CN114774339B - Whole-cell biosensor for detecting p-nitrophenol and detection method - Google Patents
Whole-cell biosensor for detecting p-nitrophenol and detection method Download PDFInfo
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- CN114774339B CN114774339B CN202210380660.1A CN202210380660A CN114774339B CN 114774339 B CN114774339 B CN 114774339B CN 202210380660 A CN202210380660 A CN 202210380660A CN 114774339 B CN114774339 B CN 114774339B
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- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 title claims abstract description 114
- 238000001514 detection method Methods 0.000 title abstract description 37
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- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 36
- 229930182823 kanamycin A Natural products 0.000 claims description 36
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- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 8
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- 101100190986 Acinetobacter baylyi (strain ATCC 33305 / BD413 / ADP1) pobR gene Proteins 0.000 abstract description 10
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1077—Pentosyltransferases (2.4.2)
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- 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
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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- 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
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- 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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/02—Pentosyltransferases (2.4.2)
- C12Y204/02001—Purine-nucleoside phosphorylase (2.4.2.1)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/245—Escherichia (G)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a whole-cell biosensor for detecting p-nitrophenol and a detection method, wherein the whole-cell biosensor for detecting p-nitrophenol comprises a host cell and a pPNP-mfp plasmid or a pPNP-amilCP plasmid positioned in the host cell, and the nucleotide sequence of the pPNP-mfp plasmid is shown as SEQ ID NO:1, the nucleotide sequence of the pPNP-amilCP plasmid is shown as SEQ ID NO:2, and the physical map of the pPNP-mfp plasmid and the pPNP-amilCP plasmid is shown in FIG. 1. When the whole-cell biosensor is used for detecting the extracted-state p-nitrophenol in soil, the combination of the p-nitrophenol and the repressor protein pobR eliminates the combination of the pobR and an operator locus (pobO), and the fluorescent protein or the chromoprotein gene in the opposite direction is transcribed to generate fluorescent protein or chromoprotein, so that the rapid and high-flux detection of the extracted-state p-nitrophenol in the soil is realized by detecting the fluorescent intensity value or the absorbance value.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a whole-cell biosensor for detecting p-nitrophenol and a detection method.
Background
The p-nitrophenol is an aromatic hydrocarbon compound widely applied to chemical raw materials and pharmaceutical intermediates, has long half-life in natural environment, threatens ecological environment and is considered as an environmental endocrine disrupter. At present, various detection means play an important role in the pollution risk prevention and control work of the p-nitrophenol, such as High Performance Liquid Chromatography (HPLC) and novel rapid detection methods (novel nanomaterial sensors). The existing methods cannot directly and timely reflect the comprehensive influence (bioavailability) of various toxic substances on living beings in complex environment media. As a microorganism-mediated and environment-friendly pollutant detection method, the whole-cell biosensor has obvious advantages in terms of use efficiency and ecological environment protection compared with a chemical detection method.
The full cell based biosensor has been a popular place for environmental contaminant detection and ecotoxicity assessment, and has the potential for environmental compatibility and in situ detection, which has attracted considerable attention from researchers. Currently, strains and cell lines of whole cell based biosensors are capable of detecting and reporting information about chemical substances and stress pressures, including organic compounds, xenobiotics, metals, radiation, changes in pH, etc. Due to its simple detection procedure, the application of whole cell based biosensors has been extended to the field of monitoring of contaminants in soil. The detection method established by Hansen et al has the defects of unstable reporter gene expression, long detection period (more than 1 day) and the like, and fails to provide reasonable pollutant concentration calculation formulas, detection limits, linear ranges and other detection indexes. Since a specific whole-cell biosensor has a specific response to only a certain class of chemicals, the detection of p-nitrophenol requires the development of novel biosensor strains. The current detection method of the microbial whole-cell biosensor can not meet the requirements of rapid, high-flux and accurate detection of the p-nitrophenol.
Accordingly, the prior art is still in need of improvement and development.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide a whole-cell biosensor for detecting p-nitrophenol and a detection method thereof, and aims to solve the problem that the existing detection method of the microbial whole-cell biosensor cannot meet the requirements of rapid, high-flux and accurate detection of the p-nitrophenol.
The technical scheme of the invention is as follows:
in a first aspect of the present invention, there is provided a whole cell biosensor for detecting p-nitrophenol, comprising a host cell and a pnpn-mfp plasmid or a pnpn-amilCP plasmid located in said host cell, said pnpn-mfp plasmid having a nucleotide sequence as set forth in SEQ ID NO:1, the nucleotide sequence of the pPNP-amilCP plasmid is shown as SEQ ID NO:2, and the physical map of the pPNP-mfp plasmid and the pPNP-amilCP plasmid is shown in FIG. 1.
Alternatively, the host cell is E.coli.
Optionally, the escherichia coli is e.coli BL21.
In a second aspect, the present invention provides a method for preparing a whole cell biosensor for detecting p-nitrophenol according to the present invention, comprising the steps of:
cloning the gene sequence of a pPNP gene fragment from a pET22b-YFP plasmid, cloning the gene sequence of a pobRO-m reporter gene fragment from a pMV_pobRO plasmid and cloning the gene sequence of a mrfp reporter gene fragment from a pUC19_mrfp plasmid by utilizing a primer polymerase chain reaction;
The mixture of the obtained amplified products of the pobRO-m reporter gene fragment and the mrfp reporter gene fragment is used as a template, and the pobRO-m-mrfp gene is obtained by utilizing a primer polymerase chain reaction;
connecting the pobRO-m-mrfp gene to a pClone007 skeleton to obtain a pClone007_pobRO-m-mrfp plasmid;
cloning the gene sequence of the pobRO-m-mrfp gene fragment from the pClone007_pobRO-m-mrfp plasmid;
connecting the gene sequence of the pobRO-m-mrfp gene fragment and the gene sequence of the pPNP gene fragment through homologous recombinase to obtain a connection product;
transferring the connection product into E.coli DH5 alpha competent cells to obtain E.coli DH5 alpha/pPNP-mrfp strain;
inoculating the E.coli DH5 alpha/pPNP-mrfp strain into a liquid culture medium containing kanamycin and p-nitrophenol, culturing, collecting thalli, extracting pPNP-mrfp plasmid, and introducing the extracted pPNP-mrfp plasmid into the E.coli BL21 strain by a plasmid heat shock conversion method to obtain the whole-cell biosensor E.coli BL21/pPNP-mrfp for detecting the p-nitrophenol.
Alternatively, primers used to amplify the pnp gene fragment:
the forward primer sequence is: GAGATCCGGCTGCTAACAAAGC;
The reverse primer sequence is: GGATCCGCGACCCATTTGC;
primers used for amplifying the pobRO-m reporter gene fragment:
the forward primer sequence is: TTATACCAGATTGCGCAGTTCGTTG;
the reverse primer sequence is: AAACCATTTTGGATTTGAATGTTATGATGGAACAACACCATCAGTATTTGG;
primers used for amplifying the mrfp reporter gene fragment:
the forward primer sequence is: CCAAATACTGATGGTGTTGTTCCATCATAACATTCAAATCCAAAATGGTTT;
the reverse primer sequence is: TCATTTATATAATTCGTCCATGCCAC;
the primer used for amplifying the homologous arm pobRO-m-mrfp gene fragment is as follows:
forward primer sequence: TTATACCAGATTGCGCAGTTCGTTG;
reverse primer sequence: GCTTTGTTAGCAGCCGGATCTCTCATTTATATAATTCGTCCATGCC.
In a third aspect of the present invention, there is provided another method for preparing a whole cell biosensor for detecting p-nitrophenol according to the present invention, comprising the steps of:
cloning the gene sequence of a pPNP gene fragment from a pET22b-YFP plasmid, cloning the gene sequence of a pobRO-a reporter gene fragment from a pMV_pobRO plasmid and cloning the gene sequence of an amilCP reporter gene fragment from a pUC19_amilCP plasmid by utilizing a primer polymerase chain reaction;
the mixture of the amplified products of the obtained pobRO-a reporter gene fragment and the amilCP reporter gene fragment is used as a template, and the pobRO-a-amilCP gene is obtained by utilizing a primer polymerase chain reaction;
Ligating the pobRO-a-amilCP gene to the pClone007 backbone to obtain pClone007_pobRO-a-amilCP plasmid;
cloning the gene sequence of the pobRO-a-amilCP gene fragment from the pClone007_pobRO-a-amilCP plasmid;
connecting the gene sequence of the pobRO-a-amilCP gene fragment and the gene sequence of the pPNP gene fragment through homologous recombinase to obtain a connection product;
transferring the connection product into E.coli DH5α competent cells to obtain E.coli DH5 α/pPNP-amilCP strain;
inoculating the E.coli DH5α/pPNP-amilCP strain into a liquid culture medium containing kanamycin and p-nitrophenol, culturing, collecting thalli, extracting pPNP-amilCP plasmid, and introducing the extracted pPNP-amilCP plasmid into the E.coli BL21 strain by a plasmid heat shock conversion method to obtain the whole cell biosensor E.coli BL21/pPNP-amilCP for detecting the p-nitrophenol.
Alternatively, primers used to amplify the pnp gene fragment:
the forward primer sequence is: GAGATCCGGCTGCTAACAAAGC;
the reverse primer sequence is: GGATCCGCGACCCATTTGC;
primers used for amplifying the pobRO-a reporter gene fragment:
forward primer sequence: TTATACCAGATTGCGCAGTTCGTTG;
Reverse primer sequence: ATGTATATCTCCTTGCTATTTTCTATTTT;
primers used for amplifying amilCP reporter gene fragment:
forward primer sequence: AAAATAGAAAATAGCAAGGAGATATACATATGAGTGTGATCGCTAAACAAATG;
reverse primer sequence: TTATTAGGCGACCACAGGTTTG;
the primer used for amplifying the homologous arm pobRO-a-amilCP gene fragment is as follows:
forward primer sequence: TTATACCAGATTGCGCAGTTCGTTG;
reverse primer sequence: GCTTTGTTAGCAGCCGGATCTCTTATTAGGCGACCACAGGTTT.
In a fourth aspect of the present invention, there is provided a method for detecting p-nitrophenol, comprising the steps of:
inoculating the whole cell biosensor disclosed by the invention into a culture medium containing kanamycin for culturing, collecting, and then adding the culture medium containing lactose and polymyxin B for resuscitation;
adding the resuscitated whole-cell biosensor into each small hole on the pore plate, wherein each small hole is filled with a standard sample with known concentration of the p-nitrophenol, then adding EDTA, placing the pore plate on a shaking table for incubation, measuring the fluorescence intensity value or absorbance value of each small hole of the pore plate, and establishing a standard curve equation between the concentration of the p-nitrophenol and the fluorescence intensity value or absorbance value;
Adding resuscitated whole-cell biosensor cells into each small hole on a pore plate, filling a to-be-detected p-nitrophenol sample into each small hole, adding EDTA (ethylene diamine tetraacetic acid), placing the pore plate on a shaking table for incubation, measuring the fluorescence intensity value or absorbance value of each small hole of the pore plate, and calculating to obtain the concentration of the to-be-detected p-nitrophenol sample through a standard curve equation between the p-nitrophenol concentration and the fluorescence intensity value or absorbance value.
Alternatively, the whole cell biosensor is inoculated into LB liquid medium containing 50mg/L kanamycin antibiotics, and collected after 4-5 hours of culture.
Alternatively, the ratio of the whole cell biosensor resuscitated by adding to the medium containing lactose and polymyxin B (PMB) is (3-4) mg: 100. Mu.L.
The beneficial effects are that: when the whole-cell biosensor provided by the invention is used for detecting the extracted-state parachlorophenol in soil, the combination of the parachlorophenol and the repressor protein pobR eliminates the combination of the pobR and an operator locus (pobO), and the reverse direction fluorescent protein or the color protein gene is transcribed to generate fluorescent protein or color protein, and the fluorescent intensity value or the absorbance value is detected, and the fluorescent intensity value or the absorbance value obtained by detecting different concentrations of the parachlorophenol is different, so that the rapid and high-flux detection of the extracted-state parachlorophenol in the soil is realized.
Drawings
FIG. 1 is a physical map of the DNA sequence of the pPNP-mrfp plasmid, pPNP-amilCP plasmid in the examples of the present invention.
FIG. 2 is a schematic diagram of the principle of action of the whole cell biosensor and p-nitrophenol in the embodiment of the invention.
FIG. 3 (a) shows the concentration induction curve of the whole cell biosensor E.collBL21/pPNP-mrfp according to example 1 of the present invention, and FIG. 3 (b) shows the concentration induction curve of the whole cell biosensor E.collBL21/pPNP-amiICP according to example 2 of the present invention.
FIG. 4 is a schematic diagram of the detection of p-nitrophenol using the whole cell biosensor of example 1 of the present invention.
Detailed Description
The invention provides a whole-cell biosensor for detecting p-nitrophenol and a detection method thereof, and the invention is further described in detail below for the purpose, technical scheme and effect of the invention to be clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The embodiment of the invention provides a whole-cell biosensor for detecting p-nitrophenol, which comprises a host cell and a pPNP-mrfp plasmid or a pPNP-amilCP plasmid positioned in the host cell, wherein the nucleotide sequence of the pPNP-mrfp plasmid is shown as SEQ ID NO:1, the nucleotide sequence of the pPNP-amilCP plasmid is shown as SEQ ID NO:2, and the physical map of the pPNP-mfp plasmid and the pPNP-amilCP plasmid is shown in FIG. 1.
When the whole-cell biosensor provided by the invention is used for detecting the extracted-state p-nitrophenol in soil, as shown in fig. 2, p-nitrophenol (p-NP) is combined with the repressor protein pobR, so that the combination of the pobR and an operator locus (pobO) is eliminated, a fluorescent protein or a chromoprotein gene in the opposite direction is transcribed, and the fluorescent intensity value or the absorbance value of the chromoprotein is detected, and the fluorescent intensity value or the absorbance value obtained by detecting different p-nitrophenol concentrations is different, so that the rapid and high-flux detection of the extracted-state p-nitrophenol in the soil is realized.
Wherein, the chemical structural formula of the p-nitrophenol is as follows:
in one embodiment, the host cell is E.coli.
In one embodiment, the E.coli is E.coli BL21.
The embodiment of the invention also provides a preparation method of the whole-cell biosensor for detecting p-nitrophenol, which is disclosed by the embodiment of the invention, and comprises the following steps:
s11, cloning the gene sequence of a pPNP gene fragment from a pET22b-YFP plasmid (the nucleotide sequence of which is shown as SEQ ID NO: 3), cloning the gene sequence of a pobRO-m reporter gene fragment from a pMV_pobRO plasmid (the nucleotide sequence of which is shown as SEQ ID NO: 4), and cloning the gene sequence of a mrfp reporter gene fragment from a pUC19_mrfp plasmid (the nucleotide sequence of which is shown as SEQ ID NO: 5) by utilizing a primer polymerase chain reaction;
s12, taking the obtained mixture of amplified products of the pobRO-m reporter gene fragment and the mrfp reporter gene fragment as a template, and obtaining the pobRO-m-mrfp gene by utilizing a primer polymerase chain reaction;
s13, connecting the pobRO-m-mrfp gene to a pClone007 skeleton to obtain a pClone007_pobRO-m-mrfp plasmid;
s14, cloning a gene sequence of a pobRO-m-mrfp gene fragment from the pClone007_pobRO-m-mrfp plasmid;
s15, connecting the gene sequence of the pobRO-m-mrfp gene segment and the gene sequence of the pPNP gene segment through homologous recombinase to obtain a connection product;
S16, transferring the connection product into E.collDH5α competent cells to obtain E.collDH5α/pPNP-mrfp strain;
s17, inoculating the E.coliDH5α/pPNP-mrfp strain into a liquid culture medium containing kanamycin and p-nitrophenol, culturing, collecting thalli, extracting pPNP-mrfp plasmid, and introducing the extracted pPNP-mrfp plasmid into the E.coliBL21 strain by a plasmid thermal shock conversion method to obtain the whole cell biosensor E.coliBL21/pPNP-mrfp for detecting the p-nitrophenol.
In step S11, in one embodiment, the gene sequence of the pPNP gene fragment (4677 bp) is cloned from the pET22b-YFP plasmid using primer Polymerase Chain Reaction (PCR), annealing temperature is 53 ℃; cloning the gene sequence of a pobRO-m reporter gene fragment (842 bp) from a pMV_pobRO plasmid, wherein the annealing temperature is 60 ℃; the gene sequence of the mrfp reporter fragment (860 bp) was cloned from the pUC19_mrfp plasmid, and the annealing temperature was 60 ℃.
Specifically, primers used for amplifying the pnp gene fragment:
the forward primer sequence is: GAGATCCGGCTGCTAACAAAGC;
the reverse primer sequence is: GGATCCGCGACCCATTTGC;
primers used for amplifying the pobRO-m reporter gene fragment:
the forward primer sequence is: TTATACCAGATTGCGCAGTTCGTTG;
The reverse primer sequence is: AAACCATTTTGGATTTGAATGTTATGATGGAACAACACCATCAGTATTTGG;
primers used for amplifying the mrfp reporter gene fragment:
the forward primer sequence is: CCAAATACTGATGGTGTTGTTCCATCATAACATTCAAATCCAAAATGGTTT;
the reverse primer sequence is: TCATTTATATAATTCGTCCATGCCAC;
in step S12, in one embodiment, the mixture of the amplified products of the pobRO-m reporter gene fragment and the mrfp reporter gene fragment is used as a template, and the primer polymerase chain reaction is utilized to obtain the pobRO-m-mrfp gene, wherein the volume ratio of the amplified products of the pobRO-m reporter gene fragment and the mrfp reporter gene fragment in the mixture is 1:1.
In step S13, in one embodiment, the pobRO-m-mrfp gene is ligated to the pClone007 backbone using the pClone007 Blunt Simple Vector Kit kit to obtain the pClone007_pobRO-m-mrfp plasmid.
In step S14, in one embodiment, the gene sequence of the pobRO-m-mrfp gene fragment (1694 bp) is cloned from the pClone 007-pobRO-m-mrfp plasmid at an annealing temperature of 64 ℃. Meanwhile, when designing the primer, a homologous fragment is added. Further, primers used for amplifying the homologous arm pobRO-m-mrfp gene fragment are as follows:
Forward primer sequence: TTATACCAGATTGCGCAGTTCGTTG;
reverse primer sequence: GCTTTGTTAGCAGCCGGATCTCTCATTTATATAATTCGTCCATGCC.
In step S15, in one embodiment, the gene sequence of the pobRO-m-mrfp gene fragment and the gene sequence of the pPNP gene fragment are connected through homologous recombination enzyme, the pobRO-m-mrfp gene fragment and the pPNP gene fragment are added into a PCR tube, the mixture is gently blown and mixed by a pipette, all the liquid is instantaneously centrifuged at a low speed to the bottom of a centrifuge tube, 10-100 ng of carrier is used, the molar ratio of carrier to insert is 1:1-1:10, the solid molar ratio of each fragment is 1:1, wherein pmoles= (mass ng×1000)/(fragment length bp×650 daltons), and the mixture is reacted at 50 ℃ for 15min.
In step S16, transferring the connection product into E.coli DH5 alpha competent cells to obtain E.coli DH5 alpha/pPNP-mrfp strain, which comprises the following steps:
taking 100 mu L of E.coliDH5α competent cells melted on ice bath, adding a connection product, lightly mixing, standing on ice for 25min, carrying out water bath heat shock at 42 ℃ for 30-45 s, rapidly transferring to the ice bath, standing for 2min, adding 500 mu L of sterile LB culture medium without antibiotics, mixing uniformly, resuscitating at 37 ℃ at 200rpm for 1h, absorbing resuscitating liquid with different volumes, uniformly coating on the LB culture medium with kanamycin antibiotics, and placing a flat plate in a 37 ℃ incubator for overnight culture;
Spot on the medium, draw lines on LB medium containing 50mg/L kanamycin antibiotic, LB solid medium containing 50mg/L kanamycin and 10mg/L p-nitrophenol respectively using sterile 10 μl pipette tip, screen strains that can grow on both media and grow color spots on LB solid medium containing 50mg/L kanamycin and 10mg/L p-nitrophenol, identify recombinant plasmid using primer PCR, annealing temperature is 60 ℃, verify core fragment of plasmid is 2815bp;
identifying the primers used:
forward primer sequence: GCAAATGGGTCGCGGATCC;
reverse primer sequence: GCTTTGTTAGCAGCCGGATCTC;
in step S17, inoculating the e.colldh5α/pnpn-mrfp strain to a liquid medium containing kanamycin and p-nitrophenol, culturing, collecting thalli, extracting pnpn-mrfp plasmid, and introducing the extracted pnpn-mrfp plasmid into the e.collbl 21 strain by a plasmid heat shock transformation method, thereby obtaining the whole cell biosensor e.collbl21/pnpn-mrfp for detecting p-nitrophenol, which specifically comprises the steps of:
inoculating the E.coli DH5α/pPNP-mrfp strain into LB liquid medium containing 50mg/L kanamycin and 10mg/L p-nitrophenol, culturing for 24 hours, centrifuging and collecting thalli by using 10000 Xg relative centrifugal force, extracting pPNP-mrfp plasmid by using a plasmid extraction box, and introducing the extracted pPNP-mrfp plasmid into the E.coli BL21 strain by a plasmid heat shock conversion method to obtain the whole cell biosensor E.coli BL21/pPNP-mrfp for detecting p-nitrophenol.
The embodiment of the invention also provides a preparation method of the whole-cell biosensor for detecting the p-nitrophenol, which comprises the following steps:
s21, cloning the gene sequence of a pPNP gene fragment from a pET22b-YFP plasmid (the nucleotide sequence of which is shown as SEQ ID NO: 3), cloning the gene sequence of a pobRO-a reporter gene fragment from a pMV_pobRO plasmid (the nucleotide sequence of which is shown as SEQ ID NO: 4), and cloning the gene sequence of an amilCP reporter gene fragment from a pUC19_amilCP plasmid (the nucleotide sequence of which is shown as SEQ ID NO: 6) by utilizing a primer polymerase chain reaction;
s22, taking the obtained mixture of amplified products of the pobRO-a reporter gene fragment and the amilCP reporter gene fragment as a template, and utilizing a primer polymerase chain reaction to obtain the pobRO-a-amilCP gene;
s23, connecting the pobRO-a-amilCP gene to a pClone007 framework to obtain a pClone007_pobRO-a-amilCP plasmid;
s24, cloning a gene sequence of a pobRO-a-amilCP gene fragment from the pClone007_pobRO-a-amilCP plasmid;
s25, connecting the gene sequence of the pobRO-a-amilCP gene fragment and the gene sequence of the pPNP gene fragment through homologous recombinase to obtain a connection product;
s26, transferring the connection product into E.collDH5α competent cells to obtain E.collDH5α/pPNP-amilCP strain;
S27, inoculating the E.coli DH5 alpha/pPNP-amilCP strain into a liquid culture medium containing kanamycin and p-nitrophenol, culturing, collecting thalli, extracting pPNP-amilCP plasmid, and introducing the extracted pPNP-amilCP plasmid into the E.coli BL21 strain by a plasmid thermal shock conversion method to obtain the whole cell biosensor E.coli BL21/pPNP-amilCP for detecting the p-nitrophenol.
In step S21, in one embodiment, the gene sequence of the pPNP gene fragment (4677 bp) is cloned from the pET22b-YFP plasmid by primer polymerase chain reaction at 53 ℃; cloning the gene sequence of a pobRO-a reporter gene fragment (949 bp) from a pMV_pobRO plasmid, wherein the annealing temperature is 55 ℃; the gene sequence of the amilCP reporter fragment (698 bp) was cloned from the pUC19_amilCP plasmid at an annealing temperature of 55 ℃.
Specifically, primers used for amplifying the pnp gene fragment:
the forward primer sequence is: GAGATCCGGCTGCTAACAAAGC;
the reverse primer sequence is: GGATCCGCGACCCATTTGC;
primers used for amplifying the pobRO-a reporter gene fragment:
forward primer sequence: TTATACCAGATTGCGCAGTTCGTTG;
reverse primer sequence: ATGTATATCTCCTTGCTATTTTCTATTTT;
Primers used for amplifying amilCP reporter gene fragment:
forward primer sequence: AAAATAGAAAATAGCAAGGAGATATACATATGAGTGTGATCGCTAAACAAATG;
reverse primer sequence: TTATTAGGCGACCACAGGTTTG.
In step S22, in one embodiment, the mixture of amplified products of the pobRO-a reporter fragment and the amilCP reporter fragment is used as a template, and the pobRO-a-amilCP gene is obtained by a primer polymerase chain reaction, wherein the volume ratio of the amplified products of the pobRO-a reporter fragment and the amilCP reporter fragment in the mixture is 1:1.
In step S23, in one embodiment, the pClone007_pobRO-a-amilCP plasmid is obtained by ligating the pobRO-a-amilCP gene to the pClone007 backbone using the pClone007 Blunt Simple Vector Kit kit.
In step S24, in one embodiment, the gene sequence of the pobRO-a-amilCP gene fragment (1640 bp) is cloned from the pClone007_pobRO-a-amilCP plasmid, annealing temperature is 60℃and, at the same time, a homologous fragment is added when designing the primer. Further, primers used for amplifying the homologous arm pobRO-a-amilCP gene fragment:
forward primer sequence: TTATACCAGATTGCGCAGTTCGTTG;
reverse primer sequence: GCTTTGTTAGCAGCCGGATCTCTTATTAGGCGACCACAGGTTT.
In step S25, in one embodiment, the gene sequence of the pobRO-m-amilCP gene fragment and the gene sequence of the pPNP gene fragment are connected through homologous recombinase, the pobRO-m-amilCP gene fragment and the pPNP gene fragment are added into a PCR tube, the mixture is gently blown and mixed by a pipette, all the liquid is instantaneously centrifuged at a low speed to the bottom of a centrifuge tube, 10-100 ng of carrier is used, the molar ratio of carrier to insert is 1:1-1:10, the solid molar ratio of each fragment is 1:1, wherein pmoles= (mass ng×1000)/(fragment length bp×650 daltons), and the mixture is reacted at 50 ℃ for 15min.
In step S26, the step of transferring the ligation product into e.colldh5α competent cells to obtain e.colldh5α/pnp-amilCP strain specifically includes:
taking 100 mu L of E.coliDH5α competent cells melted on ice bath, adding a connection product, lightly mixing, standing on ice for 25min, carrying out water bath heat shock at 42 ℃ for 30-45 s, rapidly transferring to the ice bath, standing for 2min, adding 500 mu L of sterile LB culture medium without antibiotics, mixing uniformly, resuscitating at 37 ℃ at 200rpm for 1h, absorbing resuscitating liquid with different volumes, uniformly coating on the LB culture medium with kanamycin antibiotics, and placing a flat plate in a 37 ℃ incubator for overnight culture;
Spots were picked up on the medium, lines were drawn on LB medium containing 50mg/L kanamycin antibiotic, LB solid medium containing 50mg/L kanamycin and 10mg/L p-nitrophenol, respectively, strains were screened which can grow on both media and color spots developed on LB solid medium containing 50mg/L kanamycin and 10mg/L p-nitrophenol, the recombinant plasmid was identified by primer PCR at an annealing temperature of 60℃and the core fragment of the plasmid was confirmed to be 2761bp.
Identifying the primers used:
forward primer sequence: GCAAATGGGTCGCGGATCC;
reverse primer sequence: GCTTTGTTAGCAGCCGGATCTC;
in step S27, inoculating the e.colldh5α/pnpn-amilCP strain to a liquid medium containing kanamycin and p-nitrophenol, culturing, collecting the bacterial cells, extracting the pnpn-amilCP plasmid, and introducing the extracted pnpn-amilCP plasmid into the e.collbl21 strain by a plasmid heat shock transformation method to obtain the whole cell biosensor e.collbl21/pnpn-amilCP for detecting p-nitrophenol, wherein the steps specifically comprise:
inoculating the E.coli DH5 alpha/pPNP-amilCP strain into LB liquid medium containing 50mg/L kanamycin and 10mg/L p-nitrophenol, culturing for 24 hours, centrifugally collecting thalli by using 10000 Xg relative centrifugal force, extracting pPNP-amilCP plasmid by using a plasmid extraction box, and introducing the extracted pPNP-amilCP plasmid into E.coli BL21 strain by a plasmid heat shock conversion method to obtain the whole cell biosensor E.coli BL21/pPNP-amilCP for detecting p-nitrophenol.
The embodiment of the invention also provides a detection method of the p-nitrophenol, which comprises the following steps:
s31, inoculating a whole cell biosensor E.coll DH5 alpha/pPNP-mrfp or E.coll DH5 alpha/pPNP-amilCP into a culture medium containing kanamycin for culture, collecting, and then adding into a culture medium containing lactose and polymyxin B (PMB) for resuscitation;
s32, adding the resuscitated whole-cell biosensor into each small hole on the pore plate, wherein a standard sample with known concentration of the p-nitrophenol is filled in each small hole, then adding EDTA, placing the pore plate on a shaking table for incubation, measuring the fluorescence intensity value or absorbance value of each small hole of the pore plate, and establishing a standard curve equation between the concentration of the p-nitrophenol and the fluorescence intensity value or absorbance value;
s33, adding the resuscitated whole-cell biosensor cells into each small hole on the pore plate, wherein each small hole is filled with a to-be-detected p-nitrophenol sample, then adding EDTA, placing the pore plate on a shaking table for incubation, measuring the fluorescence intensity value or absorbance value of each small hole of the pore plate, and calculating the concentration of the to-be-detected p-nitrophenol sample according to a standard curve equation between the p-nitrophenol concentration and the fluorescence intensity value or absorbance value.
In this embodiment, on the basis of constructing the whole-cell biosensor, EDTA and polymyxin B are used to reconstruct a whole-cell biosensor sensitive to p-nitrophenol, when the whole-cell biosensor is used to detect the extracted p-nitrophenol in soil, the binding of p-nitrophenol and the repressor protein pobR eliminates the binding of pobR and the operator site (pobO), fluorescent protein or chromoprotein genes in opposite directions are transcribed to generate fluorescent protein or chromoprotein, and p-nitrophenol with different concentrations can induce the whole-cell biosensor to generate different amounts of fluorescent protein or chromoprotein, and the relationship between the fluorescent intensity value or absorbance value and the concentration of p-nitrophenol is determined by detecting the fluorescent intensity value (fluorescent protein) or absorbance value (chromoprotein), so as to realize the rapid and high-throughput detection of the extracted p-nitrophenol in soil.
In step S31, the whole cell biosensor E.collDH5α/pPNP-mrfp or E.collDH5α/pPNP-amilCP is inoculated into LB liquid medium containing 50mg/L kanamycin antibiotics, cultured for 4-5 hours, and collected.
In step S32, the ratio of the whole cell biosensor (E.collDH5α/pPNP-mrfp or E.collDH 5 α/pPNP-amilCP) that was resuscitated by adding to the medium containing lactose and polymyxin B (PMB) was (3-4) mg: 100. Mu.L.
The invention is further illustrated by the following specific examples.
The strain and plasmid information used in the following examples are shown in Table 1.
TABLE 1 information on strains and plasmids
Example 1
Preparation of whole cell biosensor E.coli BL 21/pPNP-mrfp:
(1) Cloning a gene sequence of a pPNP gene fragment (4677 bp) of a pET22b plasmid skeleton structure from a pET22b-YFP plasmid by utilizing a primer Polymerase Chain Reaction (PCR), wherein the annealing temperature is 53 ℃;
primers used for amplifying the pPNP gene fragment:
forward primer sequence (pnpf): GAGATCCGGCTGCTAACAAAGC;
reverse primer sequence (pnpr): GGATCCGCGACCCATTTGC;
(2) Cloning the gene sequence of a pobRO-m reporter gene fragment (842 bp) from a pMV_pobRO plasmid by using a primer Polymerase Chain Reaction (PCR), wherein the annealing temperature is 60 ℃;
primers used for amplifying the pobRO-m reporter gene fragment:
forward primer sequence (pobR-mF): TTATACCAGATTGCGCAGTTCGTTG;
reverse primer sequence (pobR-mR): AAACCATTTTGGATTTGAATGTTATGATGGAACAACA
CCATCAGTATTTGG；
(3) Cloning the gene sequence of an mrfp reporter gene fragment (860 bp) from the pUC19_mrfp plasmid by using a primer Polymerase Chain Reaction (PCR), wherein the annealing temperature is 60 ℃;
primers used for amplifying the mrfp reporter gene fragment:
Forward primer sequence (mfpf): CCAAATACTGATGGTGTTGTTCCATCATAACATTCAAATCCAAAATGGTTT;
reverse primer sequence (mrfpR): TCATTTATATAATTCGTCCATGCCAC.
(4) The mixture (1:1, v/v) of amplified products of the pobRO-m reporter gene fragment and the mrfp reporter gene fragment is used as a template, and a pobR-mF/mrfpR primer is used for carrying out a primer polymerase chain reaction to obtain a pobRO-m-mrfp gene;
the primers used were:
forward primer sequence (pobR-mF): TTATACCAGATTGCGCAGTTCGTTG;
the reverse primer sequence is (mrfpR): TCATTTATATAATTCGTCCATGCCAC;
(5) The pClone007_pobRO-m-mrfp plasmid is obtained by connecting the pobRO-m-mrfp gene to the pClone007 skeleton using the pClone007 Blunt Simple Vector Kit kit;
(6) Cloning the gene sequence of a pobRO-m-mrfp gene fragment (1694 bp) from a pClone007_pobRO-m-mrfp plasmid, annealing at 64 ℃, and adding a homologous fragment when designing a primer;
the primer used for amplifying the homologous arm pobRO-m-mrfp gene fragment is as follows:
forward primer sequence (pobR-mrfp-F): TTATACCAGATTGCGCAGTTCGTTG
Reverse primer sequence (pobR-mrfp-R):
GCTTTGTTAGCAGCCGGATCTCTCATTTATATAATTCGTCCATGCC；
(7) Connecting the gene sequence of the pobRO-m-mfp gene fragment and the gene sequence of the pPNP gene fragment through homologous recombinase, adding the pobRO-m-mfp gene fragment and the pPNP gene fragment into a PCR tube, lightly blowing and mixing by a liquid-transfering device, instantly centrifuging all liquid to the bottom of a centrifuge tube at a low speed, using 100ng of carrier, the molar ratio of the carrier to the inserted fragment is 1:1, the solid molar ratio of each fragment is 1:1, wherein pmols= (mass ng multiplied by 1000)/(fragment length bp multiplied by 650 daltons), and reacting the mixed liquid at 50 ℃ for 15min to obtain a connecting product;
(8) Placing 100 mu L of E.coli DH5 alpha competent cells melted on ice bath into a centrifuge tube, adding the connection product, lightly mixing, standing on ice for 25min, carrying out heat shock in a 42 ℃ water bath for 45s, rapidly transferring to the ice bath, standing for 2min, adding 500 mu L of sterile LB culture medium without antibiotics into the centrifuge tube, mixing uniformly, resuscitating at 37 ℃ and 200rpm for 1h, absorbing resuscitating liquid with different volumes, uniformly coating the resuscitating liquid onto LB culture medium with 50mg/L kanamycin antibiotics, and inversely placing a flat plate into a 37 ℃ incubator for overnight culture;
(9) Spot on the medium, draw lines on LB medium containing 50mg/L kanamycin antibiotic, LB solid medium containing 50mg/L kanamycin and 10mg/L p-nitrophenol respectively using sterile 10 μl pipette tip, screen strains that can grow on both media and grow color spots on LB solid medium containing 50mg/L kanamycin and 10mg/L p-nitrophenol, identify recombinant plasmid using primer PCR, annealing temperature is 60 ℃, verify core fragment of plasmid is 2815bp;
and (3) identifying the adopted primers:
forward primer sequence (TF): GCAAATGGGTCGCGGATCC;
reverse primer sequence (TR): GCTTTGTTAGCAGCCGGATCTC;
and sequencing and verifying that the obtained plasmid map containing the recombinant plasmid strain E.coli DH5 alpha/pPNP-mrfp and pPNP-mrfp is shown in figure 1.
(10) Inoculating the strain into LB liquid medium containing 50mg/L kanamycin and 10mg/L p-nitrophenol by using a sterile inoculating loop, culturing for 24 hours, centrifuging and collecting thalli by using 10000 Xg relative centrifugal force, extracting pPNP-mrfp plasmid by using a plasmid extraction kit, and introducing the extracted pPNP-mrfp plasmid into E.coli BL21 strain by using a common plasmid thermal shock transformation test to obtain the whole cell biosensor E.coli BL21/pPNP-mrfp.
Example 2
Preparation of Whole cell biosensor E.coli BL 21/pPNP-amilCP:
(1) Cloning the gene sequence of a pPNP gene fragment (4677 bp) from pET22b-YFP plasmid by utilizing a primer polymerase chain reaction, wherein the annealing temperature is 53 ℃;
primers used for amplifying the pPNP gene fragment:
forward primer sequence (pnpf): GAGATCCGGCTGCTAACAAAGC;
reverse primer sequence (pnpr): GGATCCGCGACCCATTTGC;
(2) Cloning the gene sequence of a pobRO-a reporter gene fragment (949 bp) from a pMV_pobRO plasmid by utilizing a primer polymerase chain reaction, wherein the annealing temperature is 55 ℃;
primers used for amplifying the pobRO-a reporter gene fragment:
forward primer sequence (pobR-aF): TTATACCAGATTGCGCAGTTCGTTG;
reverse primer sequence (pobR-aR): ATGTATATCTCCTTGCTATTTTCTATTTT;
(3) Cloning the gene sequence of amilCP reporter gene fragment (698 bp) from pUC19_amilCP plasmid by utilizing primer polymerase chain reaction, wherein the annealing temperature is 55 ℃;
primers used for amplifying amilCP reporter gene fragment:
forward primer sequence (amilCPF): AAAATAGAAAATAGCAAGGAGATATACATAT
GAGTGTGATCGCTAAACAAATG；
Reverse primer sequence (amilcr): TTATTAGGCGACCACAGGTTTG;
(4) The mixture (1:1, v/v) of amplified products of the pobRO-a reporter gene fragment and the amilCP reporter gene fragment is used as a template, and the pobR-aF/amilCPR primer is used for carrying out a primer polymerase chain reaction to obtain the pobRO-a-amilCP gene;
the primers used were:
forward primer sequence (pobR-aF): TTATACCAGATTGCGCAGTTCGTTG
Reverse primer sequence (amilcr): AAAATAGAAAATAGCAAGGAGATATACATAT
GAGTGTGATCGCTAAACAAATG；
(5) The pClone007_pobRO-a-amilCP plasmid is obtained by connecting the pobRO-a-amilCP gene to the pClone007 skeleton using the pClone007 Blunt Simple Vector Kit kit;
(6) The gene sequence of the pobRO-a-amilCP (1640 bp) gene fragment was cloned from the pClone007_pobRO-a-amilCP plasmid, the annealing temperature was 60℃and the homologous fragment was added when designing the primer.
The primer used for amplifying the homologous arm pobRO-a-amilCP gene fragment is as follows:
forward primer sequence (pobR-amilCP-F): TTATACCAGATTGCGCAGTTCGTTG;
Reverse primer sequence (pobR-amilCP-R): GCTTTGTTAGCAGCCGGATCTCTTATTAGGCGACCACAGGTTT;
(7) Connecting the gene sequence of the pobRO-a-amilCP gene fragment and the gene sequence of the pPNP gene fragment through homologous recombinase, adding the pobRO-a-amilCP gene fragment and the pPNP gene fragment into a PCR tube, lightly blowing and mixing by a liquid-transfering device, instantly centrifuging all liquid to the bottom of a centrifuge tube at a low speed, using 100ng of carrier, the mole ratio of the carrier to the inserted fragment is 1:1, the mole ratio of each fragment is 1:1, wherein pmoles= (mass ng multiplied by 1000)/(fragment length bp multiplied by 650 daltons), and reacting the mixed liquid at 50 ℃ for 15min to obtain a connecting product;
(8) Taking 100 mu L of E.coli DH5 alpha competent cells melted on ice bath, adding the connection product, lightly mixing, standing on ice for 25min, carrying out heat shock in a water bath at 42 ℃ for 45s, rapidly transferring to the ice bath, standing for 2min, adding 500 mu L of antibiotic-free LB culture medium into a centrifuge tube, mixing uniformly, resuscitating at 37 ℃ and 200rpm for 1h, absorbing resuscitating liquid with different volumes, uniformly coating the resuscitating liquid onto the LB culture medium containing 50mg/L kanamycin antibiotics, and inversely placing a flat plate into a 37 ℃ incubator for overnight culture;
(9) Spots were picked up on the medium, lines were drawn on LB medium containing 50mg/L kanamycin antibiotic, LB solid medium containing 50mg/L kanamycin and 10mg/L p-nitrophenol, respectively, strains were screened which can grow on both media and color spots developed on LB solid medium containing 50mg/L kanamycin and 10mg/L p-nitrophenol, the recombinant plasmid was identified by primer PCR at an annealing temperature of 60℃and the core fragment of the plasmid was confirmed to be 2761bp.
And (3) identifying the adopted primers:
forward primer sequence (TF): GCAAATGGGTCGCGGATCC;
reverse primer sequence (TR): GCTTTGTTAGCAGCCGGATCTC;
and sequencing and verifying that the obtained recombinant plasmid strain E.coli DH5 alpha/pPNP-amilCP-containing plasmid map is shown in figure 1.
(10) Inoculating the strain into LB liquid medium containing 50mg/L kanamycin and 10mg/L p-nitrophenol by using a sterile inoculating loop, culturing for 24 hours, centrifuging and collecting thalli by using 10000 Xg relative centrifugal force, extracting pPNP-amilCP plasmid by using a plasmid extraction kit, and introducing the extracted pPNP-amilCP plasmid into E.coli BL21 strain by using a common plasmid thermal shock transformation test to obtain the whole cell biosensor E.coli BL21/pPNP-amilCP.
And (3) testing:
(1) As shown in FIG. 4, the whole cell biosensor E.coli BL21/pPNP-mrfp of example 1 was inoculated into LB liquid medium containing 50mg/L kanamycin antibiotic, cultured at 37℃for 4 hours with vigorous shaking at 150r/min, OD 600 nm Centrifuging at 4000 Xg relative centrifugal force for 10min to collect 0.450-0.500, adding into 1/4LB culture medium containing 10% lactose and 0.50 μg/mLPMB for resuscitation, wherein each 100 μl culture medium contains 4mg whole cell biosensor;
100. Mu.L of resuscitated whole cell biosensor was added to each well of a 96 well plate (Corning, america), each small Kong Fenzhuang. Mu.L of test sample (soil extract containing paranitrophenol standard, concentration of paranitrophenol 0, 10, 25, 50, 75, 100, 250, 500, 750, 1000, 2500, 5000, 7500, 10000. Mu.g/L, respectively) and 25. Mu.L of 125mmol/LEDTA was added, mixed well, and after covering the well, the 96 well plate was put on a shaker (300 rpm) at 30℃and incubated for 4.5 hours. Red fluorescence intensity values were measured for each well of the 96-well plates tested using a SpectraMaxM5 microplate reader and the equation y = 82.2x, r for a standard curve between p-nitrophenol concentration and fluorescence intensity values was established 2 =0.980 (x represents p-nitrophenol concentration)And y represents the measured fluorescence intensity value, R 2 The correlation coefficient in the case of linear fitting), and the results are shown in FIG. 3 (a), which shows that there is a linear correlation between the response value of the reporter gene of the whole-cell biosensor and the logarithm of the detection substrate concentration.
(2) Inoculating whole cell biosensor E.coli BL21/pPNP-amilCP of example 2 into LB liquid medium containing 50mg/L kanamycin antibiotic, culturing at 37deg.C for 4 hr, shaking vigorously at 150r/min, and OD 600 nm Centrifuging at 4000 Xg relative centrifugal force for 10min to collect at 0.450-0.500, and adding into 1/4LB medium containing 10% lactose and 0.50 μg/mLPMB for resuscitation;
100. Mu.L of resuscitated whole cell biosensor was added to each well of a 96 well plate (Corning, america), each small Kong Fenzhuang. Mu.L of test sample (soil extract containing a paranitrophenol standard sample, concentrations of paranitrophenol were 0, 10, 25, 50, 75, 100, 250, 500, 750, 1000, 2500, 5000, 7500, 10000. Mu.L of 12mmol/LEDTA, respectively), and 25. Mu.L of 12mmol/LEDTA was added, mixed uniformly, after covering the well, the 96 well plate was placed on a shaker (300 revolutions/minute) at 30℃for 4.5 hours, absorbance values were measured for each well of the 96 well plate by incubating using a SpectraMaxM5 enzyme-labeled instrument, and a standard curve equation y=0.310 x, R between the paranitrophenol concentration and absorbance values was established 2 =0.978 (x represents the logarithmic value of the concentration of p-nitrophenol, y represents the measured absorbance value, R 2 The correlation coefficient in the case of linear fitting), and the result is shown in (b) of fig. 3, which shows that there is a linear correlation between the response value of the reporter gene of the whole cell biosensor and the logarithm of the concentration of the detection substrate, the obtained standard curve can be used for analyzing the content of p-nitrophenol in the contaminated soil.
The Detection Limit (DL) of the whole-cell biosensor is calculated according to the following formula:
dl=3 SD/S, where SD is the standard deviation of the blank control and S is the slope of the standard curve.
The detection limits and linear ranges of the extractable p-nitrophenol in the soil are shown in table 2.
TABLE 2 Whole cell biosensor for measuring detection parameters of extractable Parnitrophenols in soil
Note that: mrfp represents the reporter gene of the whole cell biosensor E.coli BL21/pPNP-mrfp, iICP represents the reporter gene of the whole cell biosensor E.coli BL21/pPNP-amilCP, and HPLC represents high performance liquid chromatography.
(2) By adopting the method, S polluted by p-nitrophenol is randomly obtained 1-10 Soil leaching solution and two kinds of p-nitrophenol contaminated soil (S) collected in Nanjing A And S is B ) The leachate was tested using whole cell biosensors E.coli BL21/pPNP-mrfp and E.coli BL21/pPNP-amilCP, respectively, and the results are shown in Table 3.
TABLE 3 Whole cell biosensor determination of the concentration of Parafocal phenols in soil samples
Note that: BC and HPLC represent the p-nitrophenol concentration measured using a whole cell biosensor and high performance liquid chromatography, respectively, and DC represents the standard deviation between the p-nitrophenol concentration detected with the whole cell biosensor and the p-nitrophenol concentration measured by high performance liquid chromatography.
The result shows that the result of measuring the content of the p-nitrophenol in the soil by using the whole-cell biosensor is stable and reproducible, and the aromatic compound is reliable and feasible and has practical application value.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention can effectively detect the content of the p-nitrophenol in the soil;
(2) The invention can detect the p-nitrophenol in the soil without introducing any organic chemical reagent;
(3) The invention provides a powerful means for researching the biological effective state p-nitrophenol in soil.
In summary, the invention provides a whole-cell biosensor and a detection method for detecting p-nitrophenol, when the whole-cell biosensor is used for detecting the extracted p-nitrophenol in soil, the combination of the p-nitrophenol and a repressor protein pobR eliminates the combination of pobR and an operator locus (pobO), and the gene fluorescent protein or the chromoprotein in the opposite direction is transcribed to generate fluorescent protein or chromoprotein, so that the rapid and high-flux detection of the extracted p-nitrophenol in the soil is realized by detecting a fluorescent intensity value or an absorbance value.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
SEQUENCE LISTING
<110> Shenzhen university
<120> Whole-cell biosensor for detecting p-nitrophenol and detection method
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 7470
<212> DNA
<213> artificial sequence
<400> 1
tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 60
cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga 120
gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc 180
cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata 240
acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag 300
cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat 360
cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc 420
actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg 480
ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat 540
ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga 600
gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt 660
agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag 720
cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct 780
tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc 840
cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa 900
cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat 960
cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg 1020
ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt 1080
cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt 1140
tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta 1200
ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat 1260
gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga 1320
aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga 1380
tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt 1440
agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc 1500
ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg 1560
gcagcagcca tcatcatcat catcacagca gcggcctggt gccgcgcggc agccatatgg 1620
ctagcatgac tggtggacag caaatgggtc gcggatccat gcccctgaag aaccgcttgc 1680
tggcccgcct gtcctgtgtt gcggccgtgg tggccgccac ggccgccgtt gcaccgttga 1740
cgctggtgtc caccgcccgc gccgccgcac cgcaggtgcg cacctcggcc cccggctact 1800
accggatgct gctgggcgac ttcgaaatca ccgcgctgtc ggacggcacg gtggcgctgc 1860
cggtcgacaa gcggctgaac cagccggccc cgaagacgca gagcgcgctg gccaagtcct 1920
tccagaaagc gccgctcgaa acctcggtca ccggttacct cgtcaacacc ggctccaagc 1980
tggtgctggt ggacaccggc gcggccggcc tgttcggccc caccctgggc cggctggcgg 2040
ccaacctcaa ggccgcaggc tatcagcccg agcaggtcga cgagatctac atcacccaca 2100
tgcaccccga ccacgtgggc ggcttgatgg tgggtgagca actggcgttc ccgaacgcgg 2160
tggtgcgtgc ggaccagaaa gaagccgatt tctggctcag ccagaccaac ctcgacaagg 2220
ccccggacga cgagagcaaa ggcttcttca aaggcgccat ggcctcgctg aacccctatg 2280
tgaaggccgg caagttcaag cctttctcgg ggaacaccga cctggtgccc ggcatcaaag 2340
cgctggccag ccacggccac accccgggcc acaccaccta cgtggtcgaa agccaggggc 2400
aaaagctcgc cctgctcggc gacctgatac tcgtcgccgc ggtgcagttc gacgacccca 2460
gcgtcacgac ccagctcgac agcgacagca agtccgtcgc ggtggagcgc aagaaggcct 2520
tcgcggatgc cgccaagggc ggctacctga tcgcggcgtc ccacctgtcg ttccccggca 2580
tcggccacat ccgcgccgaa ggcaagggct accgtttcgt gccggtgaac tactcggtcg 2640
tcaaccccaa gtgataacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag 2700
caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa 2760
ggaggaacta tatccggatt tataccagat tgcgcagttc gttggcagtg ttgcgcagca 2820
acggtaacac ctggtcgatg aggtactggg gttgaacccg attagtctgg gacaggcagt 2880
tcagggctgc aatcgtcagc ccttgtgcgt tgaggactgg aaccgcaatg ccaatcacac 2940
cgagctcata ttcttccgtg gttaagcagt aatccgactg acgaacggca tccagggttt 3000
caagaaaggt gtgttcatcg gtgatggtat acggcgtcag gcgtttcagg ccatacttct 3060
cgatccactc gatctgtact tcgcgatcaa gcacgctaag aagcacttta ccggttgcga 3120
tagcatgcgc aggcagacga ttccctaagt gcatgccata cggactgagg cggttgtctt 3180
gctgaggcag ataggaacgg gcgactggaa ccacctcgtg ttcatccagg accacaatgg 3240
taaacgtcag actcgtctgc gcacacagca gattgaggaa agattgggcc actttcggca 3300
aatgcgctga actcagatag ctcgaagaga agcgcaaaac acgatgggtt aaccagaagt 3360
agtgttcgtc agtatccagg taacccagaa acttcagggt tttcagatag cgacgagctg 3420
ctgtacggct aatgccggtg cgttcagcta cctgtgtcac gtttaagcgc tgccgatcaa 3480
tgccaaacgc ttccagtaac gccagacctt ttgccagtcc cgcgatgtag tcctctgtac 3540
gaatctcttc gctcgaatgc ggatgcgcca aatactgatg gtgttgttcc ataacattca 3600
aatccaaaat ggttttgtcc gatcatcgga cagttgtaat gctaatcgga taattttgag 3660
ccttgattat agatgtcttt ttaatgaggc ggtactttaa aaatagaaaa tagcaaggag 3720
atatacatat gaattcagat atggtttcaa aaggggagga ggataatatg gcgattatca 3780
aggaatttat gcggtttaaa gtgcatatgg aagggtcggt caatggtcat gaatttgaga 3840
ttgaaggtga gggcgaaggg cgtccgtatg aaggtacgca gacggcgaag ttaaaagtca 3900
ctaagggagg tccgctgcca tttgcttggg atattttgag tccacagttc atgtatggat 3960
cgaaagctta cgtaaagcac cctgccgata ttccggatta tttgaagtta agttttccag 4020
aaggatttaa gtgggaacgt gttatgaact ttgaagacgg tggcgttgtt acagtaactc 4080
aagattcttc gttgcaagat ggtgaattta tttacaaggt taagttgcga ggtacgaatt 4140
ttccgtcaga tggaccagta atgcaaaaga aaactatggg ttgggaagcg agcagtgaac 4200
gcatgtatcc agaagatggc gctttgaaag gtgaaattaa acagcggttg aaattaaaag 4260
atggtggtca ctatgatgcg gaagtcaaaa cgacttataa agctaagaaa ccagttcaac 4320
tgccaggcgc atataacgtc aatattaagc tggatatcac gagtcataat gaagactata 4380
cgatcgtcga acagtatgaa cgagcagagg gacgacactc aacaggtggc atggacgaat 4440
tatataaatg agagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc 4500
caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt 4560
tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca 4620
ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 4680
gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 4740
caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 4800
cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt 4860
tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 4920
acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 4980
gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 5040
ttaacgttta caatttcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 5100
ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc 5160
atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg 5220
aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag 5280
atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc 5340
ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga 5400
gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc 5460
gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag 5520
acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg 5580
caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac 5640
ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg 5700
gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat 5760
ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc 5820
atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc 5880
ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga 5940
cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag 6000
ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 6060
ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 6120
tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 6180
ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag 6240
tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 6300
tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 6360
actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 6420
cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 6480
gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 6540
tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 6600
ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 6660
ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 6720
cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 6780
cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 6840
gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt 6900
cacaccgcat atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 6960
gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca 7020
cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 7080
accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg 7140
cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca 7200
tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg 7260
gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt 7320
ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact 7380
gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg 7440
cggcgggacc agagaaaaat cactcagggt 7470
<210> 2
<211> 5881
<212> DNA
<213> artificial sequence
<400> 2
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320
tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atgagtgtga tcgctaaaca aatgacctac 5100
aaggtttata tgtcaggcac ggtcaatgga cactactttg aggtcgaagg cgatggaaaa 5160
ggtaagccct acgaggggga gcagacggta aagctcactg tcaccaaggg cggacctctg 5220
ccatttgctt gggatatttt atcaccacag tgtcagtacg gaagcatacc attcaccaag 5280
taccctgaag acatccctga ctatgtaaag cagtcattcc cggagggcta tacatgggag 5340
aggatcatga actttgaaga tggtgcagtg tgtactgtca gcaatgattc cagcatccaa 5400
ggcaactgtt tcatctacca tgtcaagttc tctggtttga actttcctcc caatggacct 5460
gtcatgcaga agaagacaca gggctgggaa cccaacactg agcgtctctt tgcacgagat 5520
ggaatgctgc taggaaacaa ctttatggct ctgaagttag aaggaggcgg tcactatttg 5580
tgtgaattta aaactactta caaggcaaag aagcctgtga agatgccagg gtatcactat 5640
gttgaccgca aactggatgt aaccaatcac aacaaggatt acacttcggt tgagcagtgt 5700
gaaatttcca ttgcacgcaa acctgtggtc gcctaataac tcgaggatcc ggctgctaac 5760
aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc 5820
cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga 5880
t 5881
<210> 3
<211> 6055
<212> DNA
<213> artificial sequence
<400> 3
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320
tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100
agcagcggcc tggtgccgcg cggcagccat atggctagca tgactggtgg acagcaaatg 5160
ggtcgcggat ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 5220
gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 5280
gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 5340
ccctggccca ccctcgtgac caccttcggc tacggcctgc agtgcttcgc ccgctacccc 5400
gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 5460
cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 5520
ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 5580
atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac 5640
aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc 5700
gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 5760
cccgacaacc actacctgag ctaccagtcc gccctgagca aagaccccaa cgagaagcgc 5820
gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag 5880
ctgtacaagt aactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc 5940
cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg 6000
gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggat 6055
<210> 4
<211> 3158
<212> DNA
<213> artificial sequence
<400> 4
aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa 60
gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt 120
tgtaaaacga cggccagtcg aaccacgcaa tgcgtctcga tccgcagtgt cttgcgtctc 180
tcataacatt caaatccaaa atggttttgt ccgatcatcg gacagttgta atgctaatcg 240
gataattttg agccttgatt atagatgtct ttttaatgag gcggtacttt aaaaatagaa 300
aatagcaagg atgatgttat gaattcagat atggtttcaa aaggggagga ggataatatg 360
gcgattatca aggaatttat gcggtttaaa gtgcatatgg aagggtcggt caatggtcat 420
gaatttgaga ttgaaggtga gggcgaaggg cgtccgtatg aaggtacgca gacggcgaag 480
ttaaaagtca ctaagggagg tccgctgcca tttgcttggg atattttgag tccacagttc 540
atgtatggat cgaaagctta cgtaaagcac cctgccgata ttccggatta tttgaagtta 600
agttttccag aaggatttaa gtgggaacgt gttatgaact ttgaagacgg tggcgttgtt 660
acagtaactc aagattcttc gttgcaagat ggtgaattta tttacaaggt taagttgcga 720
ggtacgaatt ttccgtcaga tggaccagta atgcaaaaga aaactatggg ttgggaagcg 780
agcagtgaac gcatgtatcc agaagatggc gctttgaaag gtgaaattaa acagcggttg 840
aaattaaaag atggtggtca ctatgatgcg gaagtcaaaa cgacttataa agctaagaaa 900
ccagttcaac tgccaggcgc atataacgtc aatattaagc tggatatcac gagtcataat 960
gaagactata cgatcgtcga acagtatgaa cgagcagagg gacgacactc aacaggtggc 1020
atggacgaat tatataaatg attaacctag gctgctgcca ccgctgagca ataactagca 1080
taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaacc tcaggcattt 1140
gagaagcaca cggtcacaca tcggatcccg ggcccgtcga ctgcagaggc ctgcatgcaa 1200
gcttggcgta atcatggtca tagctgtttc ctggagacgg agtcactgcc aaccgagacg 1260
gtcatagctg tttcctgtgt gccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 1320
ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttaccc acagaatcag 1380
gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 1440
aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 1500
gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 1560
ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 1620
cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 1680
cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 1740
gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 1800
cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 1860
agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 1920
ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 1980
ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 2040
gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 2100
cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 2160
attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 2220
accaatgctt aatcagtgag gcacctatct cagcgatctg tctctttcgt tcatccatag 2280
ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 2340
gtgctgcaat aataccgcgg gacccacgct caccggctcc agatttatca gcaataaacc 2400
agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 2460
ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 2520
ttgttgccat cgctacaggc atcgtggtat cacgctcgtc gtttggtatg gcttcattca 2580
gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgcgc aaaaaagcgg 2640
ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgccgtg ttatcactca 2700
tggttatggc agcactacat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 2760
tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 2820
cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 2880
tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 2940
gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 3000
tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 3060
ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 3120
attgtctcat gagcggatac atatttgaat gtatttag 3158
<210> 5
<211> 3085
<212> DNA
<213> artificial sequence
<400> 5
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaatcatt tatataattc gtccatgcca cctgttgagt 180
gtcgtccctc tgctcgttca tactgttcga cgatcgtata gtcttcatta tgactcgtga 240
tatccagctt aatattgacg ttatatgcgc ctggcagttg aactggtttc ttagctttat 300
aagtcgtttt gacttccgca tcatagtgac caccatcttt taatttcaac cgctgtttaa 360
tttcaccttt caaagcgcca tcttctggat acatgcgttc actgctcgct tcccaaccca 420
tagttttctt ttgcattact ggtccatctg acggaaaatt cgtacctcgc aacttaacct 480
tgtaaataaa ttcaccatct tgcaacgaag aatcttgagt tactgtaaca acgccaccgt 540
cttcaaagtt cataacacgt tcccacttaa atccttctgg aaaacttaac ttcaaataat 600
ccggaatatc ggcagggtgc tttacgtaag ctttcgatcc atacatgaac tgtggactca 660
aaatatccca agcaaatggc agcggacctc ccttagtgac ttttaacttc gccgtctgcg 720
taccttcata cggacgccct tcgccctcac cttcaatctc aaattcatga ccattgaccg 780
acccttccat atgcacttta aaccgcataa attccttgat aatcgccata ttatcctcct 840
ccccttttga aaccatatct gaattcatag ctgtttcctg tgtgaaattg ttatccgctc 900
acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 960
gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 1020
tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 1080
cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 1140
gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 1200
aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 1260
gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 1320
aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 1380
gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 1440
ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 1500
cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 1560
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 1620
actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 1680
tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 1740
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 1800
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 1860
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 1920
ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 1980
tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 2040
agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 2100
gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 2160
ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 2220
gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 2280
cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 2340
acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 2400
cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 2460
cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 2520
ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 2580
tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 2640
atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 2700
tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 2760
actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 2820
aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 2880
ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 2940
ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 3000
cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat 3060
aggcgtatca cgaggccctt tcgtc 3085
<210> 6
<211> 3031
<212> DNA
<213> artificial sequence
<400> 6
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaattatt aggcgaccac aggtttgcgt gcaatggaaa 180
tttcacactg ctcaaccgaa gtgtaatcct tgttgtgatt ggttacatcc agtttgcggt 240
caacatagtg ataccctggc atcttcacag gcttctttgc cttgtaagta gttttaaatt 300
cacacaaata gtgaccgcct ccttctaact tcagagccat aaagttgttt cctagcagca 360
ttccatctcg tgcaaagaga cgctcagtgt tgggttccca gccctgtgtc ttcttctgca 420
tgacaggtcc attgggagga aagttcaaac cagagaactt gacatggtag atgaaacagt 480
tgccttggat gctggaatca ttgctgacag tacacactgc accatcttca aagttcatga 540
tcctctccca tgtatagccc tccgggaatg actgctttac atagtcaggg atgtcttcag 600
ggtacttggt gaatggtatg cttccgtact gacactgtgg tgataaaata tcccaagcaa 660
atggcagagg tccgcccttg gtgacagtga gctttaccgt ctgctccccc tcgtagggct 720
taccttttcc atcgccttcg acctcaaagt agtgtccatt gaccgtgcct gacatataaa 780
ccttgtaggt catttgttta gcgatcacac tcatagctgt ttcctgtgtg aaattgttat 840
ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 900
taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 960
aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 1020
attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 1080
cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 1140
gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 1200
ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 1260
agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 1320
tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 1380
ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag 1440
gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 1500
ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 1560
gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 1620
aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 1680
aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 1740
ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 1800
gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 1860
gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 1920
tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 1980
ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 2040
ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 2100
atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 2160
ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 2220
tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 2280
attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 2340
tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 2400
ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 2460
gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 2520
gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 2580
gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 2640
aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 2700
taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 2760
tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 2820
tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 2880
atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 2940
tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat 3000
aaaaataggc gtatcacgag gccctttcgt c 3031
Claims (6)
1. A whole cell biosensor for detecting p-nitrophenol, comprising a host cell and a pnpn-mfp plasmid or a pnpn-amilCP plasmid located in the host cell, wherein the nucleotide sequence of the pnpn-mfp plasmid is as shown in SEQ ID NO:1, the nucleotide sequence of the pPNP-amilCP plasmid is shown as SEQ ID NO:2 is shown in the figure;
the host cell is Escherichia coli;
the escherichia coli isE. coli BL21。
2. A method of preparing a whole cell biosensor for detecting p-nitrophenol according to claim 1, comprising the steps of:
cloning the gene sequence of a pPNP gene fragment from a pET22b-YFP plasmid, cloning the gene sequence of a pobRO-m reporter gene fragment from a pMV_pobRO plasmid and cloning the gene sequence of a mrfp reporter gene fragment from a pUC19_mrfp plasmid by utilizing a primer polymerase chain reaction;
the mixture of the obtained amplified products of the pobRO-m reporter gene fragment and the mrfp reporter gene fragment is used as a template, and the pobRO-m-mrfp gene is obtained by utilizing a primer polymerase chain reaction;
Connecting the pobRO-m-mrfp gene to a pClone007 skeleton to obtain a pClone007_pobRO-m-mrfp plasmid;
cloning the gene sequence of the pobRO-m-mrfp gene fragment from the pClone007_pobRO-m-mrfp plasmid;
connecting the gene sequence of the pobRO-m-mrfp gene fragment and the gene sequence of the pPNP gene fragment through homologous recombinase to obtain a connection product;
the ligation product was transferred to E.coliE was obtained in DH 5. Alpha. Competent cells.coliDH5 alpha/pPNP-mrfp strain;
and E, the E is carried out.coliDH5 alpha/pPNP-mrfp strain inoculationAfter culturing in a liquid medium containing kanamycin and p-nitrophenol, the cells were collected, and then the pPNP-mrfp plasmid was extracted, and the extracted pPNP-mrfp plasmid was introduced into E by a plasmid heat shock transformation method.coliIn BL21 strain, the whole cell biosensor E for detecting p-nitrophenol was obtained.coliBL21/pPNP-mrfp；
Wherein, the nucleotide sequence of the pET22b-YFP plasmid is shown as SEQ ID NO:3, the nucleotide sequence of the pMV_pobRO plasmid is shown as SEQ ID NO:4, the nucleotide sequence of the pUC19_mrfp plasmid is shown as SEQ ID NO:5 is shown in the figure;
primers used for amplifying the pPNP gene fragment:
the forward primer sequence is: GAGATCCGGCTGCTAACAAAGC;
the reverse primer sequence is: GGATCCGCGACCCATTTGC;
Primers used for amplifying the pobRO-m reporter gene fragment:
the forward primer sequence is: TTATACCAGATTGCGCAGTTCGTTG;
the reverse primer sequence is: AAACCATTTTGGATTTGAATGTTATGATGGAACAACACCATCAGTATTTGG;
primers used for amplifying the mrfp reporter gene fragment:
the forward primer sequence is: CCAAATACTGATGGTGTTGTTCCATCATAACATTCAAATCCAAAATGGTTT;
the reverse primer sequence is: TCATTTATATAATTCGTCCATGCCAC;
the primer used for amplifying the homologous arm pobRO-m-mrfp gene fragment is as follows:
forward primer sequence: TTATACCAGATTGCGCAGTTCGTTG;
reverse primer sequence: GCTTTGTTAGCAGCCGGATCTCTCATTTATATAATTCGTCCATGCC.
3. A method of preparing a whole cell biosensor for detecting p-nitrophenol according to claim 1, comprising the steps of:
cloning the gene sequence of a pPNP gene fragment from a pET22b-YFP plasmid, cloning the gene sequence of a pobRO-a reporter gene fragment from a pMV_pobRO plasmid and cloning the gene sequence of an amilCP reporter gene fragment from a pUC19_amilCP plasmid by utilizing a primer polymerase chain reaction;
the mixture of the amplified products of the obtained pobRO-a reporter gene fragment and the amilCP reporter gene fragment is used as a template, and the pobRO-a-amilCP gene is obtained by utilizing a primer polymerase chain reaction;
Ligating the pobRO-a-amilCP gene to the pClone007 backbone to obtain pClone007_pobRO-a-amilCP plasmid;
cloning the gene sequence of the pobRO-a-amilCP gene fragment from the pClone007_pobRO-a-amilCP plasmid;
connecting the gene sequence of the pobRO-a-amilCP gene fragment and the gene sequence of the pPNP gene fragment through homologous recombinase to obtain a connection product;
the ligation product was transferred to E.coli E was obtained in DH 5. Alpha. Competent cells.coliDH5 alpha/pPNP-amilCP strain;
and E, the E is carried out.coli DH5 alpha/pPNP-amilCP strain is inoculated in liquid culture medium containing kanamycin and p-nitrophenol, after culturing, bacterial cells are collected, pPNP-amilCP plasmid is extracted, and the extracted pPNP-amilCP plasmid is introduced into E by plasmid heat shock transformation method. coli In BL21 strain, the whole cell biosensor E for detecting p-nitrophenol was obtained.coliBL21/pPNP-amilCP; wherein, the nucleotide sequence of the pET22b-YFP plasmid is shown as SEQ ID NO:3, the nucleotide sequence of the pMV_pobRO plasmid is shown as SEQ ID NO:4, the nucleotide sequence of the pUC19_mrfp plasmid is shown as SEQ ID NO:5 is shown in the figure;
primers used for amplifying the pPNP gene fragment:
the forward primer sequence is: GAGATCCGGCTGCTAACAAAGC;
The reverse primer sequence is: GGATCCGCGACCCATTTGC;
primers used for amplifying the pobRO-a reporter gene fragment:
forward primer sequence: TTATACCAGATTGCGCAGTTCGTTG;
reverse primer sequence: ATGTATATCTCCTTGCTATTTTCTATTTT;
primers used for amplifying amilCP reporter gene fragment:
forward primer sequence: AAAATAGAAAATAGCAAGGAGATATACATATGAGTGTGATCGCTAAACAAATG;
reverse primer sequence: TTATTAGGCGACCACAGGTTTG;
the primer used for amplifying the homologous arm pobRO-a-amilCP gene fragment is as follows:
forward primer sequence: TTATACCAGATTGCGCAGTTCGTTG;
reverse primer sequence: GCTTTGTTAGCAGCCGGATCTCTTATTAGGCGACCACAGGTTT.
4. The method for detecting the p-nitrophenol is characterized by comprising the following steps of:
inoculating the whole cell biosensor in the culture medium containing kanamycin for culturing, collecting, and then adding the whole cell biosensor in the culture medium containing lactose and polymyxin B for resuscitation;
adding the resuscitated whole-cell biosensor into each small hole on the pore plate, wherein each small hole is filled with a standard sample with known concentration of the p-nitrophenol, then adding EDTA, placing the pore plate on a shaking table for incubation, measuring the fluorescence intensity value or absorbance value of each small hole of the pore plate, and establishing a standard curve equation between the concentration of the p-nitrophenol and the fluorescence intensity value or absorbance value;
Adding resuscitated whole-cell biosensor cells into each small hole on a pore plate, wherein each small hole is filled with a to-be-detected p-nitrophenol sample, then adding EDTA, placing the pore plate on a shaking table for incubation, measuring the fluorescence intensity value or absorbance value of each small hole of the pore plate, and calculating to obtain the concentration of the to-be-detected p-nitrophenol sample through a standard curve equation between the p-nitrophenol concentration and the fluorescence intensity value or absorbance value;
wherein, the standard curve equation is that y=82.2x, x represents the logarithmic value of the concentration of the p-nitrophenol, and y represents the fluorescence intensity value; or alternatively, the first and second heat exchangers may be,
the standard curve equation is y=0.310 x, x represents a logarithmic value of the concentration of p-nitrophenol, and y represents an absorbance value.
5. The method for detecting p-nitrophenol according to claim 4, wherein the whole cell biosensor is inoculated into LB liquid medium containing 50 mg/L kanamycin antibiotic, cultured for 4-5 hours and collected.
6. The method for detecting p-nitrophenol according to claim 5, wherein the ratio of the whole cell biosensor resuscitated by adding the whole cell biosensor to the culture medium containing lactose and polymyxin B is 3-4 mg: 100. Mu.L.
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