tgtacacgtt 2400
cgtcacatct catctacctc ccggttttaa tgaatacgat tttgtgccag agtccttcga 2460
tagggacaag acaattgcac tgatcatgaa ctcctctgga tctactggtc tgcctaaagg 2520
tgtcgctctg cctcatagaa ctgcctgcgt gagattctcg catgccagag atcctatttt 2580
tggcaatcaa atcattccgg atactgcgat tttaagtgtt gttccattcc atcacggttt 2640
tggaatgttt actacactcg gatatttgat atgtggattt cgagtcgtct taatgtatag 2700
atttgaagaa gagctgtttc tgaggagcct tcaggattac aagattcaaa gtgcgctgct 2760
ggtgccaacc ctattctcct tcttcgccaa aagcactctg attgacaaat acgatttatc 2820
taatttacac gaaattgctt ctggtggcgc tcccctctct aaggaagtcg gggaagcggt 2880
tgccaagagg ttccatctgc caggtatcag gcaaggatat gggctcactg agactacatc 2940
agctattctg attacacccg agggggatga taaaccgggc gcggtcggta aagttgttcc 3000
attttttgaa gcgaaggttg tggatctgga taccgggaaa acgctgggcg ttaatcaaag 3060
aggcgaactg tgtgtgagag gtcctatgat tatgtccggt tatgtaaaca atccggaagc 3120
gaccaacgcc ttgattgaca aggatggatg gctacattct ggagacatag cttactggga 3180
cgaagacgaa cacttcttca tcgttgaccg cctgaagtct ctgattaagt acaaaggcta 3240
tcaggtggct cccgctgaat tggaatccat cttgctccaa caccccaaca tcttcgacgc 3300
aggtgtcgca ggtcttcccg acgatgacgc cggtgaactt cccgccgccg ttgttgtttt 3360
ggagcacgga aagacgatga cggaaaaaga gatcgtggat tacgtcgcca gtcaagtaac 3420
aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac gaagtaccga aaggtcttac 3480
cggaaaactc gacgcaagaa aaatcagaga gatcctcata aaggccaaga agggcggaaa 3540
gatcgccgtg taagtcgacc gatgcccttg agagccttca acccagtcag ctccttccgg 3600
tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc 3660
gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc 3720
gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc 3780
ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg 3840
gcggccgacg cgctgggcta cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc 3900
cccattatga ttcttctcgc ttccggcggc atcgggatgc ccgcgttgca ggccatgctg 3960
tccaggcagg tagatgacga ccatcaggga cagcttcaag gatcgctcgc ggctcttacc 4020
agcctaactt cgatcattgg accgctgatc gtcacggcga tttatgccgc ctcggcgagc 4080
acatggaacg ggttggcatg gattgtaggc gccgccctat accttgtctg cctccccgcg 4140
ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa tggaagccgg cggcacctcg 4200
ctaacggatt caccactcca agaattggag ccaatcaatt cttgcggaga actgtgaatg 4260
cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc catctccagc agccgcacgc 4320
ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg catgatcgtg ctcctgtcgt 4380
tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac 4440
gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa 4500
tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcccct acgtgctgct 4560
gaagttgccc gcaacagaga gtggaaccaa ccggtgatac cacgatacta tgactgagag 4620
tcaacgccat gagcggcctc atttcttatt ctgagttaca acagtccgca ccgctgtccg 4680
gtagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct 4740
ttatcatgca actcgtagga caggtgccgg cagcgcccaa cagtcccccg gccacggggc 4800
ctgccaccat acccacgccg aaacaagcgc cctgcaccat tatgttccgg atctgcatcg 4860
caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctaaccgt 4920
ttttatcagg ctctgggagg cagaataaat gatcatatcg tcaattatta cctccacggg 4980
gagagcctga gcaaactggc ctcaggcatt tgagaagcac acggtcacac tgcttccggt 5040
agtcaataaa ccggtaaacc agcaatagac ataagcggct atttaacgac cctgccctga 5100
accgacgacc gggtcgaatt tgctttcgaa tttctgccat tcatccgctt attatcactt 5160
attcaggcgt agcaaccagg cgtttaaggg caccaataac tgccttaaaa aaattacgcc 5220
ccgccctgcc actcatcgca gtactgttgt aattcattaa gcattctgcc gacatggaag 5280
ccatcacaaa cggcatgatg aacctgaatc gccagcggca tcagcacctt gtcgccttgc 5340
gtataatatt tgcccatggt gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt 5400
aaatcaaaac tggtgaaact cacccaggga ttggctgaga cgaaaaacat attctcaata 5460
aaccctttag ggaaataggc caggttttca ccgtaacacg ccacatcttg cgaatatatg 5520
tgtagaaact gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt 5580
tgctcatgga aaacggtgta acaagggtga acactatccc atatcaccag ctcaccgtct 5640
ttcattgcca tacg 5654
<210> 3
<211> 91
<212> DNA
<213>The promoter region (artificial sequence) of 773 arsenite inhibiting factor operons of R
<400> 3
tgtgattaat catatgcgtt tttggttatg tgttgtttga cttaatatca gagccgagag 60
atacttgttt tctacaaagg agagggaaat g 91
<210> 4
<211> 32
<212> DNA
<213>Contain escherichia coli chromosome (arsenite inhibiting factor binding sequence artifitial in EC-BS
sequence)
<400> 4
cacattcgtt aagtcatata tgtttttgac tt 32
<210> 5
<211> 20
<212> DNA
<213>Contain A.ferrooxidans (binding sequence artificial sequence in AF-BS)
<400> 5
atccacgaat atttcttgca 20
<210> 6
<211> 301
<212> DNA
<213>The nucleotide sequence (artificial sequence) of the coding region of 1-102, ArsR amino acid
<400> 6
caactaacac cacttcagtt atttaaaaac ctgtccgatg aaacccgttt gggtatcgtg 60
ttgttgctca gggagatggg agagttgtgc gtgtgtgatc tttgcatggc actggatcaa 120
tcacagccca aaatatcccg tcatctggcg atgctacggg aaagtggaat ccttctggat 180
cgtaaacagg gaaaatgggt tcactaccgc ttatcaccgc atattccttc atgggctgcc 240
cagattattg agcaggcctg gttaagccaa caggacgacg ttcaggtcat cgcacgcaag 300
c 301
<210> 7
<211> 1643
<212> DNA
<213>Luciferase gene sequence (artificial sequence)
<400> 7
acgccaaaaa cataaagaaa ggcccggcgc cattctatcc gctggaagat ggaaccgctg 60
gagagcaact gcataaggct atgaagagat acgccctggt tcctggaaca attgctttta 120
cagatgcaca tatcgaggtg gacatcactt acgctgagta cttcgaaatg tccgttcggt 180
tggcagaagc tatgaaacga tatgggctga atacaaatca cagaatcgtc gtatgcagtg 240
aaaactctct tcaattcttt atgccggtgt tgggcgcgtt atttatcgga gttgcagttg 300
cgcccgcgaa cgacatttat aatgaacgtg aattgctcaa cagtatgggc atttcgcagc 360
ctaccgtggt gttcgtttcc aaaaaggggt tgcaaaaaat tttgaacgtg caaaaaaagc 420
tcccaatcat ccaaaaaatt attatcatgg attctaaaac ggattaccag ggatttcagt 480
cgatgtacac gttcgtcaca tctcatctac ctcccggttt taatgaatac gattttgtgc 540
cagagtcctt cgatagggac aagacaattg cactgatcat gaactcctct ggatctactg 600
gtctgcctaa aggtgtcgct ctgcctcata gaactgcctg cgtgagattc tcgcatgcca 660
gagatcctat ttttggcaat caaatcattc cggatactgc gattttaagt gttgttccat 720
tccatcacgg ttttggaatg tttactacac tcggatattt gatatgtgga tttcgagtcg 780
tcttaatgta tagatttgaa gaagagctgt ttctgaggag ccttcaggat tacaagattc 840
aaagtgcgct gctggtgcca accctattct ccttcttcgc caaaagcact ctgattgaca 900
aatacgattt atctaattta cacgaaattg cttctggtgg cgctcccctc tctaaggaag 960
tcggggaagc ggttgccaag aggttccatc tgccaggtat caggcaagga tatgggctca 1020
ctgagactac atcagctatt ctgattacac ccgaggggga tgataaaccg ggcgcggtcg 1080
gtaaagttgt tccatttttt gaagcgaagg ttgtggatct ggataccggg aaaacgctgg 1140
gcgttaatca aagaggcgaa ctgtgtgtga gaggtcctat gattatgtcc ggttatgtaa 1200
acaatccgga agcgaccaac gccttgattg acaaggatgg atggctacat tctggagaca 1260
tagcttactg ggacgaagac gaacacttct tcatcgttga ccgcctgaag tctctgatta 1320
agtacaaagg ctatcaggtg gctcccgctg aattggaatc catcttgctc caacacccca 1380
acatcttcga cgcaggtgtc gcaggtcttc ccgacgatga cgccggtgaa cttcccgccg 1440
ccgttgttgt tttggagcac ggaaagacga tgacggaaaa agagatcgtg gattacgtcg 1500
ccagtcaagt aacaaccgcg aaaaagttgc gcggaggagt tgtgtttgtg gacgaagtac 1560
cgaaaggtct taccggaaaa ctcgacgcaa gaaaaatcag agagatcctc ataaaggcca 1620
agaagggcgg aaagatcgcc gtg 1643
Claims (6)
1. a kind of arsenite inhibiting factor reporter plasmid pLHPars9 and pLLPars9, which is characterized in that pLHPars9
Nucleotide sequence such as SEQ ID NO:Shown in 1, the nucleotide sequence such as SEQ ID NO of pLLPars9:Shown in 2.
2. the construction method of arsenite inhibiting factor reporter plasmid pLHPars9 and pLLPars9 described in claim 1,
It is characterised in that it includes following steps：
(1) high copy number plasmid from pGFPuv, containing pUC replication orgins is used, with Fluc gene
The GFPuv genes at high copy number plasmid XbaI and EcoRI sites are substituted, and are digested to disappear using HindIII and PvuII
Except lac promoter sequences；
(2) segment of the promoter region containing 773 arsenite inhibiting factor operons of R, nucleotide sequence such as SEQ ID are synthesized
NO:Shown in 3, and the luciferase genes of upstream at HindIII and XbaI are cloned, obtain pLHPars4；
(3) it is the segment of the promoter region containing 773 arsenite inhibiting factor operons of R step (3) Suo Shu is sub- together with coding
The segment of the 1st~102, the amino acid of arsenate inhibiting factor clones to obtain pLHPars5 together；
(4) it is respectively synthesized containing arsenite inhibiting factor binding sequence in escherichia coli chromosome, such as SEQ ID NO:4 institutes
Show and the segment containing arsenite inhibiting factor binding sequence in Acidithiobacillus ferrooxidans strain GF chromosome, such as SEQ ID
NO:It shown in 5, and is inserted respectively between two sites of HindIII and PvuII of pLHPars5, clone obtains respectively
PLHPars7 and pLHPars10；
(5) be respectively synthesized Escherichia coli/Acidithiobacillus ferrooxidans strain GF chromosome arsenite inhibiting factor binding sequence, two parts
Arsenous in arsenite inhibiting factor binding sequence and two parts of Acidithiobacillus ferrooxidans strain GF chromosomes in escherichia coli chromosome
Hydrochlorate inhibiting factor binding sequence, and cloning respectively into pLHPars5, respectively obtain pLHPars9, pLHPars11 and
pLHPars12；
(6) amplification contains arsenite inhibiting factor binding sequence, Asia in Escherichia coli/Acidithiobacillus ferrooxidans strain GF chromosome
The coding region of arsenate inhibiting factor operon and the 1st~102, arsenite inhibiting factor amino acid is together with pLHPars9
Luciferase gene downstream segment, be then inserted between XbaI the and HindIII sites of pACYC184 plasmids, obtain
pLLPars9；
Wherein, the pACYC184 plasmids contain low copy number p15A replication orgins.
3. arsenite inhibiting factor reporter plasmid pLHPars9 and pLLPars9 described in claim 1 is examined in arsenite
Application in survey.
4. the application of arsenite inhibiting factor reporter plasmid pLHPars9 and pLLPars9 according to claim 3,
It is characterized in that, the arsenite inhibiting factor reporter plasmid pLHPars9 is additionally operable to the detection of antimonite.
5. a kind of arsenite biosensor, which is characterized in that be by arsenite inhibiting factor report described in claim 1
Gene plasmid pLHPars9 and/or pLLPars9 conversion bacillus coli DH 5 alpha is accused to obtain.
6. a kind of antimonite biosensor, which is characterized in that be by arsenite inhibiting factor report described in claim 1
Gene plasmid pLHPars9 conversion bacillus coli DH 5 alphas are accused to obtain.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/104721 WO2019061381A1 (en) | 2017-09-30 | 2017-09-30 | Class of arsenite inhibitory factor reporter gene plasmids, construction method therefor and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108064300A true CN108064300A (en) | 2018-05-22 |
| CN108064300B CN108064300B (en) | 2019-09-10 |
Family
ID=62142039
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201780001826.1A Active CN108064300B (en) | 2017-09-30 | 2017-09-30 | A kind of arsenite inhibiting factor reporter plasmid and its construction method and application |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN108064300B (en) |
| WO (1) | WO2019061381A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109231484A (en) * | 2018-09-30 | 2019-01-18 | 中南大学 | The method of organic matter collaboration microbiological treatment waste water containing trivalent arsenic |
| CN109402273A (en) * | 2018-10-29 | 2019-03-01 | 中南大学 | A kind of detection bacterium mediates the kit and method of gene expression dynamic change in figure water hydroxyl sarmientite generating process |
| CN110144362A (en) * | 2019-04-28 | 2019-08-20 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of arsenic response luciferase reporter vector and its construction method and application |
| CN110172498A (en) * | 2019-04-28 | 2019-08-27 | 广东省微生物研究所（广东省微生物分析检测中心） | A method of rapidly and efficiently analyzing transcription factor and its interaction of target DNA binding sequence |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003102223A1 (en) * | 2002-05-31 | 2003-12-11 | Eawag Eidg.Anstalt Für Wasserversorgung | Method of detecting arcenic ions with indicator bacteria |
| US20130337464A1 (en) * | 2011-02-25 | 2013-12-19 | Universite De Lorraine | Arsenic assay using elisa |
| WO2014012089A1 (en) * | 2012-07-13 | 2014-01-16 | The Florida International University Board Of Trustees | Biosensors for organic and inorganic arsenic |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894042B (en) * | 2014-12-24 | 2019-08-02 | 温州医科大学 | The escherichia coli of one plant of detection arsenic |
| EP4071252A1 (en) * | 2015-02-16 | 2022-10-12 | The Regents of the University of California | Microbial microfluidic biosensor |
-
2017
- 2017-09-30 WO PCT/CN2017/104721 patent/WO2019061381A1/en active Application Filing
- 2017-09-30 CN CN201780001826.1A patent/CN108064300B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003102223A1 (en) * | 2002-05-31 | 2003-12-11 | Eawag Eidg.Anstalt Für Wasserversorgung | Method of detecting arcenic ions with indicator bacteria |
| US20130337464A1 (en) * | 2011-02-25 | 2013-12-19 | Universite De Lorraine | Arsenic assay using elisa |
| WO2014012089A1 (en) * | 2012-07-13 | 2014-01-16 | The Florida International University Board Of Trustees | Biosensors for organic and inorganic arsenic |
Non-Patent Citations (2)
| Title |
|---|
| BUTCHER，B.G.等: "GenBank登录号：AF173880.1", 《GENBANK数据库》 * |
| FRANCISCO F. ROBERTO等: "Evaluation of a GFP reporter gene construct for environmental arsenic detection", 《TALANTA》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109231484A (en) * | 2018-09-30 | 2019-01-18 | 中南大学 | The method of organic matter collaboration microbiological treatment waste water containing trivalent arsenic |
| CN109402273A (en) * | 2018-10-29 | 2019-03-01 | 中南大学 | A kind of detection bacterium mediates the kit and method of gene expression dynamic change in figure water hydroxyl sarmientite generating process |
| CN109402273B (en) * | 2018-10-29 | 2021-08-06 | 中南大学 | Kit and method for detecting dynamic change of gene expression in process of generating bacterium-mediated map cyrtomium-arsenicum |
| CN110144362A (en) * | 2019-04-28 | 2019-08-20 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of arsenic response luciferase reporter vector and its construction method and application |
| CN110172498A (en) * | 2019-04-28 | 2019-08-27 | 广东省微生物研究所（广东省微生物分析检测中心） | A method of rapidly and efficiently analyzing transcription factor and its interaction of target DNA binding sequence |
| CN110144362B (en) * | 2019-04-28 | 2020-10-27 | 广东省微生物研究所（广东省微生物分析检测中心） | Arsenic response luciferase report vector and construction method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108064300B (en) | 2019-09-10 |
| WO2019061381A1 (en) | 2019-04-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108064300B (en) | A kind of arsenite inhibiting factor reporter plasmid and its construction method and application | |
| Azam et al. | Translational regulation by bacterial small RNAs via an unusual Hfq-dependent mechanism | |
| Yamaguchi‐Iwai et al. | Iron‐regulated DNA binding by the AFT1 protein controls the iron regulon in yeast. | |
| Christensen et al. | Establishment of new genetic traits in a microbial biofilm community | |
| Engebrecht et al. | Bacterial bioluminescence: isolation and genetic analysis of functions from Vibrio fischeri | |
| Fernandez-Lopez et al. | Unsaturated fatty acids are inhibitors of bacterial conjugation | |
| Thanbichler et al. | A comprehensive set of plasmids for vanillate-and xylose-inducible gene expression in Caulobacter crescentus | |
| Shao et al. | A codon-optimized luciferase from Gaussia princeps facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtii | |
| Geisenberger et al. | Monitoring the conjugal transfer of plasmid RP4 in activated sludge and in situ identification of the transconjugants | |
| Labella et al. | PipY, a member of the conserved COG0325 family of PLP-binding proteins, expands the cyanobacterial nitrogen regulatory network | |
| Zhang et al. | Impacts of nitrate and nitrite on physiology of Shewanella oneidensis | |
| Jones et al. | Shewanella oneidensis MR‐1 mutants selected for their inability to produce soluble organic‐Fe (III) complexes are unable to respire Fe (III) as anaerobic electron acceptor | |
| Ueki et al. | Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens | |
| Müller et al. | The FtsZ-like protein FtsZm of Magnetospirillum gryphiswaldense likely interacts with its generic homolog and is required for biomineralization under nitrate deprivation | |
| Mahlen et al. | Analyses of ampC gene expression in Serratia marcescens reveal new regulatory properties | |
| Kim et al. | Effect of iron concentration on the growth rate of Pseudomonas syringae and the expression of virulence factors in hrp-inducing minimal medium | |
| KR20020089462A (en) | Cellular Arrays for the Identification of Altered Gene Expression | |
| Yu et al. | Identification of a MarR subfamily that regulates arsenic resistance genes | |
| US5776681A (en) | Method for determining a metal present in a sample | |
| Liu et al. | Identification and application of a panel of constitutive promoters for gene overexpression in Staphylococcus aureus | |
| Garg et al. | Genes linking copper trafficking and homeostasis to the biogenesis and activity of the cbb 3-Type cytochrome c oxidase in the enteric pathogen Campylobacter jejuni | |
| Klotz et al. | A synthetic cumate-inducible promoter for graded and homogenous gene expression in Pseudomonas aeruginosa | |
| Olivan‐Muro et al. | Unbalancing Zur (FurB)‐mediated homeostasis in Anabaena sp. PCC7120: Consequences on metal trafficking, heterocyst development and biofilm formation | |
| Papadopoulos et al. | Characterisation of a putative M23-domain containing protein in Mycobacterium tuberculosis | |
| Pandeya et al. | Biotinylation as a tool to enhance the uptake of small molecules in Gram-negative bacteria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |