Engineering bacteria for adsorbing chlorpyrifos and degradation products thereof and construction method of engineering bacteria

Abstract

The invention discloses engineering bacteria for adsorbing chlorpyrifos and degradation products thereof and a construction method of the engineering bacteria. The engineering bacterium disclosed by the invention contains a coding gene of an InaPb-ChpR fusion protein and can express the InaPb-ChpR fusion protein, the InaPb-ChpR fusion protein is obtained by connecting an InaPb protein and a ChpR protein, the InaPb protein is as shown in the 1st-165th site of SEQ ID No.7, and the ChpR protein is as shown in the 171st-702nd site of SEQ ID No.7. The engineering bacterium disclosed by the invention has the advantages that the engineering bacterium can express the InaPb-ChpR fusion protein; experiments prove that the prepared microorganism can be used for adsorbing chlorpyrifos or a degradation product 3, 5, 6-trichloro-2-pyridinol of chlorpyrifos, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to an engineering bacterium for adsorbing chlorpyrifos and its degradation products in the field of biotechnology and a construction method thereof. Background Art

[0002] Pesticides are widely used around the world to control insects, weeds and plant pathogens to achieve agronomic purposes and increase crop productivity to meet food needs. Chlorpyrifos is a broad-spectrum chlorinated organophosphorus insecticide. Its long-term and irregular application has caused large-scale pollution of soil, sediment, water, atmosphere and other ecosystems. It can be lethal to fish, algae, crustaceans and shellfish even at low concentrations, and can be toxic to humans through three pathways: inhibition of acetylcholinesterase, oxidative stress and endocrine disruption, seriously endangering the survival and health of soil organisms, aquatic organisms and other organisms.

[0003] The accumulation rate of pesticides is much higher than the self-purification capacity of the environmental medium. To accelerate the degradation rate of pesticides in the environment, the main remediation technologies so far include physical, chemical, and biological remediation methods, including ultrasonic treatment, biochar adsorption, titanium dioxide photocatalysis, and synthetic nanocomposites. However, these methods are difficult and costly, and are prone to produce more toxic secondary pollutants. Therefore, they are not suitable for large-scale environmental governance of pesticide pollution. Compared with the first two, bioremediation through microorganisms is an economical, efficient, clean, and environmentally friendly method. Microbial surface display technology can use engineered microbial surfaces to display pesticide binding proteins to achieve efficient degradation and selective enrichment, which is environmentally friendly and sustainable. Customized treatment plans, diversified applications, and ecological restoration can be provided for different pesticides, providing innovative solutions for pesticide pollution control.

[0004] Natural microorganisms such as bacteria have gradually evolved a class of mechanisms that can resist extreme harmful conditions. Previous studies have shown that the chp operon identified on the endogenous plasmid pSymB of Sinorhizobium melilotii can induce specific binding to chlorpyrifos and its degradation products. Utilizing this property, an engineered strain was constructed based on the typical experimental model Escherichia coli as a microbial cell adsorbent for the removal of the organophosphate chlorpyrifos and its degradation product 3,5,6-trichloro-2-pyridinol. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to adsorb chlorpyrifos and its degradation products.

[0006] To solve the above technical problems, the present invention first provides a microorganism, which contains a gene encoding an InaPb-ChpR fusion protein and can express the InaPb-ChpR fusion protein, wherein the InaPb-ChpR fusion protein is obtained by connecting the InaPb protein and the ChpR protein.

[0007] In the above microorganism, the InaPb protein may be as shown in positions 1-165 of SEQ ID No.7, and the ChpR protein may be as shown in positions 171-702 of SEQ ID No.7.

[0008] The above-mentioned microorganism may contain an InaPb-ChpR expression cassette, wherein the InaPb-ChpR expression cassette contains a gene encoding the InaPb-ChpR fusion protein and a promoter for driving the expression of the encoding gene.

[0009] In the above microorganism, in the coding gene of the InaPb-ChpR fusion protein, the coding gene of the InaPb protein can be shown as positions 4-498 of SEQ ID No. 3. The coding gene of the ChpR protein can be shown as positions 8-1596 of SEQ ID No. 4.

[0010] In the above-mentioned microorganism, the promoter may be shown as SEQ ID No.12, SEQ ID No.11, SEQ ID No.10 or SEQ ID No.9.

[0011] The microorganisms are bacteria, yeast, algae or fungi. The bacteria may be Escherichia coli

[0012] The present invention also provides a method for preparing a recombinant microorganism, which comprises: introducing a gene encoding the InaPb-ChpR fusion protein into a recipient microorganism, and expressing the gene encoding the InaPb-ChpR fusion protein, thereby obtaining the target recombinant microorganism.

[0013] The present invention also provides a method for adsorbing chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol, which comprises: culturing the microorganism in a sample to be tested to achieve adsorption of chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol.

[0014] The culturing may be performed at 16°C.

[0015] The present invention also provides any of the following applications:

[0016] Y1. Application of the microorganism in the adsorption of chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol; Y2. Application of the microorganism in the preparation of a product adsorbing chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol; Y3. Application of the preparation method of the recombinant microorganism in the preparation of a product adsorbing chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol.

[0017] Experiments have shown that the microorganism prepared by the present invention can be used to adsorb chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol, and has a good application prospect.

[0018] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The design and molecular mechanism diagram of the E. coli cell surface display system. A is the construction of a series of anchor proteins LOT, InaPb, MipA surface display mechanism. B is the construction of E. coli that independently expresses ChpR protein intracellularly. C is the construction of a series of chlorpyrifos and 3,5,6-trichloro-2-pyridinol adsorption engineering bacteria, the anchor proteins LOT, InaPb, MipA and ChpR protein coding genes are fused, and the His-tag label is introduced at the C-terminus of the fusion protein, which is transformed into E. coli to express the recombinant protein.

[0020] Figure 2 The expression analysis of anchored proteins. A is a 15% SDS-PAGE analysis of E. coli BL21 (DE3) containing three anchoring components of pMipA, pLOT, and pInapb. B is a Western Blot analysis of E. coli BL21 (DE3) containing three anchoring components of pMipA, pLOT, and pInapb. IPTG (-) means that IPTG was not added, and IPTG (+) means that a final concentration of 1mM IPTG was added to induce protein expression. The molecular weights of the fusion proteins of the anchoring proteins MipA, LOT, and Inapb and the His-tag label are 17.8Kda, 22Kda, and 20Kda, respectively.

[0021] Figure 3For the expression analysis of intracellular ChpR protein. A is 8% SDS-PAGE analysis of E. coli BL21 (DE3) containing pET-ChpR components. B is Western Blot analysis of E. coli BL21 (DE3) containing pET-ChpR components. Lane 1 represents bacterial protein, lane 2 represents the supernatant of cells after disruption, and lane 3 represents the precipitate of cells after disruption.

[0022] Figure 4 The expression analysis of fusion proteins. A is SDS-PAGE analysis of E. coli BL21 (DE3) containing three pMipA-ChpR, pLOT-ChpR, and pInapb-ChpR. B is the outer membrane localization analysis of the fusion protein Inapb-ChpR, and SDS-PAGE analysis and Western Blot analysis were performed on E. coli BL21 (DE3) containing Inapb-ChpR. The molecular weights of the fusion proteins MipA-ChpR, LOT-ChpR, and Inapb-ChpR are 76Kda, 80Kda, and 78Kda, respectively.

[0023] Figure 5 Verification of the adsorption function of the engineered bacteria BL21(DE3) / pInaPb-ChpR. A is the residual chlorpyrifos content in the supernatant after the engineered bacteria adsorption was determined by sensor cells. B is the residual 3,5,6-trichloro-2-pyridinol content in the supernatant after the engineered bacteria adsorption was determined by sensor cells. C is the quantification of 3,5,6-trichloro-2-pyridinol residue by gas chromatography-mass spectrometry. The representative images of n-butanol extracts are shown on the right of A and B. The data are shown as the mean ± standard deviation of independent experiments (n = 3).

[0024] Figure 6 The design and molecular mechanism of the E. coli cell surface display system for environmental applications. A is an engineering bacterium constructed with E. coli as a vector, T7lac as a promoter, Inapb-ChpR as a membrane protein display, and mCherry in series. B is an engineering bacterium constructed with E. coli as a vector, a multi-level combinatorial promoter, Inapb-ChpR as a membrane protein display, and mCherry in series.

[0025] Figure 7The expression of BL21 (DE3) / pInaPb-ChpR fluorescent protein with T7lac as promoter. A. Adding different concentrations of IPTG to induce the production of ChpR protein and mcherry red fluorescent protein for SDS-PAGE analysis. B. The immunofluorescence intensity of ChpR protein and mcherry red fluorescent protein expression induced by different concentrations of IPTG. The data are shown as the mean ± standard deviation of independent experiments (n = 3). C. The photos under bright field and corresponding fluorescence microscope after the expression of ChpR protein and mcherry red fluorescent protein induced by different concentrations of IPTG, the scale bar is 5μm.

[0026] Figure 8 The expression of fusion proteins and fluorescent proteins of different gradients expressed by promoters of different strengths. A is the fluorescence expression intensity value of recombinant bacteria TOP10 / p302-InChpR-R, TOP10 / p479-InChpR-R, and TOP10 / p637-InChpR-R after cultivation. B is the bright field and corresponding fluorescence microscope photos of recombinant bacteria TOP10 / p302-InChpR-R, TOP10 / p479-InChpR-R, and TOP10 / p637-InChpR-R after protein expression, with a scale of 5 μm. The control represents the fluorescence intensity of pure TOP10 bacteria.

[0027] Fig. 9 This is to verify the adsorption function of the constitutive promoter engineered bacteria. This is to determine the remaining 3,5,6-trichloro-2-pyridinol content in the supernatant after the engineered bacteria are adsorbed by sensor cells. The control means that no bacteria are added. DETAILED DESCRIPTION

[0028] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative tests in the following examples were all repeated at least three times, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0029] Example 1: Construction of a whole-cell biosorbent for chlorpyrifos and 3,5,6-trichloro-2-pyridinol based on chpR protein

[0030] 1. Construction of recombinant plasmids pMipA, pLOT, pInaPb, pET-ChpR, pMipA-ChpR, pLOT-ChpR, and pInaPb-ChpR

[0031] Construction of recombinant plasmid pMipA: Synthesize a DNA fragment with NdeI and BamHI recognition sequences and MipA gene at both ends, digest with NdeI and BamHI, connect the obtained digested fragment with the vector backbone obtained by double digestion of pET-21a(+) vector with NdeI and BamHI, and the obtained recombinant vector containing MipA gene with correct sequence is pMipA. The sequence of the DNA fragment used is as follows (the underlined are the recognition sequences of NdeI and BamHI, and the 4th to 424th positions are the MipA gene):

[0032] CATATG ACCAAACTGAAACTGCTGGCGCTGGGCGTTCTGATCGCGACCAGCGCGGGCGTTGCACGCCGGAAGGTAAATTCAGCCTGGGCGCGGGCGTTGGCGTTGTGGAACACCCGTATAAGGATTACGACACCGACGTTTACCCAGTTCCGGTTATCAACTACGAGGGGGACAACTTCTGGTTCCGCGGTCTGGGTGGCGGTTACTATCTGTG GAACGACGCTACCGATAAGCTGTCGATTACCGCGTACTGGAGCCCGCTGTACTTCAAAGCGAAAGATTCCGGCGACCACCAGATGCGTCATCTGGATGACCGTAAATCTACCATGATGGCGGGTCTGAGCTACGCGCACTTCACCCAGTACGGTTACCTGCGTACCACTCTGGCTGGCGATACCCTGGATAACTCTAACGGCATTGTTGC GGATCC (SEQ ID No.1).

[0033] The recombinant plasmid pMipA can express the following MipA fusion protein, where positions 1-140 represent MipA proteins. The MipA fusion protein is as follows:

[0034] MTKLKLLLALGVLIATSAGVAHAEGKFSLGAGVGVVEHPYKDYDTDVYPVPVINYEGDNFWFRGLGGGYYLWNDATDKLSITAYWSPL YFKAKDSGDHQMRHLDDRKSTMMAGLSYAHFTQYGYLRTTLAGDTLDNSNGIV ADP NSSSVDKLAAALEHHHHHH .

[0035] Construction of recombinant plasmid pLOT: Synthesize a DNA fragment with NdeI and BamHI recognition sequences and LOT gene at both ends, digest with NdeI and BamHI, connect the obtained fragment with the vector backbone obtained by double digestion of pET-21a(+) vector with NdeI and BamHI, and the obtained recombinant vector containing LOT gene with correct sequence is pLOT. The sequence of the DNA fragment used is as follows (the underlined are the recognition sequences of NdeI and BamHI, and the 4th to 552nd positions are the LOT gene):

[0036] CATATG AAAGCGACCAAACTGGTTCTGGGCGCGGTTATCCTGGGTAGCACCCTGCTGGCGGGTTGCAGTAGCAACGCGAAAATCGATCAGGGCATCTTCACCCCGGATAACATCAATGCGGATATCAGCCTGGGCACCCTGAG CGGTAAAACCAAAGAACGTGTTTACCTGGCGGAAGAAGGCGGTCGTAAAGTTAGCCAGCTGGACTGGAAATTCAACAACGCGGCGATCATCAAAGGCCGATCAACTGGGATCTGATGCCGCAGATCTCTATCGGTG CGGCGGGCTGGACCACCCTGGGTAGCCGTGGCGGTAACATGGTCGATCAGGATTGGATGGATTCTAGCAACCCAGGCACCTGGACCGATGAAAGCCGTCACCCGGATACCCAATTGAACTACGCTAATGAATTTGAT CTGAACATTAAGGGGTGGCTGCTGAATGAACCTAACTACCGTCTGGGTCTGATGGCGGGTTACCAGGAAAGCCGTTACAGCTTCACCGCGCGTGGCGGTAGCTACATCTACTCTTCTGAAGAAGGCTTCCGTGATGC GGATCC (SEQ ID No. 2).

[0037] The recombinant plasmid pLOT can express the following LOT fusion protein, where positions 1-183 represent LOT protein. The LOT fusion protein is as follows:

[0038] MKATKLVLGAVILGSTLLAGCSSNAKIDQGIFTPDNINADISLGTLSGKTKERVYLAEEGGRKVSQLDWKFNNAAIIKGAINWDLMP QISIGAAGWTTLGSRGGNMVDQDWMDSSNPGTWTDESRHPDTQLNYANEFDLNIKGWLLNEPNYRLGLMAGYQESRYSFTARGGSYIYSSE EGFRD ADPNSSSVDKLAAALEHHHHHH .

[0039] Construction of recombinant plasmid pInaPb: Synthesize a DNA fragment with NdeI and BamHI recognition sequences and InaPb gene at both ends, digest with NdeI and BamHI, connect the obtained fragment with the vector backbone obtained by double digestion of pET-21a(+) vector with NdeI and BamHI, and the obtained recombinant vector containing InaPb gene with correct sequence is pInaPb. The sequence of the DNA fragment used is as follows (the underlined are the recognition sequences of NdeI and BamHI, and the 4th to 498th positions are the InaPb gene):

[0040] CATATG AACGATGATAAAGTTCTGGTTCTGCGTACCTGCGCGAACAACATGGCGGATCACTGCGGCCAGATCTGGCCGGTTAGCGGCGTTGTAGAATGCAAATACTGGGAACCGACCCGTAAACTGGAAAACGGCCTGGCGGGCCTGCTGTGGGGCAAAGGCGCGAGCACCCACCTGAACATGCAAGCTGACGCCCGCTGGGTGATCTGTGAAGTAGCAGTGTCTGACATTATCTTCCTGGACGCCCAGGGTG GCGTGAAGTTCCCGCGCGCTGAAGTGGTTCACGTAGGCACCCGTAACAGCGCGGCGGGTTACATCAGCGCGAACATCGCGAGCTACGCGAGCAGCACCGTTGCGCTGAACGAAACCTTCGTTTTCCCGGAAGTTCGTACCGAAACCAAAGTTGACTTCCCGGCGAGCCCGGCGACCGCGGATAGCACCTTCGATATTGATCGTCACGCGACCATCCAGGGCCCGCAGACCCTGGAAACCGCGGTTGC GGATCC (SEQ ID No. 3).

[0041] The recombinant plasmid pInaPb can express the following InaPb fusion protein, where positions 1-165 represent the InaPb protein, and the InaPb fusion protein is as follows:

[0042] MNDDKVLVLRTCANNMADHCGQIWPVSGVVECKYWEPTRKLENGLAGLLWGKGASTHLNMQADARWVICEVAVSDIIFLDAQGGVKF PRAEVVHVGTRNSAAGYISANIASYASSTVALNETFVFPEVRTETKVDFPASPATADSTFDIDRHATIQGPQTLETAV ADPNSSSVDKLAA ALEHHHHHH .

[0043] Construction of recombinant plasmid pET-ChpR: Synthesize a DNA fragment with NdeI and HindIII recognition sequences and ChpR gene at both ends, digest with NdeI and HindIII, connect the obtained digested fragment with the vector backbone obtained by double digestion of pET-21a(+) vector with NdeI and HindIII, and the obtained recombinant vector containing ChpR gene with correct sequence is pET-ChpR. The sequence of the DNA fragment used is as follows (the underlined are the recognition sequences of NdeI and HindIII, and the 4th to 424th positions are the ChpR gene):

[0044] CATATG AAGCTT .

[0045] Construction of recombinant plasmids pMipA-ChpR, pLOT-ChpR, and pInaPb-ChpR: Synthesize DNA fragments with EcoRI and XhoI recognition sequences and ChpR gene at both ends, digest with EcoRI and XhoI, and connect the obtained digested fragments to the vector backbone obtained by double digestion of pMipA, pLOT, and pInaPb vectors with EcoRI and XhoI, respectively. The obtained recombinant vectors with correct sequences containing MipA, LOT or InaPb genes and ChpR genes are pMipA-ChpR, pLOT-ChpR, and pInaPb-ChpR. The sequences of the DNA fragments used are as follows (underlined are the recognition sequences of EcoRI and XhoI, and the 8th to 1596th positions are the ChpR gene):

[0046] GAATTCG CTCGAG (SEQ ID No.4).

[0047] pMipA-ChpR can express the MipA-ChpR fusion protein shown in SEQ ID No.5, wherein positions 1-140 represent MipA protein, positions 146-677 represent ChpR protein, and positions 680-685 represent HIS tag. The sequence of MipA-ChpR fusion protein is as follows:

[0048]

[0049] pLOT-ChpR can express the LOT-ChpR fusion protein shown in SEQ ID No.6, where positions 1-183 represent LOT protein, positions 189-720 represent ChpR protein, and positions 723-728 represent HIS tag. The sequence of LOT-ChpR fusion protein is as follows:

[0050]

[0051] pInaPb-ChpR can express the InaPb-ChpR fusion protein shown in SEQ ID No.7, where positions 1-165 represent the InaPb protein, positions 171-702 represent the ChpR protein, and positions 705-710 represent the HIS tag. The sequence of the InaPb-ChpR fusion protein is as follows:

[0052]

[0053] Molecular mechanism diagram Figure 1 shown.

[0054] 2. Construction of recombinant bacteria BL21(DE3) / pLOT, BL21(DE3) / pMipA, BL21(DE3) / pInaPb, BL21(DE3) / pET-ChpR, BL21(DE3) / pMipA-ChpR, BL21(DE3) / pLOT-ChpR, BL21(DE3) / pInaPb-ChpR

[0055] The recombinant plasmids pMipA, pLOT, pInaPb, pET-ChpR, pMipA-ChpR, pLOT-ChpR, and pInaPb-ChpR constructed in step 1 were respectively introduced into the Escherichia coli chassis strain E. coli BL21 (DE3) to obtain recombinant bacteria BL21 (DE3) / pMipA, BL21 (DE3) / pLOT, BL21 (DE3) / pInaPb, BL21 (DE3) / pET-ChpR, BL21 (DE3) / pMipA-ChpR, BL21 (DE3) / pLOT-ChpR, and BL21 (DE3) / pInaPb-ChpR, respectively.

[0056] 3. Verification of protein expression in cell surface display system

[0057] (I) Screening and verification of three anchoring proteins

[0058] 1. Seed liquid culture: The three recombinant bacteria BL21(DE3) / pMipA, BL21(DE3) / pLOT, and BL21(DE3) / pInaPb in step 2 were inoculated at 1% (volume ratio) in a Luria-Bertani (LB) medium centrifuge tube, and cultured overnight at 37°C, 250 rpm, with shaking for 12-16 hours to obtain seed culture liquid;

[0059] 2. Add IPTG to induce culture: Take the seed culture solution and transfer it to a centrifuge tube containing 1 mL of LB medium containing 50 μg / mL ampicillin resistance at a 1% (volume ratio) inoculation amount, and culture it at 37°C, 250 rpm for 3 hours. Add IPTG to the centrifuge tube to a final concentration of 1 mM, and continue to culture at 37°C, 250 rpm for 6 hours;

[0060] 3. Collection of bacteria: Centrifuge the bacterial solution at 6500 rpm for 10 min, and wash the collected bacteria with distilled water three times;

[0061] 4. Protein electrophoresis: The cells obtained in step 3 were subjected to 15% (wt / vol) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE);

[0062] 5. Protein immunoblotting: The electrophoresis samples were transferred to polyvinylidene difluoride membranes at 30 mA for 20 min, and anti-6×His-Tag mouse monoclonal antibody was added at a dilution of 1:1000. The membranes were incubated at room temperature for 3 h and at 4°C overnight. The membranes were then rinsed three times with 0.05% (v / v) phosphate buffered saline Tween-20 (PBST) for 10 min each time. Secondary goat anti-mouse immunoglobulin IgG-horseradish peroxidase HRP conjugate was then added at a dilution of 1:2000 and incubated at room temperature for 1 h. The membranes were washed three times with PBST for 10 min each time. The antigen-antibody conjugates were detected by using ECL developer (A and B solution mixed in a 1:1 ratio) and reacted in the dark for 30 s to observe and detect the antigen-antibody conjugates.

[0063] The results are as follows Figure 2 As shown in the figure, the recombinant bacteria BL21(DE3) / pMipA, BL21(DE3) / pLOT, and BL21(DE3) / pInaPb can express the fusion proteins of the anchoring proteins MipA, LOT, Inapb and 6×His, respectively.

[0064] (II) Independent expression of chpR protein in E. coli

[0065] 1. Seed liquid culture: Inoculate BL21(DE3) / pET-ChpR in step 2 at a 1% (volume ratio) inoculum into a centrifuge tube based on LB culture medium, and culture overnight at 37°C, 250 rpm, with shaking for 12-16 hours to obtain seed culture liquid;

[0066] 2. Add IPTG to induce culture: same as step 2 in step (a);

[0067] 3. Collection of bacteria: centrifuge the bacterial solution at 6500 rpm for 10 min, wash the collected bacteria with purified water three times, and resuspend the bacteria in 10 mM Tris-HCl buffer (pH 8.8);

[0068] 4. Protein electrophoresis: The bacteria obtained in step 3 were ultrasonically disrupted for 30 min until the bacteria were transparent, centrifuged at 12000 rpm for 5 min, the supernatant and precipitate were collected, and then subjected to 8% (wt / vol) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE);

[0069] 5. Western blotting: Same as step 5 in step (a).

[0070] The results are as follows Figure 3 As shown, BL21(DE3) / pET-ChpR can express the fusion protein of ChpR and 6×His, and it is mainly concentrated in the supernatant.

[0071] (III) Screening and verification of three fusion proteins

[0072] 1. Seed liquid culture: BL21(DE3) / pMipA-ChpR, BL21(DE3) / pLOT-ChpR, and BL21(DE3) / pInaPb-ChpR prepared in step 2 were inoculated at 1% (volume ratio) in LB culture medium in a centrifuge tube, and cultured overnight at 37°C, 250 rpm, with shaking for 12-16 hours to obtain seed culture liquid;

[0073] 2. Induction culture: same as step 2 in step (a);

[0074] 3. Collection of bacteria: Same as step 3 in step (I);

[0075] 4. Protein electrophoresis: by 10% (wt / vol) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE);

[0076] The BL21(DE3) / pInaPb-ChpR cells were ultrasonically disrupted for 30 min until the cells were transparent, centrifuged at 12000 rpm for 2 min, the supernatant was centrifuged for 30 min, the supernatant and the precipitate were collected, and subjected to 10% (wt / vol) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE);

[0077] 5. Western blotting: Same as step 5 in step (a).

[0078] The results are as follows Figure 4 As shown, compared with BL21(DE3) / pMipA-ChpR and BL21(DE3) / pLOT-ChpR, the target fusion protein of BL21(DE3) / pInaPb-ChpR was successfully expressed.

[0079] 4. Verification of adsorption function of cell surface display system

[0080] (I) Adsorption of chlorpyrifos by BL21(DE3) / pInaPb-chpR fusion protein strain

[0081] 1. Preparation of whole-cell biosorbent: BL21(DE3) / pInaPb and BL21(DE3) / pInaPb-ChpR were inoculated into LB medium at 1% (volume ratio), and cultured overnight at 37°C, 250rpm, for 12-16h to obtain seed culture solution. Then, the seed culture solution was inoculated into LB medium containing 50μg / mL ampicillin resistance at 1% (volume ratio), and cultured for 3h at 37°C, 250rpm; after 3h, isopropyl β-D-thiogalactoside (IPTG) was added at a final concentration of 1mM, and cultured overnight at 16°C, 200rpm, for 12-16h. The bacterial culture solution was centrifuged at 4°C, 6500rpm for 10 minutes, and the bacteria were washed 3 times with saline, and resuspended with saline to adjust OD600 to about 0.6 to obtain a bacterial suspension.

[0082] 2. Adsorption co-culture: Take 2 ml of the obtained bacterial suspension and add it to a centrifuge tube. Centrifuge at 37°C, 3000 rpm for 10 min and discard the saline. Prepare chlorpyrifos (CPF) at a concentration of 0, 25, 50, 100, 200, 400 or 800 μmol / L by 2-fold dilution method, add 2 ml to the centrifuge tube and fully resuspend the cells. Cultivate at 16°C, 200 rpm for 1 hour to obtain the adsorbed bacterial suspension.

[0083] 3. Determination of residual chlorpyrifos content in the supernatant: (1) Whole-cell sensing detection: Centrifuge the adsorbed bacterial suspension at 4°C and 12000rpm for 10 minutes, and take out 1.9ml of the supernatant. Add 900μl of the supernatant to a centrifuge tube, add 100μl of 10×LB medium, and inoculate sensor cells TOP10 / pPseu-DV at a ratio of 1:100 (inoculated in LB medium at 1% (volume ratio), 37°C, 250rpm, shake culture overnight for 12-16h to obtain seed culture solution), and continue to culture for 4h at 37°C and 250rpm. Measure the cell density OD600 and the absorbance OD570 after extraction with n-butanol using an ELISA reader.

[0084] The preparation steps of TOP10 / pPseu-DV are as follows:

[0085] Construction of pET-vioABCE: A DNA fragment containing the vioABCE gene cluster (deoxyviolacein, DV) was synthesized by full gene synthesis, and digested with NdeI and SacI. The obtained digested fragment was connected to the vector backbone obtained by double digestion of the pET-21a(+) vector with NdeI and SacI, and the recombinant vector with the correct sequence was obtained, namely pET-vioABCE. The sequence of the DNA fragment containing the vioABCE gene cluster is as follows (the underlines at the beginning and the end are the recognition sequences of NdeI and SacI, respectively, and the 4th to 6207th positions show the vioABCE gene cluster):

[0086] CATATG GAGCTC .

[0087] Construction of pPseu-DV: The DNA fragment derived from Pseudomonas spB21-015 was synthesized by full gene synthesis, and digested with BglII and XbaI. The obtained digested fragment was connected to the vector backbone obtained by double digestion of pET-vioABCE vector with BglII and XbaI. The obtained recombinant vector containing the vioABCE gene cluster and the following DNA fragment with the correct sequence was pEadh-DV. The sequence of the DNA fragment used is as follows (underlined is the recognition sequence of BglII and XbaI, the 7th to 1464th position shows the ChpR gene, and the 1465th to 1848th position shows the chpR-chpA spacer):

[0088] AGATCT TCTAGA .

[0089] The recombinant plasmid pPseu-DV constructed in step 1 was introduced into the E. coli chassis strain E. coli TOP10 to obtain the recombinant bacteria TOP10 / pPseu-DV.

[0090] When chlorpyrifos or 3,5,6-trichloro-2-pyridinol molecules appear, the transcription and translation of four genes (vioA, vioB, vioC, vioE) are activated. The four pigment synthases produced use tryptophan as a substrate to catalyze the biosynthesis of deoxyviolacein (DV), which can produce purple pigment.

[0091] The results are shown in Table 1. Figure 5 As shown. The sensor TOP10 / pPseu-DV was used to measure the residues in the supernatant after co-culture of CPF. The results showed that the supernatant of the co-culture of the simple anchor protein strain BL21(DE3) / pInaPb was consistent with the artificially added pollution standard control, with no statistical difference (P>0.05), and had no adsorption ability. The fusion protein strain BL21(DE3) / InaPb-ChpR did not detect CPF residues in the supernatant at 25μM, and CPF residues were detected at 50-100μM, which was statistically different from the standard control (P<0.05). The fusion protein strain BL21(DE3) / InaPb-ChpR has the ability to adsorb chlorpyrifos.

[0092] Table 1. Results of A570 sensing of the adsorption supernatant residue of chlorpyrifos by the recombinant bacteria BL21(DE3) / pInaPb-ChpR

[0093]

[0094] (II) Adsorption of 3,5,6-trichloro-2-pyridinol by InaPb-chpR fusion protein strain

[0095] 1. Preparation of whole-cell biosorbent: same as step 1 in step (I);

[0096] 2. Adsorption co-culture: Take 2 ml of the obtained bacterial suspension and add it to a centrifuge tube. Centrifuge at 37°C, 3000 rpm for 10 min and discard the saline. Prepare 3,5,6-trichloro-2-pyridinol (TCP) concentrations of 0, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 μmol / L by 2-fold dilution method, add 2 ml to the centrifuge tube respectively, and fully resuspend the cells. Cultivate at 16°C, 200 rpm for 1 hour to obtain the adsorbed product.

[0097] 3. Determination of the residual content of 3,5,6-trichloro-2-pyridinol in the supernatant: (1) Whole cell sensing detection: Same as step (a) 3 (1); (2) Liquid chromatography-mass spectrometry detection, the detection conditions are as follows: Centrifuge the adsorbed product at 4°C and 12000rpm for 10 minutes, and take 1.9ml of the supernatant. Take 1ml of the supernatant and use liquid chromatography-mass spectrometry for detection. Add 5ml of ethyl acetate to 1ml of the co-culture supernatant for liquid-liquid extraction, add 0.5g Nacl, vortex for 10min, take the upper organic phase, blow nitrogen at 40° for 15min to make the volume 1ml, take 200μl, add 15μl of trimethylsilyl trifluoroacetamide (BSTFA) derivatizer, and vortex for 10min.

[0098] The results are shown in Table 2 and Table 3. Figure 5 As shown. The sensor TOP10 / pPseu-DV was used to measure the residue in the supernatant after CPF co-culture. The results showed that the simple anchor protein strain BL21(DE3) / pInaPb had no adsorption ability, while the fusion protein strain BL21(DE3) / InaPb-ChpR could adsorb TCP. TCP was not detected before 0.4μM, and TCP was completely adsorbed. The adsorption reached saturation after 1.6μM, and TCP residues were detected in the supernatant, indicating that the strain gradually reached saturation in the adsorption of TCP. As the concentration increased, the recombinant bacteria could complete the adsorption to a certain extent.

[0099] Table 2. Results of A570 sensing of the adsorption supernatant residue of 3,5,6-trichloro-2-pyridinol by the recombinant bacteria BL21(DE3) / pInaPb-chpR

[0100]

[0101] Table 3. Gas chromatography-mass spectrometry results of the adsorption of 3,5,6-trichloro-2-pyridinol by the recombinant bacteria BL21(DE3) / pInaPb-chpR in the supernatant (μmol / L)

[0102]

[0103] Example 2: Construction of a whole-cell biosorbent composed of chlorpyrifos and 3,5,6-trichloro-2-pyridinol based on chpR protein for environmental applications

[0104] 1. Construction of recombinant plasmids pT7lac-InChpR-R, p302-InChpR-R, p479-InChpR-R, and p637-InChpR-R

[0105] The insertion sequence between NdeI and Xhol of pInaPb-ChpR in Example 1 is as follows:

[0106] CATATG AACGATGATAAAGTTCTGGTTCTGCGTACCTGCGCGAACAACATGGCGGATCACTGCGGCCAGATCTGGCCGGTTAGCGGC

[0107] GTTGTAGAATGCAAATACTGGGAACCGACCCGTAAACTGGAAAACGGCCTGGCGGGCCTGCTGTGGGGCAAAGGCGCGAGCACCCACCTGA

[0108] ACATGCAAGCTGACGCCCGCTGGGTGATCTGTGAAGTAGCAGTGTCTGACATTATCTTCCTGGACGCCCAGGGTGGCGTGAAGTTCCCGCG

[0109] CGCTGAAGTGGTTCACGTAGGCACCCGTAACAGCGCGGCGGGTTACATCAGCGCGAACATCGCGAGCTACGCGAGCAGCACCGTTGCGCTG

[0110] AACGAAACCTTCGTTTTCCCGGAAGTTCGTACCGAAACCAAAGTTGACTTCCCGGCGAGCCCGGCGACCGCGGATAGCACCTTCGATATTG

[0111] ATCGTCACGCGACCATCCAGGGCCCGCAGACCCTGGAAACCGCGGTTGCGGATCC GAATTCG ATGGTGCAATTCATGTTTGGAGACTACGT

[0112] GCTCGACCAGGAGCGCCGGGAACTCACCCGGCGCGGACAGGTCGTGTCCGTCGGACCGCAGGTCTTCGACCTCCTGCTGCATCTGGTCGGC

[0113] ACCCGCGACCGTGTCGTCAGCAAGGACGAGTTGCTCCAGGCGGTGTGGGGCGGCCGCATCGTCTCGGAATCGACGATCACCAGCCACATCA

[0114] ATGCGGTTCGCAAAGCCATCGGCGACACCGGCGGGGAGCAGCGCCTGATCCGGACAGTCGCCCGCAAGGGCTTCCGCTTCGTCGGCGACAT

[0115] CAGGATCGGCGGGATCGGAGAAGTCCGACAGCCCGTCGGGCCAGGCGCCGCGTTACAGGCCTCCGGCGGATCGGGAGAAACAGCCTCCGCG

[0116] CTCGTCCTTCCGGACAAACCCTCCATCACCGTCCTGCCTTTTCAGAATCTCAGCGGTGATCCGGAGCAGGAATATTTCGCAGACGGCATCG

[0117] TGGAGGACATCATCACGGCCCTCTCGCGCATCCGCTGGCTGTTCGTCATCGCCCGCAATTCGAGTTTTAGGTATAAAGGCCGGGCGGTGGA

[0118] GGTGAAGGACGTCGGTAGGGAGCTTGGCGTGCGCTATGTGCTGGAAGGCAGCGTGCGCAAATCCGGAAACAGGGTGCGCATAACCGGGCAG

[0119] CTCATCGACGCGACGACGGGGACGCATCTCTGGGCGGAGCGCTTCGAGGGCACCCTGGACGACATTTTCGAGCTGCAGGACCGGATGGCGG

[0120] AAAGCGTCGTCGGCGCTATCGCACCTCAGGTCGAGCGGGCGGAAATGGAGCGCGCCAAGCGCAAGCCGACGGAAAGCCTCGACGCCCACGA

[0121] CTATTATCTGCGTGGAATGGCAAAACTGCACAGCGGAACCCATGAAGCAATCGAGGCGGCACTGCCCTTGTTCTATCGGGCGATCGAGCTC

[0122] GATCAGGAATTTGCGTCTGCCTATGCCGGGGCCGCCTGGTGTTACTTCTGGCGCAAGTTGAATGGCTGGATGGTCGATCGGGCCGAGGAGA

[0123] TAGCCGAGGGCGCGCGCCTGGCGCGGCGGGCAGTGGAACTTGGCCGGGACGATGCGGTGGCGCTGACAAGAAGCGGTCACGCGCTCGGACA

[0124] TCTCGTCGGCGACCTGGACGGCGGCATTGCGCTCATCGACCGGGCACGGCTGCTCAACCCCAACTTCGCCCCCGCCTGGTTCCTCGGCGGC

[0125] TTCCTGCGCGTTTTCCGCGGTGAACCGGAGAGTGCGATCGAGCATATCGAGCACGCCGCACGTCTGAGCCCCCTGGACCAGGAAATGTTCC

[0126] GGATGCAGGCCGGAACGGCACTCGCGCATTTCTTCGCCGGTCGCTTCGACTCCGCCCTGGTCTGGGCGGAAAGGGCGCTGGGAAACCTGCC

[0127] CAGCCTGCTCGTCGCGGTCGCCCTAGTGGCGGCGAGCCACGCGCTTGCCGGGCGGACGGAGGAAGCGCGAAAGACGATGCAGCGCCTGCGG

[0128] GCGCTCGACCCGTCATTGCGCGTGTCCACCCTTCGGGACTGGCTGCCGATTCATCGCCCGGAAGACCTCGCCCGATTTGCGGACGGACTGC

[0129] GGCTGGCCGGATTGCCCGAG CTCGAG 。

[0130] A sequence was inserted into the NdeI and XhoI sites of pInaPb-ChpR in Example 1 using XhoI to obtain the recombinant plasmid pT7lac-InChpR-R, wherein the DNA fragment between NdeI and Xhol of pInaPb-ChpR was replaced with the following fragment:

[0131] CATATG AACGATGATAAAGTTCTGGTTCTGCGTACCTGCGCGAACAACATGGCGGATCACTGCGGCCAGATCTGGCCGGTTAGCGGCGTTGTAGAATGCAAATACTGGGAACCGACCCGTAAACTGGAAAA CGGCCTGGCGGGCCTGCTGTGGGGCAAAGGCGCGAGCACCCACCTGAACATGCAAGCTGACGCCCGCTGGGTGATCTGTGAAGTAGCAGTGTCTGACATTATCTTCCTGGACGCCCAGGGTGGCG TGAAGTTCCCGCGCGCTGAAGTGGTTCACGTAGGCACCCGTAACAGCGCGGCGGGTTACATCAGCGCGAACATCGCGAGCTACGCGAGCAGCACCGTTGCGCTGAACGAAACCTTCGTTTTCCCGGAAGTTCGTACCGAAACCAAAGTTGACTTCCCGGCGAGCCCGGCGACCGCGGATAGCACCTTCGATATTGATCGTCACGCGACCATCCAGGGCCCGCAGACCCTGGAAACCGCGGTTGCGGATCC GAATTCG CTCGAG CACCACCACCACCACCACTGAGAAGGAGATATACATATGGTCTCTAAAGGCGAGGAAGACAACATGGCAATCATCAAAGAGTTCATGCGTTTCAAAGTGCACATGGAGGGTAGCGTCAACGGTCACGAATTTGAAATCGAAGGTGAGGGTGAAGGTCGCCCGTACGAAGGTACCCAAACCGCTAAA CTGAAAGTGACGAAAGGTGGTCCGCTGCCATTCGCATGGGATATCCTGTCTCCACAGTTCATGTACGGTTCTAAAGCGTACGTGAAACACCCGGCTGACATTCCTGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTGATGAACTTCGAGGACGGTGGCGTAGTTACTGTTA CCCAGGACTCTTCCCTGCAGGATGGTGAGTTTATCTACAAGGTTAAACTGCGTGGCACTAACTTTCCGTCCGACGGCCCGGTTATGCAGAAGAAGACTATGGGCTGGGAAGCATCTAGCGAACGTATGTATCCGGAAGATGGTGCTCTGAAAGGCGAAATCAAACAGCGTCTGAAACTGAAAGACGG CGGCCATTATGATGCGGAAGTTAAGACGACCTACAAAGCCAAGAAACCGGTTCAGCTGCCGGGCGCCTATAATGTAAACATCAAACTGGATATTACCTCCCACAACGAAGATTACACCATTGTAGAACAATATGAACGCGCGGAAGGCCGCCATAGCACCGGCGGCATGGACGAACTGTACAAAtaa CTCGAG (SEQ ID No.8).

[0132] In SEQ ID No. 8, positions 2152-2862 show the mCherrry red fluorescent protein encoding gene.

[0133] pT7lac-InChpR-R can express the InaPb-ChpR fusion protein shown in SEQ ID No.7 and the mCherrry red fluorescent protein, and the expression of the coding genes of the two proteins is driven by the T7 promoter.

[0134] The DNA fragment between the BglII and XbaI recognition sequences in pT7lac-InChpR-R (sequence: CGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCC (SEQ ID No. 9), containing T7 promoter and lac operator) was replaced with p302 promoter, p479 promoter and p637 promoter, respectively, to obtain recombinant plasmids p302-InChpR-R, p479-InChpR-R and p637-InChpR-R, respectively.

[0135] p302-InChpR-R, p479-InChpR-R, and p637-InChpR-R can all express the InaPb-ChpR fusion protein shown in SEQ ID No.7 and the mCherrry red fluorescent protein, and the expression of the coding genes of these two proteins is driven by the p302 promoter, the p479 promoter, and the p637 promoter, respectively.

[0136] p302 promoter: CAGGCCTTAGACTTTATGCTTCCGGCTCGTATGTTGTGTGG (SEQ ID No. 10).

[0137] p479 promoter: TCGAGTTAAGACTTTATGCTTCCGGCTCGTATAATGTGTGG (SEQ ID No. 11).

[0138] p637 promoter: TCGAGATTACACTTTATGCTTCCGGCTCGTATAATGTGTGG (SEQ ID No. 12).

[0139] Molecular mechanisms such as Figure 6 shown.

[0140] 2. Construction of recombinant bacteria BL21(DE3) / pT7lac-InChpR-R, TOP10 / p302-InChpR-R, TOP10 / p479-InChpR-R, and TOP10 / p637-InChpR-R

[0141] The recombinant plasmid pT7lac-InChpR-R constructed in step 1 was introduced into the Escherichia coli chassis strain E. coli BL21 (DE3) to obtain BL21 (DE3) / pT7lac-InChpR-R; the recombinant plasmids p302-InChpR-R, p479-InChpR-R, and p637-InChpR-R were respectively introduced into the Escherichia coli chassis strain E. coli TOP10 to obtain TOP10 / p302-InChpR-R, TOP10 / p479-InChpR-R, and TOP10 / p637-InChpR-R, respectively.

[0142] 3. Verification of protein expression in cell surface display system

[0143] (I) Expression and verification of adsorption protein and fluorescent protein induced by pT7lac promoter

[0144] 1. Seed liquid culture: Inoculate BL21(DE3) / pT7lac-InChpR-R in step 2 at a volume ratio of 1% (volume ratio) in LB culture medium in a centrifuge tube, and culture overnight at 37°C, 250 rpm with shaking for 12-16 hours to obtain seed culture liquid;

[0145] 2. Add IPTG to induce culture: Take the seed culture solution according to the inoculation amount of 1% (volume ratio), transfer it to a centrifuge tube containing 1mL of LB medium containing 50μg / mL ampicillin resistance, and culture it at 37℃, 250rpm for 3h. Experiment (1) Add IPTG to the centrifuge tube to the final concentration of 0, 0.001, 0.01, 0.1, 0.2, 0.4, 0.8, 1mM, and continue to culture at 37℃, 250rpm for 6h; Experiment (2) Add IPTG to the centrifuge tube to the final concentration of 0, 0.001, 0.1mM, and continue to culture at 37℃, 250rpm for 6h;

[0146] 3. Collection of bacteria: The bacterial suspensions of experiments (1) and (2) were centrifuged at 6500 rpm for 10 min respectively, and the collected bacteria were washed three times with distilled water; the bacteria of experiment (2) were resuspended with an equal volume of distilled water to obtain a bacterial suspension;

[0147] 4. Protein electrophoresis: The bacteria from experiment (1) were subjected to 15% (wt / vol) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE);

[0148] 5. Observation under fluorescence microscope: Take 20 μL of the bacterial suspension from experiment (2) and spread it evenly. Let it stand for 10 minutes and observe the bacteria under bright field and corresponding fluorescence microscope.

[0149] 6. Fluorescence intensity analysis: Take 1 ml of the bacterial suspension from experiment (2) and place it in a cuvette. Use a fluorescence spectrophotometer to measure the relative fluorescence value of the sample. The excitation wavelength of red fluorescent protein is 587 nm, and the emission wavelength is 610 nm. Take 100 μl of the bacterial suspension and place it in a 96-well plate. Use an ELISA reader to measure the bacterial concentration OD600 of the sample. Relative fluorescence value = relative fluorescence intensity / bacterial concentration OD600.

[0150] The results are shown in Table 4. Figure 7 As shown in Figure 3, the target protein was well expressed in BL21(DE3) / pT7lac-InChpR-R.

[0151] Table 4. Fluorescence intensity of recombinant bacteria BL21(DE3) / pT7lac-InChpR-R under induction of different concentrations of IPTG

[0152] IPTG (mM) <![CDATA[Relative fluorescence value (relative fluorescence intensity / OD 600 )]]> Standard Deviation 0 2320.63025 1813.77358 0.001 53946.92946 61676.9541 0.1 55359.48563 39934.54787

[0153] (II) Expression and verification of adsorption proteins and fluorescent proteins regulated by promoters of different strengths

[0154] 1. Seed liquid culture: inoculate the cells TOP10 / p302-InChpR-R, TOP10 / p479-InChpR-R, and TOP10 / p637-InChpR-R in step 2 at a 1% (volume ratio) inoculum in LB culture medium in a centrifuge tube, and culture overnight at 37°C, 250 rpm with shaking for 12-16 hours to obtain seed culture liquid;

[0155] 2. Transfer culture: Take the seed culture solution according to 1% (volume ratio) inoculation amount, transfer it to a centrifuge tube containing 1 mL of LB medium containing 50 μg / mL ampicillin resistance, and culture it at 37°C, 250 rpm for 12 hours;

[0156] 3. Collection of bacteria: Centrifuge the bacterial solution at 6500 rpm for 10 min, wash the collected bacteria with distilled water three times, and resuspend with an equal volume of distilled water to obtain a bacterial suspension;

[0157] 4. Observation under fluorescence microscope: Pipette 20 μL of bacterial suspension and spread evenly, let it stand for 10 minutes, and observe the bacteria under bright field and corresponding fluorescence microscope.

[0158] 5. Fluorescence intensity analysis: Take 1 ml of bacterial suspension in a cuvette and use a fluorescence spectrophotometer to measure the relative fluorescence value of the sample. The excitation wavelength of red fluorescent protein is 585 nm, the emission wavelength is 610 nm, and the absorbance wavelength is 600 nm; take 100 μl of bacterial suspension in a 96-well plate and use an enzyme-labeled instrument to measure the bacterial concentration OD600 of the sample. Relative fluorescence value = relative fluorescence intensity / bacterial concentration OD600.

[0159] The results are shown in Table 5. Figure 8 As shown in Figure 2, as the promoter strength increases, the expression level of the fusion protein increases, and the intensity of the tandem red fluorescent protein indicates the expression level of the fusion protein.

[0160] Table 5. Fluorescence intensity of recombinant bacteria after culture with promoters of different strengths

[0161]

[0162]

[0163] 4. Verification of adsorption function of cell surface display system

[0164] (I) Adsorption of 3,5,6-trichloro-2-pyridinol by strains with different promoter fusion protein strengths

[0165] 1. Preparation of whole-cell biosorbent: Inoculate the recombinant bacteria TOP10 / p302-InChpR-R, TOP10 / p479-InChpR-R, and TOP10 / p637-InChpR-R in LB medium at 1% (volume ratio), and culture overnight at 37°C, 250rpm, with shaking for 12-16h to obtain seed culture solution. Transfer to a centrifuge tube containing 1mL LB medium containing 50μg / mL ampicillin resistance at 1% (volume ratio), and culture for 12h at 37°C, 250rpm. Centrifuge the bacterial solution at 4°C, 6500rpm for 10 minutes, wash three times with saline, and resuspend in saline to adjust OD600 to about 0.6.

[0166] 2. Adsorption co-culture: Add 2 ml of the obtained adsorbed cells to a centrifuge tube, centrifuge at 37°C, 3000 rpm, centrifuge for 10 min, and discard the saline. Prepare 3,5,6-trichloro-2-pyridinol at a concentration of 0, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 or 3.2 μmol / L by 2-fold dilution method, add 2 ml to the centrifuge tube, and fully resuspend the cells. Cultivate at 37°C, 250 rpm for 1 hour to obtain the adsorbed product.

[0167] 3. Determination of residual chlorpyrifos content in the supernatant: Whole cell sensing detection: The adsorbed product was centrifuged at 4°C and 12000rpm for 10 minutes, 1.9ml of the supernatant was aspirated, 900μl of the supernatant was added to a centrifuge tube, 100μl of 10×LB medium was added, and sensor cells TOP10 / pPseu-DV were inoculated at a ratio of 1:100 (inoculated in LB medium at 1% (volume ratio), 37°C, 250rpm, overnight shaking culture for 12-16h to obtain seed culture solution), and culture was continued for 3h, 37°C, 250rpm. The cell density OD600 and the absorbance OD570 were measured by an ELISA instrument after extraction with n-butanol.

[0168] The results are shown in Table 6. Fig. 9 As shown in the figure, with the increase of promoter strength and the increase of fusion protein expression content, the adsorption capacity of 3,5,6-trichloro-2-pyridinol gradually increased, among which TOP10 / p637-InChpR-R had the best adsorption effect on 3,5,6-trichloro-2-pyridinol.

[0169] Table 6. Adsorption of 3,5,6-trichloro-2-pyridinol by recombinant bacteria with different promoter strengths

[0170]

[0171] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A microorganism characterized by: The microorganism contains a gene encoding an InaPb-ChpR fusion protein and can express the InaPb-ChpR fusion protein. The InaPb-ChpR fusion protein is obtained by connecting the InaPb protein and the ChpR protein.

2. The microorganism according to claim 1, characterized in that: The InaPb protein is shown as positions 1-165 of SEQ ID No.7, and the ChpR protein is shown as positions 171-702 of SEQ ID No.

7.

3. The microorganism according to claim 1 or 2, characterized in that: The microorganism contains an InaPb-ChpR expression cassette, and the InaPb-ChpR expression cassette contains a gene encoding the InaPb-ChpR fusion protein and a promoter for driving the expression of the encoding gene.

4. The microorganism according to claim 3, characterized in that: In the coding gene of the InaPb-ChpR fusion protein, the coding gene of the InaPb protein is shown in positions 4-498 of SEQ ID No. 3; And / or, the gene encoding the ChpR protein is shown in positions 8-1596 of SEQ ID No.

4.

5. The microorganism according to claim 3 or 4, characterized in that: The promoter is shown as SEQ ID No.12, SEQ ID No.11, SEQ ID No.10 or SEQ ID No.

9.

6. The microorganism according to any one of claims 1 to 5, characterized in that: The microorganism is bacteria, yeast, algae or fungi; Furthermore, the bacteria is Escherichia coli.

7. A method for preparing a recombinant microorganism, comprising: The gene encoding the InaPb-ChpR fusion protein is introduced into a recipient microorganism, and the gene encoding the InaPb-ChpR fusion protein is expressed.

8. A method for adsorbing chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol, comprising: The microorganism described in any one of claims 1 to 6 is cultured in a sample to be tested to achieve adsorption of chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol.

9. Any of the following applications: Y1. Use of the microorganisms according to claims 1 to 6 in the adsorption of chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol; Y2. Use of the microorganisms described in claims 1 to 6 in the preparation of products adsorbing chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol; Y3. Use of the method described in claim 7 in the preparation of a product adsorbing chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol.

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