CN115896154A - Microbial fluorescence sensor for detecting p-nitrophenol and preparation method and application thereof - Google Patents
Microbial fluorescence sensor for detecting p-nitrophenol and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a microbial fluorescence sensor and a preparation method and application thereof. The microbial fluorescence sensor comprises a promoter P C1 And a sensing element of a regulatory gene pnPR and a reporter element containing green fluorescent protein GFP. The microbial fluorescence sensor can specifically respond to p-nitrophenol with different concentrations,fluorescence at different RFU values is generated and the p-nitrophenol concentration is coupled to the self-luminescence RFU value. The microbial fluorescence sensor can realize specific identification and detection of p-nitrophenol, is simple and convenient to operate and high in sensitivity, and can be used for linearly detecting pollutant molecules in a low concentration range, so that the microbial fluorescence sensor has a good application prospect.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and molecular biology, and particularly relates to a microbial fluorescence sensor for detecting p-nitrophenol, and a preparation method and application thereof.
Background
p-Nitrophenol (PNP) mainly comes from the fields of pesticide, dye, medicine, rubber and the like, has a 'three-cause' effect, is considered as an environmental endocrine disruptor and is listed as a priority pollutant control in China. Therefore, the effective detection of the p-nitrophenol has important significance for the health of people and the environmental protection.
At present, the common methods for measuring p-nitrophenol mainly comprise: the chemical analysis methods such as the spectroscopy, the electrochemical method, the gas chromatography, the liquid chromatography, the chromatography-mass spectrometry and the like have the problems of complicated operation, expensive instruments, high analysis cost and the like. In recent years, the microbial sensor has the characteristics of good selectivity, high sensitivity, simple and convenient use, low cost, continuous online monitoring in a complex system and the like, and becomes a hotspot of research as a novel technology of environmental monitoring. The microorganism is genetically modified by utilizing a genetic engineering means, so that the microorganism reacts to certain special toxic substances or physical stress in the environment to generate a measurable signal, and finally, the detection and quantification of specific chemical substances in the air, soil or water are realized.
The microbial sensor mainly comprises a sensing element and a reporter gene, wherein the sensing element usually comprises a specific recognition protein gene and a promoter element related to a detected compound, and the specificity of the system is determined; while the reporter gene does not require specific recognition, its expression level and intensity are responsive to the sensitivity and detection threshold of the system. At present, although degradation characteristics, metabolic pathways and regulation mechanisms of p-nitrophenol degradation strains are researched more, the development of p-nitrophenol microbial sensors is limited by the discovery of p-nitrophenol specific response elements.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method and application of a microbial fluorescence sensor synthesized by utilizing a specific response element of an efficient degradation strain, wherein the microbial fluorescence sensor contains regulatory protein, a corresponding promoter thereof and green fluorescent protein, can realize specific linear detection of nitrophenol, has high sensitivity, is suitable for environmental monitoring, and has wide application prospect.
The invention is realized by adopting the following technical scheme:
a recombinant plasmid is prepared by using pBBR-MCS2 as starting vector, and inserting P-nitrophenol inductive element P between BamH I and Xho I sites R -pnpR-P C1 And green fluorescent protein gene gfp, wherein promoter P R The nucleotide sequence of (A) is shown as SEQ ID NO.1, the nucleotide sequence of regulatory protein PnpR is shown as SEQ ID NO.2, and the promoter P C1 The nucleotide sequence of (A) is shown in SEQ ID NO. 3.
As a preferable preference of the invention, the gfp sequence of the green fluorescent protein gene is shown as SEQ ID No. 4.
As a preferred aspect of the present invention, the P-nitrophenol sensing element P R -pnpR-P C1 The nucleotide sequence of (A) is shown in SEQ ID NO. 5.
As a further preferred embodiment of the present invention, the recombinant plasmid is constructed mainly by the following method:
(1) Amplifying P-nitrophenol response element P by taking total DNA of P-nitrophenol efficient degradation strain DLL-E4 genome as template R -pnpR-P C1 ；
(2) Amplifying a Green Fluorescent Protein (GFP) gene by PCR;
(3) With recovered P R -pnpR-P C1 And gfp as template to amplify the fusion fragment P R -pnpR-P C1 -gfp, purified again for recovery;
(4) Carrying out double digestion on the plasmid vector pBBR-MCS2 by using BamH I and Xho I, and carrying out seamless connection on a plasmid digestion fragment and a fusion fragment according to the molar ratio of 1: 2 to obtain the plasmid vector.
As a further preferred aspect of the present invention, P is amplified R -pnpR-P C1 The primer sequences of (1) are shown as SEQ ID NO.7 and SEQ ID NO.8.
A preparation method of a microbial fluorescence sensor for detecting p-nitrophenol comprises the following steps:
(1) The recombinant plasmid is transformed into KT2440 competent cells, and positive clones are screened on an LB solid plate containing antibiotics to obtain pBBR-P containing the recombinant plasmid R -pnpR-P C1 -gfp engineering bacteria PNPgfp-1;
(2) Culturing and activating the engineering bacterium PNPgfp-1 in an LB liquid culture medium containing kanamycin to grow to OD 600 And =0.2, finally obtaining the microbial fluorescence sensor.
The microbial fluorescence sensor capable of linearly responding to p-nitrophenol is obtained by the preparation method.
The microbial fluorescence sensor disclosed by the invention is applied to real-time detection of p-nitrophenol.
Preferably, the microbial fluorescence sensor and a sample to be detected are mixed uniformly according to the volume ratio of 1.
As a preferred mode of the present invention, the concentration of the contaminant molecules sensed by the microbial fluorescence sensor is 10ppm to 100ppm.
Furthermore, the microbial fluorescence biosensor can sense pollutant molecules with different concentrations, so that fluorescence with different RFU values is generated, and the concentration of the pollutant molecules and the self-luminous RFU value are coupled to achieve the purpose of monitoring the pollutant molecules in real time.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention takes a pollutant tolerant strain KT2440 as a chassis cell and takes p-nitrophenol as a substrate cellScreening the high-efficiency degradation strain DLL-E4 to obtain a regulation and control unit pnPR-P C1 The biosensor can specifically respond to p-nitrophenol in a short time, so that fluorescence with different RFU values is generated, and a linear relation is formed in a certain concentration range, thereby realizing the fluorescence detection of pollutant molecules. Compared with the existing p-nitrophenol detection method, the biosensor has the advantages of quick response, high specificity and sensitivity and strong environmental adaptability, thereby having wider application prospect in the field of p-nitrophenol detection.
Drawings
FIG. 1 shows the growth of E.coli DH 5. Alpha. At various p-nitrophenol concentration levels.
FIG. 2 shows the growth of Pseudomonas putida KT2440 at different p-nitrophenol concentration levels.
FIG. 3 shows the recombinant plasmid pBBR-P R -pnpR-P C1 Plasmid map of gfp.
FIG. 4 shows the recombinant plasmid pBBR-P R1 -pnpR1-P A Plasmid map of gfp.
FIG. 5 shows fluorescence detection result data of the constructed sensor strain PNPgfp-1.
FIG. 6 shows fluorescence detection result data of the constructed sensor strain PNPgfp-2.
FIG. 7 shows fluorescence detection data of the constructed sensor strain PNPgfp-1 under the condition of fixed pollutant concentration within 12 h.
FIG. 8 shows the result data of the pollutant-specific fluorescence detection of the constructed sensor strain PNPgfp-1.
FIG. 9 shows fluorescence detection data of the constructed sensor strain PNPgfp-1 under different pH conditions.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.
The examples do not show the specific techniques or conditions, and the techniques described in the literature in the field or the product specifications are followed. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase.
Example 1: chassis cell screening
Preparing p-nitrophenol mother liquor (120000ppm, 60000ppm and 30000ppm) with different concentrations; the solvent used was absolute ethanol.
Preparing p-nitrophenol culture solutions with different concentrations: 4mL of LB culture medium, 1mL of bacterial liquid and 5 mu L of p-nitrophenol mother liquor with corresponding concentration.
1% inoculum size was inoculated into OD 600 DH 5. Alpha. And KT2440 strains (ATCC 47057) of =1 were cultured in constant temperature shaking tables (37 ℃ and 30 ℃,180 rpm), respectively.
Sampling at regular time, adding 200 μ L of the sample into a transparent enzyme label plate, performing 3 parallels, and measuring OD at different time 600 And detecting the growth conditions of different strains.
Results as shown in fig. 1 and fig. 2, strain KT2400 was able to tolerate high concentrations of p-nitrophenol compared to strain DH5 α, and therefore KT2440 was selected as the underplate cell to construct a biosensor.
Example 2: gene acquisition and vector construction
1. Acquisition of genes
Characteristic of efficiently degrading strain DLL-E4 (Liuzhi, hongqing and the like. Methyl parathion degrading strain DLL-E4 for degrading p-nitrophenol [ J)]Chinese environmental science, 004 th stage 2003, 435-439) to respectively amplify P R pnPR and P C1 The nucleotide sequence of the gene is shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, P R1 pnPR1 and P A The nucleotide sequence of the gene is shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, the primers are R-F, R-R and P C1 -F and P C1 -R, R1-F and R1-R, P A -F and P A -R, taking DLL-E4 total DNA as a template, and carrying out Polymerase Chain Reaction (PCR), wherein a PCR amplification system is shown as follows:
the PCR procedure was: 5min at 95 ℃;32 cycles X (94 ℃ 30s,55 ℃ 30s,72 ℃ 45 s); 5min at 72 ℃; infinity at 4 ℃.
The primer sequences are shown below:
R-F：
5’-cgctctagaactagtggatccGGAAAGTGGCCGCTTCAAG-3’；
R-R：
5’-TCAGACGACCCAGCCACG-3’。
P C1 -F：
5’-CATTGAGCAGTGTTCGCGTTT-3’；
P C1 -R：
5’-CGCATTCACCGCACTTGTT-3’。
R1-F：
5’-cgctctagaactagtggatccCGGGATTTGTGGGCCATC-3’；
R1-R：
5’-TCAGCAGTTTGCAAGGGCA-3’。
P A -F：
5’-GTGAATTCCTGAATACGTCATTGTTAG-3’；
P A -R：
5’-GTAGGCGACTGCCCGCGG-3’。
the PCR product was gel recovered and purified using a gel recovery and purification kit (Vazyme).
Then recovering P R -pnPR and P C1 The gene is used as a template and R-F and P are used C1 -R is a primer to carry out Polymerase Chain Reaction (PCR) and amplify the fusion fragment P R -pnpR-P C1 The nucleotide sequence is shown as SEQ ID NO.5, and the PCR amplification system is shown as follows:
the PCR procedure was: 5min at 95 ℃;32 cycles X (94 ℃ 30s,55 ℃ 30s,72 ℃ 70 s); 5min at 72 ℃; infinity at 4 ℃.
Then using the above-mentioned method to make P R1 -pnPR1 and P A As template, with R1-F and P A -R is a primer, and the fusion fragment P is amplified R1 -pnpR1-P A Nucleosides of sameThe sequence is shown as SEQ ID NO.9.
The PCR product was gel recovered and purified using a gel recovery and purification kit (Vazyme).
The GFP gene was amplified from plasmid pMD19-T-GFP containing green fluorescent protein GFP, which was purchased from wuhan 28156ling biotechnology limited, and the nucleotide sequence thereof is shown in SEQ ID No.4, and the primers GFP-F and GFP-R were used to perform Polymerase Chain Reaction (PCR), and the PCR amplification system thereof is shown below:
the PCR procedure was: 5min at 95 ℃;35 cycles X (94 ℃ 30s,55 ℃ 30s,72 ℃ 30 s); 5min at 72 ℃; infinity at 4 ℃.
The primer sequences are shown below:
primer 1 (gfp-F):
5’-ATGAGTAAAGGAGAAGAACTTTTCACTG-3’；
primer 2 (gfp-R):
5’-ggtaccgggccccccctcgagGTGTATCCAGGAGCTGTTACAACTCA-3’。
then recovering P R -pnpR-P C1 And gfp gene as template, and R-F and gfp-R as primers to amplify fusion fragment P R -pnpR-P C1 -gfp, as P R1 -pnpR1-P A And gfp as a template, and R1-F and gfp-R as primers to amplify the fusion fragment P R1 -pnpR1-P A Gfp, performing Polymerase Chain Reaction (PCR) with the PCR amplification system as follows:
the PCR procedure was: 5min at 95 ℃;32 cycles X (94 ℃ 30s,55 ℃ 30s,72 ℃ 70 s); 5min at 72 ℃; infinity at 4 ℃.
The PCR product was gel recovered and purified using a gel recovery and purification kit (Vazyme).
2、pBBR-P R -pnpR-P C1 Gfp and pBBR-P R1 -pnpR1-P A Construction of-gfp expression vector
(1) The pBBR-MCS2 plasmid was double-digested with restriction enzyme 1BamH I (Takara Bio, cat. No. 1010S) and restriction enzyme 2Xho I (Takara Bio, cat. No. 1094S) in the following manner:
the enzyme digestion system is incubated for 3h at 37 ℃ for gel recovery and purification.
P cloning Using seamless cloning R -pnpR-P C1 -gfp and P R1 -pnpR1-P A The gfp fragment was cloned into the pBBR-MCS2 plasmid in the following system:
the ligation was incubated at 37 ℃ for 30min. Transformation of E.coli DH5 alpha competence by ligation products, plating on LB solid plate containing 50mg/L kanamycin, screening positive clones, and extracting recombinant plasmid pBBR-P from positive clones R -pnpR-P C1 Gfp (FIG. 3) and pBBR-P R1 -pnpR1-P A Gfp (FIG. 4), identified by restriction and sequencing.
Example 3: construction of p-nitrophenol whole-cell microbial sensor
The recombinant plasmid was transformed into Pseudomonas putida KT2440 competent cells by high-pressure electroporation, spread on LB solid plate containing 100mg/L kanamycin, and positive clones were obtained by PCR screening, thereby obtaining pBBR-P containing the vector R -pnpR-P C1 -gfp engineering strain PNPgfp-1 and vector pBBR-P R1 -pnpR1-P A -engineering strain of gfp PNPgfp-2.
Example 4: application of microbial fluorescence sensor in detection of p-nitrophenol
1. Strain activation and culture
Engineering strains PNPgfp-1 and PNPgfp-2 with correct sequencing are respectively transferred to an LB liquid culture medium containing 100mg/L kanamycin and cultured overnight at 30 ℃ to obtain a bacterial liquid.
2. Dose effect detection for sensors
Preparing p-nitrophenol mother liquor with different concentrations (100000ppm, 80000ppm,60000ppm,50000ppm,40000 ppm,30000ppm,20000ppm and 10000ppm); the solvent used was absolute ethanol.
Preparing p-nitrophenol culture solutions with different concentrations: 4mL of LB medium, 1mL of bacterial liquid and 5. Mu.L of p-nitrophenol mother liquor with corresponding concentration to obtain the culture solution with p-nitrophenol concentrations of 100ppm,80ppm,60ppm,50ppm,40ppm,30ppm,20ppm and 10ppm respectively.
The culture was carried out for 6h in a constant temperature shaker (30 ℃,180 rpm).
Samples were taken at 200. Mu.L and added to a black well plate, 3 replicates for each experimental group.
3. Specific detection of sensors
40000ppm (benzene, hydroquinone, catechol, p-nitrophenol and p-nitrobenzene) of different structurally similar pollutant mother liquor is prepared; the solvent used was absolute ethanol.
Preparing culture solutions of different pollutants: 4mL of LB culture medium, 1mL of bacterial liquid and 5 mu L of corresponding pollutant mother liquor are used for obtaining culture solutions with different structures and similar pollutants, and the final concentration of the culture solutions is 40 ppm.
The culture was carried out for 6h in a constant temperature shaker (30 ℃,180 rpm).
Samples were taken at 200. Mu.L and added to a primed black plate, 3 replicates per concentration.
3. Response time detection of sensors
Preparing a pollutant culture solution: 4mL of LB medium +1mL of the broth + 5. Mu.L of p-nitrophenol mother liquor (40000 ppm).
The culture was carried out for 12 hours in a constant temperature shaker (30 ℃,180 rpm) and samples were taken every two hours for detection.
A200. Mu.L sample was added to a black well plate and 3 replicates were run for each experimental group.
4. pH adaptation detection of sensor
LB medium was prepared at different pH (pH adjusted using NaOH and HCl).
Preparing culture solutions of different pollutants: 4mL of LB culture medium with different pH values, 1mL of bacterium solution and 5 mu L of 40000ppm of p-nitrophenol pollutant mother solution
The culture was carried out for 6h in a constant temperature shaker (30 ℃,180 rpm).
Samples were taken at 200. Mu.L and added to a black well plate, 3 replicates for each experimental group.
5. Fluorescence detection
The OD concentration of the bacterial liquid was measured at 30 ℃ by a microplate reader (Biotek) 600 。
Detecting the fluorescence intensity (FU) of the PNPgfp-1 strain at constant temperature of 30 ℃ by using a microplate reader, and calculating RFU = FU/OD 600 -FU CK /OD’ 600
The results are shown in fig. 5 and fig. 6, compared with the constructed PNPgfp-2 recombinant strain, the PNPgfp-1 recombinant strain constructed by the invention, as a microbial fluorescence sensor for sensing pollutant molecules, has different changes of RFU value under the action of different concentrations of p-nitrophenol, and the higher the concentration of p-nitrophenol is, the higher the RFU value is, the more obvious the detection effect is, and the linear relation is shown in 10-100 ppm. As can be seen from FIG. 7, the PNPgfp-1 recombinant strain produced a rapid response at 2 hours and the fluorescence intensity increased with time. The incubation time of the microbial fluorescence sensor was selected to be 6 hours based on the response time and fluorescence intensity. The results are shown in FIG. 8 and FIG. 9, and the PNPgfp-1 recombinant strain can specifically respond to p-nitrophenol and express strong fluorescent signals, and can maintain the pollutant responsiveness of the strain in a wider pH range.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. A recombinant plasmid is characterized in that pBBR-MCS2 is used as a starting vector, and a P-nitrophenol sensing element P is inserted between BamH I and Xho I sites R -pnpR-P C1 And green fluorescent protein gene gfp, wherein the promoter P R The nucleotide sequence of (A) is shown as SEQ ID NO.1, the nucleotide sequence of regulatory protein PnpR is shown as SEQ ID NO.2, and the promoter P C1 The nucleotide sequence of (A) is shown in SEQ ID NO. 3.
2. The recombinant plasmid of claim 1, wherein the gfp sequence of the green fluorescent protein gene is shown as SEQ ID No. 4.
3. The recombinant plasmid of claim 1, wherein the P-nitrophenol sensing element P R -pnpR-P C1 The nucleotide sequence of (A) is shown in SEQ ID NO. 5.
4. The recombinant plasmid according to any one of claims 1 to 3, wherein the recombinant plasmid is constructed mainly by:
(1) Amplifying P-nitrophenol inductive element P by taking total DNA of P-nitrophenol efficient degradation strain DLL-E4 genome as template R -pnpR-P C1 ；
(2) Artificially synthesizing or amplifying a green fluorescent protein gfp gene by PCR;
(3) With recovered P R -pnpR-P C1 And gfp as template to amplify the fusion fragment P R -pnpR-P C1 -gfp, purified again for recovery;
(4) The plasmid vector pBBR-MCS2 is subjected to double digestion by BamHI and Xho I, and the plasmid digestion fragment and the fusion fragment are subjected to seamless connection according to the molar ratio of 1: 2 to obtain the plasmid vector.
5. The recombinant plasmid according to claim 4, wherein P is amplified R -pnpR-P C1 The primer sequence of (A) is shown as SEQ ID NO.12, SEQ ID NO.13,SEQ ID NO.14 and SEQ ID NO.15.
6. A preparation method of a microbial fluorescence sensor capable of linearly responding to p-nitrophenol is characterized by comprising the following steps:
(1) The recombinant plasmid of any one of claims 1 to 5 is transformed into pseudomonas putida KT2440 competent cells, and positive clones are screened on an antibiotic-containing LB solid plate to obtain a recombinant plasmid-containing pBBR-P R -pnpR-P C1 -gfp engineering bacteria PNPgfp-1;
(2) Culturing engineering bacteria PNPgfp-1 in LB liquid culture medium containing kanamycin, activating and growing to OD 600 And =0.2, finally obtaining the microbial fluorescence sensor.
7. The microbial fluorescence sensor capable of linearly responding to p-nitrophenol, obtained by the preparation method according to claim 6.
8. The use of the microbial fluorescence sensor of claim 6 for the real-time detection of p-nitrophenol.
9. The application of the fluorescent probe as claimed in claim 8, wherein the microbial fluorescence sensor and a sample to be detected are mixed uniformly according to a volume ratio of 1.
10. Use of the microbial fluorescence sensor of claim 8 for the detection of contaminant molecules, wherein the microbial fluorescence sensor exhibits a linear response in the contaminant molecule concentration range of 10ppm to 100ppm.
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