CN116555143A - Short-chain chlorinated hydrocarbon induced biosensor and application thereof - Google Patents
Short-chain chlorinated hydrocarbon induced biosensor and application thereof Download PDFInfo
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- 150000008280 chlorinated hydrocarbons Chemical class 0.000 title claims abstract description 83
- 230000001580 bacterial effect Effects 0.000 claims abstract description 70
- 238000001514 detection method Methods 0.000 claims abstract description 29
- 230000006698 induction Effects 0.000 claims abstract description 21
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 18
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- 238000000034 method Methods 0.000 claims description 8
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- 239000000047 product Substances 0.000 claims description 7
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- QPFMBZIOSGYJDE-UHFFFAOYSA-N 1,1,2,2-tetrachloroethane Chemical compound ClC(Cl)C(Cl)Cl QPFMBZIOSGYJDE-UHFFFAOYSA-N 0.000 claims description 4
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- 238000012257 pre-denaturation Methods 0.000 claims description 3
- UOCLXMDMGBRAIB-UHFFFAOYSA-N 1,1,1-trichloroethane Chemical compound CC(Cl)(Cl)Cl UOCLXMDMGBRAIB-UHFFFAOYSA-N 0.000 claims description 2
- CYTYCFOTNPOANT-UHFFFAOYSA-N Perchloroethylene Chemical group ClC(Cl)=C(Cl)Cl CYTYCFOTNPOANT-UHFFFAOYSA-N 0.000 claims description 2
- NEHMKBQYUWJMIP-NJFSPNSNSA-N chloro(114C)methane Chemical compound [14CH3]Cl NEHMKBQYUWJMIP-NJFSPNSNSA-N 0.000 claims description 2
- HRYZWHHZPQKTII-UHFFFAOYSA-N chloroethane Chemical compound CCCl HRYZWHHZPQKTII-UHFFFAOYSA-N 0.000 claims description 2
- 229960003750 ethyl chloride Drugs 0.000 claims description 2
- VHHHONWQHHHLTI-UHFFFAOYSA-N hexachloroethane Chemical compound ClC(Cl)(Cl)C(Cl)(Cl)Cl VHHHONWQHHHLTI-UHFFFAOYSA-N 0.000 claims description 2
- -1 monochloroethylene, dichloroethylene, trichloroethylene Chemical group 0.000 claims description 2
- BNIXVQGCZULYKV-UHFFFAOYSA-N pentachloroethane Chemical compound ClC(Cl)C(Cl)(Cl)Cl BNIXVQGCZULYKV-UHFFFAOYSA-N 0.000 claims description 2
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- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 2
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- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 1
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 1
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- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
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- FBBDOOHMGLLEGJ-UHFFFAOYSA-N methane;hydrochloride Chemical compound C.Cl FBBDOOHMGLLEGJ-UHFFFAOYSA-N 0.000 description 1
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Abstract
A short-chain chlorinated hydrocarbon induction type biosensor and application thereof relate to the technical field of genetic engineering biosensors, and the induction type biosensor specifically comprises Top10 engineering bacteria containing a C50-AlkS-EGFP vector, wherein AlkS and an operon gene thereof have the nucleotide sequence shown in SEQ ID NO:1, and a nucleotide sequence shown in the specification. The chlorohydrocarbon inducible operon adopted by the invention is a mutant of a wild alkane inducible promoter, and the chlorohydrocarbon inducible bacterial biosensor optimized based on the operon successfully realizes the detection of various short-chain chlorohydrocarbons. The biosensor is easy to operate in detection of short-chain chlorinated hydrocarbon, and the sample preparation process is simple and quick. The detection limit is improved, and the repeatability is good. Greatly improves the detection efficiency and reduces the reagent cost. The chlorinated hydrocarbon biosensor has the advantages of low background fluorescence leakage and high induced fluorescence expression, and shows extremely high sensitivity and specificity.
Description
Technical Field
The invention relates to the technical field of genetic engineering biosensors, in particular to a short-chain chlorinated hydrocarbon induction type biosensor and application thereof.
Background
Volatile chlorinated hydrocarbons are short-chain hydrocarbons containing at least one covalent chlorine atom, including methyl chloride, ethyl chloride, vinyl chloride, etc., which, due to their polarity, rapid evaporation and low flammability, have been widely used in machinery, electronics, leather, dry cleaning and chemical industries (groston, a., & Edwards, e.a. a1, 1-trichloroethane-degrading anaerobic mixed microbial culture enhances biotransformation of mixtures of chlorinated ethenes and ethane.applied and environmental microbiology,2006,72 (12); 7849-7856.).
It is counted that short-chain chlorinated hydrocarbon pollution exists in many countries worldwide at present, and serious threat is caused to the environment and human health. The detection results of the U.S. environmental protection agency on the groundwater supply sources and the common sites of 39 small towns show that 11 volatile chlorinated hydrocarbons are detected in untreated or treated groundwater, wherein the detection rates of trichloroethylene and trichloromethane are 36% and 31% respectively (Lu Jie, li Menggong, pan Jiafen. The treatment and repair of groundwater environment polluted by organic chlorinated hydrocarbons [ J]University of eastern university of science, 2008 (04): 27-30. He Jiangtao and the like are used for investigating organic pollutants in shallow groundwater in a certain city in north China. The survey results show that serious chlorinated hydrocarbon contamination occurs in some localized areas. The primary contaminants include Carbon Tetrachloride (CT), tetrachloroethylene (PCE), trichloroethylene (TCE), and trichloromethane (CF). Wherein, the pollution degree of TCE and PCE is most serious, and the highest concentration respectively reaches 63.74 mug.L -1 And 487.55. Mu.g.L -1 (He Jiangtao, cheng Dong, korean ice, cui Xuehui. Estimation of Natural decay Rate of chlorinated hydrocarbon contamination of shallow groundwater [ J)]The geodesic front, 2006 (01): 140-144.).
Short-chain chlorinated hydrocarbons are Persistent Organic Pollutants (POPs), are difficult to degrade in the environment, are easily accumulated in the food chain, and pose a threat to the ecosystem and biodiversity; has toxicity and carcinogenicity, and is harmful to human health, especially to liver and kidney (Wang Hailan. Occupational hazard and protection of chloroform [ J ]. Modern occupational safety, 2014 (05): 110-111.); in addition, short-chain chlorinated hydrocarbon is easy to volatilize, and can enter human bodies through the approaches of atmosphere, water, food and the like, so that the harm is caused.
In order to prevent the spread of chlorinated hydrocarbon contamination and the potential threat to human health, an urgent and critical rapid detection of chlorinated hydrocarbons is required. Only if the quick and accurate detection is carried out, effective measures can be taken to reduce chlorinated hydrocarbon pollution and protect the environment and human health. Traditional chlorinated hydrocarbon detection mainly depends on physical and chemical means, including gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC-MS), inductively coupled plasma mass spectrometry (ICP-MS) and the like. These methods, while accurate and sensitive, require complex pretreatment and operation, requiring more specialized personnel investment and high capital equipment. Therefore, development of a novel chlorinated hydrocarbon detection means which is more convenient, cheaper and easier to operate is particularly urgent.
In recent years, biosensors have demonstrated excellent performance in contaminant detection and have great potential for use in environmental risk assessment. The biosensor is a product based on genetic engineering, can sense target chemical substances in the environment, can generate detectable electrochemical or optical signals (Dhyani, R, jain, S, bhatt, A, kumar, P, & Navani, N.K. genetics regulatory element based whole-cell biosensors for the detection of metabolic distders. Biosensors & bioelectricics, 2022,199,113869), can establish a concentration gradient relation between the concentration of the to-be-detected substance and the detectable signals through a biological sensing element, can judge the concentration or toxicity of the pollutants in the environment through the strength of the expression signals, and has great development potential and prospect in the analysis of the pollutants. Compared with the traditional physicochemical detection method, the bacterial biosensor has the advantages of quick reaction time, capability of analyzing the concentration of the target substance in one hour or even a plurality of minutes, and easiness in operation. The core of the bacterial biosensor is bacteria, so that the bacterial biosensor is easy to culture and extremely low in cost, and is beneficial to popularization and application. More importantly, the bacterial biosensor not only can detect the content of chlorinated hydrocarbon in the environment, but also can accurately reflect the amount of chlorinated hydrocarbon entering the organism in the environment and the biotoxicity thereof, and can carry out guiding evaluation on the toxicity of chlorinated hydrocarbon. Compared with the traditional method, the biosensor has the advantages of rapidness, economy, simple operation and the like, and is an ideal tool for detecting the biological effectiveness of pollutants in the environment.
In view of the severity of short-chain chlorinated hydrocarbon contamination, and the great promise and superiority of bacterial biosensors for chlorinated hydrocarbon contamination detection. The invention obtains the induced operon capable of detecting chlorinated hydrocarbon through directed evolution means, thereby establishing the bacterial biosensor capable of detecting short-chain chlorinated hydrocarbon.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a short-chain chlorinated hydrocarbon induction type biosensor and application thereof, so as to solve the problem that the short-chain chlorinated hydrocarbon cannot be detected by the bacterial biosensor constructed based on transcription factors in the prior art.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a short-chain chlorinated hydrocarbon induction type biosensor, in particular to Top10 engineering bacteria containing a C50-AlkS-EGFP carrier, wherein AlkS and an operon gene thereof have the nucleotide sequence shown in SEQ ID NO:1, and a nucleotide sequence shown in the specification.
The invention also provides a preparation method of the short-chain chlorinated hydrocarbon induced biosensor, which comprises the following steps:
1) Obtaining wild-type chlorinated hydrocarbon inducible operon gene:
plasmid pCOM8-Alks is used as a template, and SEQ ID NO:2 and SEQ ID NO:3, performing PCR amplification by using the primer to obtain EGFP genes and alkane operon genes comprising promoters palkS, palkB and regulatory protein AlkS;
2) Construction of wild-type chlorinated hydrocarbon-induced recombinant vector:
cutting the pUC18 vector by using EcoRI and XhoI, and connecting the PCR product obtained by amplification in the step 1) by using T4 ligase to enable a wild type chlorinated hydrocarbon inducible operon to replace the original lac promoter in the pUC18 vector so as to obtain a wild type chlorinated hydrocarbon inducible recombinant vector AlkS-EGFP;
synchronously constructing AID-EGFP blank plasmids, and replacing chlorinated hydrocarbon inducible operon in the AlkS-EGFP plasmids with (MCS) non-functional sequence AID containing multiple cloning sites, wherein the AID sequence is represented by SEQ ID NO:4 and SEQ ID NO:5 is a primer, and pCI-mAID is used as a template for cloning to obtain the primer;
3) Obtaining the chlorinated hydrocarbon induced biosensor through directed evolution:
error-prone PCR is carried out on alkane-induced operon genes by taking AlkS-EGFP plasmid as a template to obtain a random mutant library, and then the mutant genes are used for replacing AID genes in AID-EGFP plasmids to construct a recombinant mutant library, wherein in a connection system, the molar ratio of an inserted fragment to a carrier is 4:1, or 50ng of the carrier and 200ng of the fragment are added into each 100ul connection system, and the connection reaction condition is that the connection is carried out for 30min at 22 ℃; the connection product is electrically transformed and is led into Top10 competent cells to obtain a flow screening library for flow high-throughput screening; the primer of the error-prone PCR is shown as SEQ ID NO:6 and SEQ ID NO: shown in figure 7;
finally, after three-turn high-flux screening, the Top10 engineering bacteria containing the C50-AlkS-EGFP plasmid vector after evolution is finally obtained, namely the bacterial biosensor after evolution, wherein AlkS and an operon gene thereof have the nucleotide sequence shown in SEQ ID NO:1, and a nucleotide sequence shown in the specification.
Preferably, the reaction system of the error-prone PCR in the preparation method is as follows:
the error-prone PCR reaction procedure is: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 45s, extension at 72 ℃ for 2.5min, and after 25 cycles, extension is continued for 10min at 72 ℃ and then the mixture is kept at 4 ℃ for standby.
The invention also provides application of the inductive biosensor in detecting short-chain chlorinated hydrocarbon, comprising the following steps:
1) Inoculating a bacterial biosensor C50-AlkS-EGFP on an ampicillin-resistant LB solid medium plate, and culturing at 37 ℃ overnight; meanwhile, inoculating a wild chlorinated hydrocarbon induction type sensor AID-EGFP as a control;
2) Picking single colonies of the wild type and the evolved sensor respectively, inoculating the single colonies into 1mL of LB liquid medium containing ampicillin resistance, and culturing overnight at 37 ℃ and 200rpm to obtain detection bacterial liquid;
3) Diluting the detection bacterial liquid by 50 times by using the LB liquid culture medium containing ampicillin resistance to obtain diluted bacterial liquid, and continuously culturing until the logarithmic phase;
4) Preparing a series of short-chain chlorinated hydrocarbon standard substances;
5) Adding chlorinated hydrocarbon standard substance with final concentration of 5mg/L into the log phase bacterial liquid to be used as an induction group; synchronously taking the log phase bacterial liquid, adding the same amount of deionized water, and taking the same amount of deionized water as a blank control; culturing for 1h at 37 ℃ and 200rpm to obtain an induced bacterial liquid;
6) Placing the induced bacterial liquid in a centrifuge for centrifugation at 5000rpm for 3min, and discarding the supernatant;
7) After resuspension with 1×m9 buffer, centrifugation was performed again, rinsing was repeated 3 times, and finally resuspension was performed with 1×pbs, and fluorescence expression was detected by flow cytometry.
The induction type biosensor provided by the invention has strong substrate specificity, responds to short-chain chlorinated hydrocarbon and does not respond to alkane. Short-chain chlorinated hydrocarbons such as methane chloride, methylene chloride, chloroform, ethylene chloride, dichloroethane, trichloroethane, tetrachloroethane, pentachloroethane, hexachloroethane, vinyl chloride, ethylene dichloride, trichloroethylene, tetrachloroethylene, and the like can be detected.
Compared with the prior art, the invention has the beneficial effects that:
1. the chlorohydrocarbon inducible operon adopted by the invention is a mutant of a wild alkane inducible promoter, and the chlorohydrocarbon inducible bacterial biosensor optimized based on the operon successfully realizes the detection of various short-chain chlorohydrocarbons at the same time.
2. The chlorinated hydrocarbon biosensor obtained through directional evolution and a flow type high-throughput bidirectional screening strategy has the advantages of low background fluorescence leakage and high induced fluorescence expression, shows extremely high sensitivity and specificity, responds to short-chain chlorinated hydrocarbon and no longer responds to original alkane (the wild type biosensor before evolution can respond to alkane but does not respond to chlorinated hydrocarbon).
3. The biosensor is easy to operate in detecting chlorinated hydrocarbon, and the sample preparation process is simple and quick. The detection limit is high, and the repeatability is good. Greatly improves the detection efficiency and reduces the reagent cost.
Drawings
FIG. 1 shows plasmid maps of C50-Alks-EGFP (A) and AID-EGFP (B), respectively.
FIG. 2 shows the response of wild-type bacterial biosensor (WT-AlkS) and of the bacterial biosensor after evolution (C50-AlkS) to short-chain chlorinated hydrocarbons.
FIG. 3 shows the response of the bacterial biosensor to medium-long chain alkanes after evolution.
FIG. 4 shows the response of the bacterial biosensor after evolution to an induced concentration gradient of short-chain chlorinated hydrocarbons (in the case of dichloroethane).
FIG. 5 shows the response of the bacterial biosensor after evolution to the induction time gradient of short-chain chlorinated hydrocarbons (in the case of dichloroethane).
Detailed Description
The following describes the embodiments of the present invention in detail, and the embodiments and specific operation procedures are given on the premise of the technical solution of the present invention, so that those skilled in the art can better understand the present invention, but the protection scope of the present invention is not limited to the following embodiments.
The preparation method of each LB culture medium comprises the following steps:
LB liquid medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride.
LB solid medium: agar 15g was added to each liter of LB liquid medium.
Ampicillin-resistant LB solid medium: the prepared LB solid culture medium is heated to be completely dissolved, and ampicillin with the total weight of 1 per mill is added when the temperature is reduced to about 55 ℃.
Example 1
The preparation method of the short-chain chlorinated hydrocarbon induction type biosensor comprises the following steps:
1) Obtaining wild-type chlorinated hydrocarbon inducible operon gene:
plasmid pCOM8-Alks is used as a template, and SEQ ID NO:2 and SEQ ID NO:3, performing PCR amplification by using the primer to obtain EGFP genes and alkane operon genes comprising promoters palkS, palkB and regulatory protein AlkS.
2) Construction of wild-type chlorinated hydrocarbon-induced recombinant vector:
and (2) cutting the pUC18 vector by using EcoRI and XhoI, and connecting the PCR product obtained by amplification in the step (1) by using T4 ligase, so that the wild-type chlorinated hydrocarbon inducible operon replaces the original lac promoter in the pUC18 vector, and the wild-type chlorinated hydrocarbon inducible recombinant vector AlkS-EGFP is obtained.
Synchronously constructing AID-EGFP blank plasmids, and replacing chlorinated hydrocarbon inducible operon in the AlkS-EGFP plasmids with (MCS) non-functional sequence AID containing multiple cloning sites, wherein the AID sequence is represented by SEQ ID NO:4 and SEQ ID NO:5 is a primer, and pCI-mAID is used as a template for cloning.
3) Obtaining the chlorinated hydrocarbon induced biosensor through directed evolution:
error-prone PCR is carried out on alkane-induced operon genes by taking AlkS-EGFP plasmid as a template to obtain a random mutant library, and then the mutant genes are used for replacing AID genes in AID-EGFP plasmids to construct a recombinant mutant library, wherein in a connection system, the molar ratio of an inserted fragment to a carrier is 4:1, or 50ng of the carrier and 200ng of the fragment are added into each 100ul connection system, and the connection reaction condition is that the connection is carried out for 30min at 22 ℃; the connection product is electrically transformed and is led into Top10 competent cells to obtain a flow screening library for flow high-throughput screening; the primer of the error-prone PCR is shown as SEQ ID NO:6 and SEQ ID NO: shown at 7.
The error-prone PCR reaction system is as follows:
the error-prone PCR reaction procedure is: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 45s, extension at 72 ℃ for 2.5min, and after 25 cycles, extension is continued for 10min at 72 ℃ and then the mixture is kept at 4 ℃ for standby.
Finally, after three-turn high-flux screening, the Top10 engineering bacteria containing the C50-AlkS-EGFP plasmid vector after evolution is finally obtained, namely the bacterial biosensor after evolution, wherein AlkS and an operon gene thereof have the nucleotide sequence shown in SEQ ID NO:1, and the plasmid spectrum of the nucleotide sequence is shown in figure 1.
Example 2
The bacterial biosensor after evolution detects a variety of short-chain chlorinated hydrocarbons:
1) Inoculating the evolved bacterial biosensor C50-AlkS-EGFP on an ampicillin-resistant LB solid medium plate, and culturing at 37 ℃ overnight; meanwhile, a wild-type chlorinated hydrocarbon induction sensor AID-EGFP is inoculated as a control.
2) The single colony of the wild type sensor and the single colony of the sensor after evolution are respectively picked up, inoculated into 1mL of LB liquid medium containing ampicillin resistance, and cultured overnight at 37 ℃ and 200rpm to obtain detection bacterial liquid.
3) The test bacterial liquid was diluted 50 times with the above-mentioned LB liquid medium containing ampicillin resistance to obtain a diluted bacterial liquid, and the culture was continued until the logarithmic phase.
4) A series of short chain chlorinated hydrocarbon standards were prepared and purchased from sigma aldrich (Shanghai) trade company, inc.
5) Adding chlorinated hydrocarbon standard substance with final concentration of 5mg/L into the log phase bacterial liquid to be used as an induction group; synchronously taking the log phase bacterial liquid, adding the same amount of deionized water, and taking the same amount of deionized water as a blank control; culturing for 1h at 37 ℃ and 200rpm to obtain an induced bacterial liquid;
6) The induced bacterial liquid is placed in a centrifuge for centrifugation at 5000rpm for 3min, and the supernatant is discarded.
7) After resuspension with 1×m9 buffer, centrifugation was performed again, rinsing was repeated 3 times, and finally resuspension was performed with 1×pbs, and fluorescence expression was detected by flow cytometry.
The obtained response is shown in fig. 2, and it can be seen that the wild-type chlorinated hydrocarbon response biosensor has almost no response to all short-chain chlorinated hydrocarbons, and the evolved chlorinated hydrocarbon response biosensor not only realizes the detection of chlorinated hydrocarbons, but also can detect various short-chain chlorinated hydrocarbons.
Example 3
The bacterial biosensor after evolution detects a variety of medium-long chain alkanes:
1) The bacterial biosensor C50-AlkS-EGFP after evolution was inoculated on ampicillin-resistant LB solid medium plates and cultured overnight at 37 ℃.
2) The single colony of the sensor after evolution is picked, inoculated in 1mL of LB liquid medium containing ampicillin resistance, and cultured overnight at 37 ℃ and 200rpm to obtain detection bacterial liquid.
3) The test bacterial liquid was diluted 50 times with the above-mentioned LB liquid medium containing ampicillin resistance to obtain a diluted bacterial liquid, and the culture was continued until the logarithmic phase.
4) A series of medium-long chain alkane standards were prepared and purchased from sigma aldrich (Shanghai) trade limited.
5) Adding chlorinated hydrocarbon standard substance with final concentration of 5mg/L into the log phase bacterial liquid to be used as an induction group; synchronously taking the log phase bacterial liquid, adding the same amount of deionized water, and taking the same amount of deionized water as a blank control; culturing for 1h at 37 ℃ and 200rpm to obtain an induced bacterial liquid;
6) The induced bacterial liquid is placed in a centrifuge for centrifugation at 5000rpm for 3min, and the supernatant is discarded.
7) After resuspension with 1×m9 buffer, centrifugation was performed again, rinsing was repeated 3 times, and finally resuspension was performed with 1×pbs, and fluorescence expression was detected by flow cytometry.
The results of the detection are shown in fig. 3, and the results show that the bacterial biosensor after evolution has little response to the medium-long chain alkane.
Example 4
Gradient concentration induction test of bacterial biosensors after evolution on short-chain chlorinated hydrocarbons (in the case of dichloroethane):
1) The single colony of C50-AlkS-EGFP is picked, inoculated into 1mL of LB liquid medium containing ampicillin resistance, and cultured overnight at 37 ℃ and 200rpm to obtain detection bacterial liquid.
2) The test bacterial liquid was diluted 50 times with the above-mentioned LB liquid medium containing ampicillin resistance to obtain a diluted bacterial liquid, and the culture was continued until the logarithmic phase.
3) Adding dichloroethane standard with final concentration of 0-40mg/L into the log phase bacterial liquid to be used as an induction group; synchronously taking the log phase bacterial liquid, adding the same amount of deionized water, and taking the same amount of deionized water as a blank control; culturing at 37 deg.C and 200rpm for 1 hr to obtain induced bacterial liquid.
4) The induced bacterial liquid is placed in a centrifuge for centrifugation at 5000rpm for 3min, and the supernatant is discarded.
5) After resuspension with 1×m9 buffer, centrifugation was performed again, rinsing was repeated 3 times, and finally resuspension was performed with 1×pbs, and fluorescence expression was detected by flow cytometry.
The results of the detection are shown in fig. 4, and the results show that the fluorescence response of the bacterial biosensor after evolution is enhanced along with the increase of the concentration of chlorinated hydrocarbon.
Example 5
Gradient time induction test of bacterial biosensors after evolution on short chain chlorinated hydrocarbons (in the case of dichloroethane):
1) Single colonies were picked up, inoculated into 1mL of LB liquid medium containing ampicillin resistance, and cultured overnight at 37℃and 200rpm to obtain a test bacterial liquid.
2) Diluting the detection bacterial liquid by 50 times by using the LB liquid culture medium containing ampicillin resistance to obtain diluted bacterial liquid, and continuously culturing until the logarithmic phase;
3) Adding dichloroethane standard with the final concentration of 5mg/L into the log phase bacterial liquid to be used as an induction group; synchronously taking the log phase bacterial liquid, adding the same amount of deionized water, and taking the same amount of deionized water as a blank control; culturing at 37deg.C and 200rpm for 1-8 hr to obtain induced bacterial liquid.
4) The induced bacterial liquid is placed in a centrifuge for centrifugation at 5000rpm for 3min, and the supernatant is discarded.
5) After resuspension with 1×m9 buffer, centrifugation was performed again, rinsing was repeated 3 times, and finally resuspension was performed with 1×pbs, and fluorescence expression was detected by flow cytometry.
The results of the detection are shown in fig. 5, and the results show that the fluorescence response of the bacterial biosensor after evolution is enhanced with the increase of the chlorinated hydrocarbon induction time.
The foregoing is merely illustrative and explanatory of the principles of the invention, as various modifications and additions may be made to the specific embodiments described, or similar thereto, by those skilled in the art, without departing from the principles of the invention or beyond the scope of the appended claims.
Claims (6)
1. A short-chain chlorinated hydrocarbon induction type biosensor, in particular to Top10 engineering bacteria containing a C50-AlkS-EGFP carrier, wherein AlkS and an operon gene thereof have the nucleotide sequence shown in SEQ ID NO:1, and a nucleotide sequence shown in the specification.
2. A method of making the short-chain chlorinated hydrocarbon-induced biosensor of claim 1, comprising the steps of:
1) Obtaining wild-type chlorinated hydrocarbon inducible operon gene:
plasmid pCOM8-Alks is used as a template, and SEQ ID NO:2 and SEQ ID NO:3, performing PCR amplification by using the primer to obtain EGFP genes and alkane operon genes comprising promoters palkS, palkB and regulatory protein AlkS;
2) Construction of wild-type chlorinated hydrocarbon-induced recombinant vector:
cutting the pUC18 vector by using EcoRI and XhoI, and connecting the PCR product obtained by amplification in the step 1) by using T4 ligase to enable a wild type chlorinated hydrocarbon inducible operon to replace the original lac promoter in the pUC18 vector so as to obtain a wild type chlorinated hydrocarbon inducible recombinant vector AlkS-EGFP;
synchronously constructing AID-EGFP blank plasmids, and replacing chlorinated hydrocarbon inducible operon in the AlkS-EGFP plasmids with (MCS) non-functional sequence AID containing multiple cloning sites, wherein the AID sequence is represented by SEQ ID NO:4 and SEQ ID NO:5 is a primer, and pCI-mAID is used as a template for cloning to obtain the primer;
3) Obtaining the chlorinated hydrocarbon induced biosensor through directed evolution:
error-prone PCR is carried out on alkane-induced operon genes by taking AlkS-EGFP plasmid as a template to obtain a random mutant library, and then the mutant genes are used for replacing AID genes in AID-EGFP plasmids to construct a recombinant mutant library, wherein in a connection system, the molar ratio of an inserted fragment to a carrier is 4:1, or 50ng of the carrier and 200ng of the fragment are added into each 100ul connection system, and the connection reaction condition is that the connection is carried out for 30min at 22 ℃; the connection product is electrically transformed and is led into Top10 competent cells to obtain a flow screening library for flow high-throughput screening; the primer of the error-prone PCR is shown as SEQ ID NO:6 and SEQ ID NO: shown in figure 7;
finally, after three-turn high-flux screening, the Top10 engineering bacteria containing the C50-AlkS-EGFP plasmid vector after evolution is finally obtained, namely the bacterial biosensor after evolution, wherein AlkS and an operon gene thereof have the nucleotide sequence shown in SEQ ID NO:1, and a nucleotide sequence shown in the specification.
3. The method of claim 2, wherein the error-prone PCR reaction system is:
the error-prone PCR reaction procedure is: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 45s, extension at 72 ℃ for 2.5min, and after 25 cycles, extension is continued for 10min at 72 ℃ and then the mixture is kept at 4 ℃ for standby.
4. Use of an inducible biosensor according to claim 1 for detecting short-chain chlorinated hydrocarbons, characterized by the following steps:
1) Inoculating a bacterial biosensor C50-AlkS-EGFP on an ampicillin-resistant LB solid medium plate, and culturing at 37 ℃ overnight; meanwhile, inoculating a wild chlorinated hydrocarbon induction type sensor AID-EGFP as a control;
2) Picking single colonies of the wild type and the evolved sensor respectively, inoculating the single colonies into 1mL of LB liquid medium containing ampicillin resistance, and culturing overnight at 37 ℃ and 200rpm to obtain detection bacterial liquid;
3) Diluting the detection bacterial liquid by 50 times by using the LB liquid culture medium containing ampicillin resistance to obtain diluted bacterial liquid, and continuously culturing until the logarithmic phase;
4) Preparing a series of short-chain chlorinated hydrocarbon standard substances;
5) Adding chlorinated hydrocarbon standard substance with final concentration of 5mg/L into the log phase bacterial liquid to be used as an induction group; synchronously taking the log phase bacterial liquid, adding the same amount of deionized water, and taking the same amount of deionized water as a blank control; culturing for 1h at 37 ℃ and 200rpm to obtain an induced bacterial liquid;
6) Placing the induced bacterial liquid in a centrifuge for centrifugation at 5000rpm for 3min, and discarding the supernatant;
7) After resuspension with 1×m9 buffer, centrifugation was performed again, rinsing was repeated 3 times, and finally resuspension was performed with 1×pbs, and fluorescence expression was detected by flow cytometry.
5. The method according to claim 4, wherein the short-chain chlorohydrocarbon is methyl chloride, methylene chloride, trichloromethane, ethyl chloride, dichloroethane, trichloroethane, tetrachloroethane, pentachloroethane, hexachloroethane, monochloroethylene, dichloroethylene, trichloroethylene or tetrachloroethylene.
6. The use according to claim 4 or 5, wherein the inducible biosensor has a strong substrate specificity, is responsive to short-chain chlorinated hydrocarbons and is no longer responsive to alkanes.
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