Whole-cell biosensor for indicating chlorpyrifos and degradation products thereof and application of whole-cell biosensor

Abstract

The invention discloses a whole-cell biosensor for indicating chlorpyrifos and degradation products thereof and application of the whole-cell biosensor. The whole-cell biosensor disclosed by the invention has the following characteristics: A1) the whole-cell biosensor contains coding genes of vioA, vioB, vioC and vioE proteins and can express the vioA, vioB, vioC and vioE proteins, the vioA protein is as shown in SEQ ID No.2, the vioB protein is as shown in SEQ ID No.3, the vioC protein is as shown in SEQ ID No.4, and the vioE protein is as shown in SEQ ID No.5; and A2) a coding gene containing the ChpR protein and capable of expressing the ChpR protein as shown in SEQ ID No.7. Experiments prove that the prepared microorganism can be used for indicating 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 a whole-cell biosensor for indicating chlorpyrifos and its degradation products in the field of biotechnology and its application. Background Art

[0002] Chlorpyrifos is a broad-spectrum chlorinated organophosphorus insecticide commonly used for pest control in agriculture and public health. The long-term and irregular application of chlorpyrifos has caused large-scale pollution of soil, sediment, water, atmosphere and other ecosystems. At the same time, chlorpyrifos and its main intermediate metabolite 3,5,6-trichloro-2-pyridinol (3,5,6-trichloro-2-pyridinol) gradually accumulate in the food chain through biomagnification, which has a negative impact on the ecosystem and affects the survival and health of soil organisms, aquatic organisms and other organisms.

[0003] The current detection methods are mainly standard analytical chromatography techniques, such as thin layer chromatography, high performance liquid chromatography, liquid chromatography-mass spectrometry combined with various detectors, etc. These traditional technologies have good selectivity, reliability, accuracy and repeatability. However, there are limitations such as slow response time, high instrument cost, and complex sample processing, which reduce its ease of use. Therefore, it is imperative to explore and develop a fast, convenient, feasible and reliable method for detecting pesticide residues in the environment. Whole-cell sensors are a flexible, simple and economical method that can simulate the actual uptake of chlorpyrifos by environmental microorganisms, and are expected to become a powerful supplement to mainstream instrumental detection methods.

[0004] Biosensing mainly uses natural pigment synthesis gene clusters as reporter gene modules to break through the bottleneck of traditional biosensor signals that are overly dependent on instrument reading. Violet bacterium is a fat-soluble pigment produced by Chromobacterium violaceum. In recent years, with the elucidation of the synthesis pathway of violet bacterium, its heterologous synthesis has become possible. There are few reports on the low-cost, fast-response, and instrument-reduced chlorpyrifos biosensor based on color change. However, there are no reports on the detection of whole-cell biosensors for chlorpyrifos and its environmental applications. Summary of the invention

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

[0006] In order to solve the above technical problems, the present invention first provides a microorganism having the following characteristics:

[0007] A1) contains the coding genes of vioA, vioB, vioC and vioE proteins and can express vioA, vioB, vioC and vioE proteins;

[0008] A2) contains the gene encoding the ChpR protein and can express the ChpR protein.

[0009] The above-mentioned microorganism may contain a vioABCE expression cassette and a ChpR expression cassette, wherein the vioABCE expression cassette contains the coding genes of vioA, vioB, vioC, and vioE proteins and a promoter for driving the expression of each coding gene, and the ChpR expression cassette contains the coding gene of the ChpR protein and a promoter for driving the expression of the coding gene of the ChpR protein.

[0010] In the above-mentioned microorganism, the vioA protein may be shown as SEQ ID No.2, the vioB protein may be shown as SEQ ID No.3, the vioC protein may be shown as SEQ ID No.4, and the vioE protein may be shown as SEQ ID No.5.

[0011] The ChpR protein may be as shown in SEQ ID No.7.

[0012] Specifically, the gene encoding the vioA protein may be as shown at positions 4-1260 of SEQ ID No.1, the gene encoding the vioB protein may be as shown at positions 1278-4274 of SEQ ID No.1, the gene encoding the vioC protein may be as shown at positions 4292-5614 of SEQ ID No.1, and the gene encoding the vioE protein may be as shown at positions 5632-6207 of SEQ ID No.1.

[0013] The gene encoding the ChpR protein may be shown as the reverse complementary sequence of positions 7 to 1464 of SEQ ID No.6.

[0014] Further, the promoter used to drive the expression of each coding gene may be as shown in positions 1465-1848 of SEQ ID No.6;

[0015] The promoter for driving the expression of the gene encoding the ChpR protein may be as shown in positions 1465-1848 of SEQ ID No.6.

[0016] The DNA fragment shown at positions 1465-1848 of SEQ ID No.6 is a bidirectional promoter, which drives the expression of vioA, vioB, vioC, and vioE genes in the direction from position 1465 to position 1848 of SEQ ID No.6, and drives the expression of ChpR gene in the opposite direction.

[0017] The microorganism can be bacteria, yeast, algae or fungi. The bacteria can be Escherichia coli.

[0018] The present invention also provides a method for preparing a recombinant microorganism, the method comprising:

[0019] B1) introducing the coding genes of vioA, vioB, vioC and vioE proteins into a recipient microorganism, and expressing the coding genes of vioA, vioB, vioC and vioE proteins;

[0020] B2) The gene encoding the ChpR protein is introduced into a recipient microorganism, and the gene encoding the ChpR protein is expressed.

[0021] The above-mentioned receptor microorganism can be bacteria, yeast, algae or fungi. The bacteria can be Escherichia coli.

[0022] The present invention also provides a method for detecting chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol, which comprises: culturing the microorganism in a sample to be tested to obtain a culture solution; extracting the obtained culture solution with n-butanol, and detecting the obtained organic phase. If the organic phase is purple, the sample to be tested contains or may contain chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol; if the organic phase is not purple, the sample to be tested does not contain or may not contain chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol.

[0023] The culturing time may be 4 hours.

[0024] The detection of the obtained organic phase can be carried out by detecting the absorbance value at 570 nm.

[0025] The present invention also provides any of the following applications:

[0026] Y1 Use of the microorganism described in the indicator of chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol;

[0027] Y2. Use of the microorganism in the preparation of an indicator product of chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol;

[0028] Y3. Application of the method in the preparation of an indicator product of chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol.

[0029] Experiments have shown that the microorganism prepared by the present invention can be used to indicate chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol, and has a good application prospect.

[0030] 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

[0031] Figure 1 Diagram of the sensing molecular mechanism of the recombinant bacteria in response to chlorpyrifos and its degradation product 3,5,6-trichloro-2-pyridinol.

[0032] Figure 2 The responses of the four recombinant bacteria to various organophosphorus. A shows the responses of the four recombinant bacteria TOP10 / pSmel-DV, TOP10 / pSfre-DV, TOP10 / pEadh-DV, and TOP10 / pPseu-DV to various organophosphorus gradient concentration pigments; B shows the photos of the extracts of the four recombinant bacteria TOP10 / pSmel-DV, TOP10 / pSfre-DV, TOP10 / pEadh-DV, and TOP10 / pPseu-DV.

[0033] Figure 3 The preliminary dose-response relationship of the two recombinant bacteria to chlorpyrifos and 3,5,6-trichloro-2-pyridinol. A is the dose-response curve of the two recombinant bacteria TOP10 / pSmel-DV and TOP10 / pPseu-DV to chlorpyrifos; B is a representative photo of the extracts of the two recombinant bacteria in response to chlorpyrifos. C is the dose-response curve of the two recombinant bacteria TOP10 / pSmel-DV and TOP10 / pPseu-DV in response to 3,5,6-trichloro-2-pyridinol. D is a representative photo of the extracts of the two recombinant bacteria in response to 3,5,6-trichloro-2-pyridinol.

[0034] Figure 4 The time-dose-response relationship of the recombinant bacteria TOP10 / pPseu-DV to 3,5,6-trichloro-2-pyridinol. A is the time-pigment accumulation curve of the recombinant bacteria TOP10 / pPseu-DV at different 3,5,6-trichloro-2-pyridinol concentrations; B is a representative photo of the recombinant bacteria extract.

[0035] Figure 5 The dose-response relationship of the recombinant bacteria TOP10 / pPseu-DV to chlorpyrifos. A is the dose-pigment accumulation curve of the TOP10 / pPseu-DV recombinant bacteria at different chlorpyrifos concentrations; B is the standard dose curve of the recombinant bacteria's response to chlorpyrifos after linear fitting; C is a representative photo of the recombinant bacteria extract.

[0036] Figure 6The dose-response relationship of the recombinant bacteria TOP10 / pPseu-DV to 3,5,6-trichloro-2-pyridinol. A is the dose-pigment accumulation curve of the recombinant bacteria TOP10 / pPseu-DV at different 3,5,6-trichloro-2-pyridinol concentrations; B is the standard dose curve of the recombinant bacteria to 3,5,6-trichloro-2-pyridinol after linear fitting; C is a representative photo of the recombinant bacteria extract.

[0037] Figure 7 The dose-response relationship of the recombinant bacteria TOP10 / pPseu-DV to 3,5,6-trichloro-2-pyridinol in tap water. A is the dose-pigment accumulation curve of the recombinant bacteria TOP10 / pPseu-DV at different 3,5,6-trichloro-2-pyridinol concentrations; B is the standard dose curve of the recombinant bacteria to 3,5,6-trichloro-2-pyridinol after linear fitting; C is a representative photo of the recombinant bacteria extract.

[0038] Figure 8 The dose-response relationship of the recombinant bacteria TOP10 / pPseu-DV to 3,5,6-trichloro-2-pyridinol in seawater. A is the dose-pigment accumulation curve of the recombinant bacteria TOP10 / pPseu-DV at different 3,5,6-trichloro-2-pyridinol concentrations; B is the standard dose curve of the recombinant bacteria to 3,5,6-trichloro-2-pyridinol after linear fitting; C is a representative photo of the recombinant bacteria extract. DETAILED DESCRIPTION

[0039] 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.

[0040] Example 1: Construction of bacterial whole-cell biosensor for chlorpyrifos and its major degradation product 3,5,6-trichloro-2-pyridinol using deoxypurpurogenous bacillus pigment as output signal

[0041] 1. Construction of recombinant plasmids pSmel-DV, pSfre-DV, pEadh-DV, and pPseu-DV

[0042] 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):

[0043] CATATG GAGCTC (SEQ ID No.1).

[0044] Construction of pSmel-DV: The DNA fragment derived from Sinorhizobiummeliloti 2011 plasmidpSymB was synthesized, and the fragment obtained after restriction digestion was connected to the vector backbone obtained by double restriction digestion of BglII and NdeI of pET-vioABCE vector. The recombinant vector containing the vioABCE gene cluster and the following DNA fragments with the correct sequence was pSmel-DV. The sequence of the DNA fragment used is as follows (underlined is the recognition sequence of BglII and NdeI, the 7th to 1602th position shows the ChpR gene, and the 1603th to 1853rd position shows the chpR-chpA spacer):

[0045] AGATCT CATATG .

[0046] Construction of pSfre-DV: The DNA fragment derived from SinorhizobiumfrediiUSDA 257 was synthesized by full gene synthesis, and was 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 pSfre-DV. The sequence of the DNA fragment used is as follows (underlined is the recognition sequence of BglII and XbaI, the 7th to 1587th position shows the ChpR gene, and the 1588th to 1831st position shows the chpR-chpA spacer):

[0047] AGATCT TCTAGA .

[0048] Construction of pEadh-DV: The whole gene was synthesized from a DNA fragment derived from EnsiferadhaerensOV14 plasmid pOV14b, which was digested with BglII and XbaI, and the obtained 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 fragments 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 1602nd position shows the ChpR gene, and the 1603rd to 1848th position shows the chpR-chpA spacer):

[0049] AGATCT TCTAGA .

[0050] 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):

[0051] AGATCT TCTAGA (SEQ ID No.6).

[0052] The molecular mechanism of recombinant plasmids pSmel-DV, pSfre-DV, pEadh-DV, and pPseu-DV is shown in the figure below. Figure 1 When chlorpyrifos or 3,5,6-trichloro-2-pyridinol molecules appear, the transcription and translation of four genes (vioA, vioB, vioC, vioE) are activated, and the four pigment synthases produced use tryptophan as a substrate to catalyze the biosynthesis of deoxyviolacein (DV). After extraction with n-butanol, purple pigment can be produced.

[0053] 2. Construction of recombinant bacteria TOP10 / pSmel-DV, TOP10 / pSfre-DV, TOP10 / pEadh-DV, and TOP10 / pPseu-DV

[0054] The recombinant plasmids pSmel-DV, pSfre-DV, pEadh-DV, and pPseu-DV constructed in step 1 were respectively introduced into the Escherichia coli chassis strain E. coli TOP10 to obtain recombinant bacteria TOP10 / pSmel-DV, TOP10 / pSfre-DV, TOP10 / pEadh-DV, and TOP10 / pPseu-DV, respectively.

[0055] 3. Screening of recombinant bacteria

[0056] (I) Selective detection of organophosphates in four whole-cell sensor chassis

[0057] 1. Centrifuge tube seed solution culture: The four whole-cell biosensors TOP10 / pSmel-DV, TOP10 / pSfre-DV, TOP10 / pEadh-DV, and TOP10 / pPseu-DV in step 2 were inoculated in LB culture medium in centrifuge tubes at a volume ratio of 1%, and cultured overnight at 37°C, 250rpm, with shaking for 12-16h to obtain seed culture solution;

[0058] 2. Organophosphorus and herbicide culture: Take the seed culture solution according to 1% (volume ratio) inoculation amount and transfer it to a centrifuge tube containing 1 mL of LB medium containing 50 μg / mL ampicillin resistance. Different concentrations of organophosphorus and herbicides (including chlorpyrifos, 3,5,6-trichloro-2-pyridinol, dichlorvos, diazinon, ethion, fenitrothion, methyl parathion, parathion in methanol, triclopyr, triclopyr 2-butoxyethyl ester, fluroxypyr, etc.) are added to the test tubes to make the concentration of each organophosphorus and herbicide 62.5 μM, 125 μM, and 250 μM, respectively. The tubes are cultured at 37°C and 250 rpm for 4 h.

[0059] 3. Response detection of four whole-cell biosensors: (1) Determination of bacterial concentration. Take 100 μL of bacterial solution in a 96-well plate and measure the absorbance at 600 nm on an ELISA reader; (2) Determination of bacterial DV pigment signal intensity. Take 900 μL of bacterial solution and add 360 μL of n-butanol (ratio 1:4), vortex for 30 seconds, centrifuge at 12000 rpm for 5 minutes, take 100 μL of the supernatant organic phase, place it in a 96-well plate, and measure the absorbance at 570 nm. The results are shown in Tables 1, 2, 3, 4 and Figure 2 shown.

[0060] Table 1. Response results of recombinant bacteria TOP10 / pSmel-DV to gradient concentrations of different organic phosphorus (cultured for 6 hours)

[0061]

[0062] Table 2. Response results of recombinant bacteria TOP10 / pSfre-DV to gradient concentrations of different organic phosphorus

[0063]

[0064] Table 3. Response results of recombinant bacteria TOP10 / pEadh-DV to gradient concentrations of different organic phosphorus

[0065]

[0066] Table 4. Response results of recombinant bacteria TOP10 / pPseu-DV to gradient concentrations of different organophosphates

[0067]

[0068] The above results show that among the four whole-cell biosensors constructed, TOP10 / pSfre-DV and pEadh-DV respond to chlorpyrifos and a variety of organophosphates and herbicides with low response signals, TOP10 / pSmel-DV responds specifically to chlorpyrifos and 3,5,6-trichloro-2-pyridinol, but not to other organophosphates, and TOP10 / pPseu-DV responds specifically to chlorpyrifos and 3,5,6-trichloro-2-pyridinol with the strongest response signal, but also responds to high concentrations of fenitrothion.

[0069] (II) Response range of two whole-cell sensor chassis cells to chlorpyrifos and 3,5,6-trichloro-2-pyridinol

[0070] 1. Centrifuge tube seed culture: The two whole-cell biosensors TOP10 / pSmel-DV and TOP10 / pPseu-DV 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 solution;

[0071] 2. Cultivation of different concentrations of chlorpyrifos: Inoculate 1% (volume ratio) of overnight culture solution into fresh LB medium (containing 50 μg / mL ampicillin), add chlorpyrifos by 2-fold dilution method to make the final concentrations of 0, 0.122, 0.244, 0.488, 0.977, 1.953, 3.906, 7.812, 15.625, 31.25, 62.5, 125, 250, 500 or 1000 μmol / L, and culture at 37°C, 250 rpm with shaking for 4 h.

[0072] 3. Cultivation of different concentrations of 3,5,6-trichloro-2-pyridinol: inoculate 1% (volume ratio) of overnight culture solution into fresh LB medium (containing 50 μg / mL ampicillin), add 3,5,6-trichloro-2-pyridinol by 2-fold dilution method to the final concentration of 0, 0.008, 0.015, 0.031, 0.061, 0.122, 0.244, 0.488, 0.977, 1.953, 3.906, 7.812, 15.625, 31.25, 62.5, 125, 250, 500 or 1000 μmol / L, and culture at 37°C, 250 rpm for 4 h.

[0073] 4. Response detection of two whole-cell biosensors: (1) Determination of bacterial concentration. Take 100 μL of bacterial solution in a 96-well plate and measure the absorbance at a wavelength of 600 nm on an ELISA reader; (2) Determination of bacterial DV pigment signal intensity. Take 900 μL of bacterial solution and add 360 μL of n-butanol (ratio 1:4), vortex for 30 seconds, centrifuge at 12000 rpm for 5 minutes, take 100 μL of the supernatant organic phase, place it in a 96-well plate, and measure the absorbance at 570 nm. The results are shown in Tables 5, 6 and Figure 3 shown.

[0074] Table 5. Dose-response relationship of two whole-cell biosensors to chlorpyrifos

[0075]

[0076] Note: “-” means no such data.

[0077] Table 6. Dose-response relationship of two whole-cell biosensors to 3,5,6-trichloro-2-pyridinol

[0078]

[0079] The above results show that TOP10 / pSmel-DV and TOP10 / pPseu-DV have a dose-response relationship for chlorpyrifos and 3,5,6-trichloro-2-pyridinol, and TOP10 / pPseu-DV has a higher sensitivity, so the recombinant bacteria TOP10 / pPseu-DV will be used in subsequent experiments. In addition, the results show that the recombinant bacteria TOP10 / pPseu-DV is more sensitive to 3,5,6-trichloro-2-pyridinol than chlorpyrifos, and subsequent studies will focus on the degradation product of chlorpyrifos, 3,5,6-trichloro-2-pyridinol.

[0080] IV. Time-dose-response relationship of TOP10 / pPseu-DV whole-cell biosensor to 3,5,6-trichloro-2-pyridinol

[0081] 1. Centrifuge tube seed culture: The whole-cell biosensor TOP10 / pPseu-DV screened in step 3 was inoculated at 1% (volume ratio) in LB culture medium in a centrifuge tube, and cultured overnight at 37°C, 250rpm, with shaking for 12-16h to obtain seed culture solution;

[0082] 2. Cultivation of 3,5,6-trichloro-2-pyridinol at different time periods: Inoculate 1% (volume ratio) of overnight culture solution into fresh LB medium (containing 50 μg / mL ampicillin), add 3,5,6-trichloro-2-pyridinol to final concentrations of 0, 0.05, and 0.5 μmol / L, respectively, and culture at 37°C, 250 rpm shaking, take samples every 1h, and store at 4°C. The sampling time is 0, 1, 2, 3, 4, 5, 6, 7, and 8 hours, respectively.

[0083] 3. Response detection of target whole-cell biosensor: (1) Determine bacterial concentration. Take 100 μL of bacterial solution in a 96-well plate and measure the absorbance at 600 nm on an ELISA reader; (2) Determine bacterial DV pigment signal intensity. Take 900 μL of bacterial solution and add 360 μL of n-butanol (ratio 1:4), vortex for 30 seconds, centrifuge at 12000 rpm for 5 minutes, take 100 μL of supernatant organic phase, place in a 96-well plate, and measure the absorbance at 570 nm. The results are shown in Table 7 and Figure 4 shown.

[0084] Table 7. Time-dose-response relationship of 3,5,6-trichloro-2-pyridinol at different time points

[0085]

[0086] The above studies show that the recombinant bacteria TOP10 / pPseu-DV has a time-response and dose-response relationship to 3,5,6-trichloro-2-pyridinol. As the induction time increases, the pigment production gradually increases, with a statistical difference at 4 hours. After 4 hours, the pigment accumulates too much, which is not conducive to direct reading and detection, so 4 hours is selected as the detection time.

[0087] 5. Dose-response relationship of TOP10 / pPseu-DV whole-cell biosensor to different concentrations of chlorpyrifos and 3,5,6-trichloro-2-pyridinol

[0088] 1. Centrifuge tube seed culture: The whole-cell biosensor TOP10 / pPseu-DV screened in step 3 was inoculated at 1% (volume ratio) in LB culture medium in a centrifuge tube, and cultured overnight at 37°C, 250rpm, with shaking for 12-16h to obtain seed culture solution;

[0089] 2. Cultivation of different concentrations of chlorpyrifos: (1) Inoculate 1% (volume ratio) of overnight culture into fresh LB medium (containing 50 μg / mL ampicillin), add chlorpyrifos by 2-fold dilution method to make the final concentration of 0, 0.122, 0.244, 0.488, 0.977, 1.953, 3.906, 7.812, 15.625, 31.25, 62.5, 125, 250, 500 or 1000 μmol / L, and culture at 37°C with shaking at 250 rpm for 4 h.

[0090] 3. Cultivation of different concentrations of 3,5,6-trichloro-2-pyridinol: inoculate 1% (volume ratio) of overnight culture solution into fresh LB medium (containing 50 μg / mL ampicillin), add 3,5,6-trichloro-2-pyridinol by 2-fold dilution method to the final concentration of 0, 0.0002, 0.0004, 0.0009, 0.0019, 0.0038, 0.0076, 0.015, 0.061, 0.122, 0.244, 0.488, 0.977, 1.953, 3.906, 7.812, 15.625, 31.25, 62.5, 125, 250, 500 or 1000 μmol / L, and culture at 37°C, 250 rpm for 4 h.

[0091] 4. Response detection of target whole-cell biosensor: (1) Determine bacterial concentration. Take 100 μL of bacterial solution in a 96-well plate and measure the absorbance at 600 nm on an ELISA reader; (2) Determine bacterial DV pigment signal intensity. Take 900 μL of bacterial solution and add 360 μL of n-butanol (ratio 1:4), vortex for 30 seconds, centrifuge at 12000 rpm for 5 minutes, take 100 μL of supernatant organic phase, place in a 96-well plate, and measure the absorbance at 570 nm. The results are shown in Table 8 and Figure 5 , 6 shown.

[0092] Table 8. Dose-response relationship of recombinant bacteria TOP10 / pPseu-DV to chlorpyrifos and 3,5,6-trichloro-2-pyridinol

[0093]

[0094] The above results show that the recombinant bacteria TOP10 / pPseu-DV responds to chlorpyrifos and its degradation product 3,5,6-trichloro-2-pyridinol, and there is an obvious dose-effect relationship. With the increase of the concentration of chlorpyrifos and 3,5,6-trichloro-2-pyridinol, the pigment production first gradually increases and then decreases. The highest point of chlorpyrifos response is about 125μmol / L, and there is a good linear relationship in the concentration range of 1.953-15.625nmol / L. Compared with 0 concentration, the difference is statistically significant from 0.488μmol / L (two-sided T test, P<0.05). The highest point of 3,5,6-trichloro-2-pyridinol response is about 1.953μmol / L, and there is a good linear relationship in the concentration range of 3.814-61.024nmol / L. Compared with 0 concentration, the difference is statistically significant from 0.95nmol / L (two-sided T test, P<0.05).

[0095] VI. Dose response of biosensors to 3,5,6-trichloro-2-pyridinol in environmental samples

[0096] 1. Centrifuge tube seed culture: The recombinant bacteria TOP10 / pPseu-DV was 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 solution;

[0097] 2. Prepare liquid LB culture medium: tap water and seawater are used as the matrix. The tap water culture system is composed of 90% volume of tap water and 10% volume of 10×LB culture medium; the seawater culture system is composed of 50% volume of seawater, 40% volume of pure water, and 10% volume of 10×LB culture medium.

[0098] 3. Cultivation of different concentrations of 3,5,6-trichloro-2-pyridinol: inoculate 1% (volume ratio) of overnight culture solution into the above two LB medium (containing 50 μg / mL ampicillin), add 3,5,6-trichloro-2-pyridinol by 2-fold dilution method to the final concentration of 0, 0.977, 1.953, 3.906, 7.812, 15.625, 31.25, 62.5, 125, 250, 500, 1000 or 2000 nmol / L, and culture at 37°C, 250 rpm with shaking for 4 h.

[0099] 4. Response detection of target whole-cell biosensor: (1) Determine bacterial concentration. Take 100 μL of bacterial solution in a 96-well plate and measure the absorbance at 600 nm on an ELISA reader; (2) Determine bacterial DV pigment signal intensity. Take 900 μL of bacterial solution and add 360 μL of n-butanol (ratio 1:4), vortex for 30 seconds, centrifuge at 12000 rpm for 5 minutes, take 100 μL of supernatant organic phase, place in a 96-well plate, and measure absorbance at 570 nm. The results are shown in Table 9 and Figure 7 , 8 shown.

[0100] Table 9. Effect of the ability of recombinant bacteria to respond to 3,5,6-trichloro-2-pyridinol in different environmental samples

[0101]

[0102] The above results show that the recombinant bacteria can respond to 3,5,6-trichloro-2-pyridinol in tap water and seawater environmental samples. There is a good linear relationship in the concentration range of 15.625-125nmol / L in tap water. Compared with 0 concentration, the difference is statistically significant from 1.95nmol / L (two-sided T test, P<0.05). In seawater, there is a good linear relationship in the concentration range of 7.628-61.024nmol / L. Compared with 0 concentration, the difference is statistically significant from 3.91nmol / L (two-sided T test, P<0.05), which has the potential for quantitative detection of environmental pollution.

[0103] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Microorganisms, Features: The microorganism has the following characteristics: A1) contains the coding genes of vioA, vioB, vioC and vioE proteins and can express vioA, vioB, vioC and vioE proteins; A2) contains the gene encoding the ChpR protein and can express the ChpR protein.

2. The microorganism according to claim 1, Features: The microorganism contains a vioABCE expression cassette and a ChpR expression cassette. The vioABCE expression cassette contains coding genes for vioA, vioB, vioC, and vioE proteins and a promoter for driving the expression of each coding gene. The ChpR expression cassette contains a coding gene for the ChpR protein and a promoter for driving the expression of the coding gene for the ChpR protein.

3. The microorganism according to claim 2, Features: The vioA protein is shown in SEQ ID No.2, the vioB protein is shown in SEQ ID No.3, the vioC protein is shown in SEQ ID No.4, and the vioE protein is shown in SEQ ID No.5; The ChpR protein is shown as SEQ ID No.

7.

4. The microorganism according to claim 2 or 3, Features: The coding gene of the vioA protein is shown in positions 4 to 1260 of SEQ ID No. 1, the coding gene of the vioB protein is shown in positions 1278 to 4274 of SEQ ID No. 1, the coding gene of the vioC protein is shown in positions 4292 to 5614 of SEQ ID No. 1, and the coding gene of the vioE protein is shown in positions 5632 to 6207 of SEQ ID No. 1; The gene encoding the ChpR protein is shown in the reverse complementary sequence of positions 7-1464 of SEQ ID No.6; further, The promoter used to drive the expression of each coding gene is shown in positions 1465-1848 of SEQ ID No.6; And / or, the promoter for driving the expression of the gene encoding the ChpR protein is as shown in positions 1465-1848 of SEQ ID No.

6.

5. A microorganism according to any one of claims 1 to 4, Features: The microorganism is bacteria, yeast, algae or fungi.

6. Method for preparing recombinant microorganisms, include: B1) introducing the coding genes of vioA, vioB, vioC and vioE proteins into a recipient microorganism, and expressing the coding genes of vioA, vioB, vioC and vioE proteins; B2) The gene encoding the ChpR protein is introduced into a recipient microorganism, and the gene encoding the ChpR protein is expressed.

7. A method for detecting chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol, include: The microorganism described in any one of claims 1 to 5 is cultured in a sample to be tested to obtain a culture solution; the obtained culture solution is extracted with n-butanol, and the obtained organic phase is detected. If the organic phase is purple, the sample to be tested contains or may contain chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol; if the organic phase is not purple, the sample to be tested does not contain or may not contain chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol.

8. Any of the following applications: Y1. Use of the microorganisms according to claims 1 to 5 in indicating chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol; Y2. Use of the microorganisms described in claims 1 to 5 in the preparation of an indicator product of chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol; Y3. Use of the method described in claim 6 in the preparation of an indicator product of chlorpyrifos or its degradation product 3,5,6-trichloro-2-pyridinol.

Images