CN115896154A - A microbial fluorescence sensor for detecting p-nitrophenol and its preparation method and application - Google Patents

Abstract

The invention discloses a microbial fluorescence sensor, a preparation method and an application thereof. The microorganism fluorescence sensor comprises a sensing element containing a promoter P _C1 and its regulatory gene pnpR and a reporter element containing a green fluorescent protein GFP. The microbial fluorescence sensor can specifically respond to different concentrations of p-nitrophenol to generate fluorescence with different RFU values, and couple the p-nitrophenol concentration with the self-luminous RFU value. The microbial fluorescence sensor of the invention can realize the specific identification and detection of p-nitrophenol, has the advantages of simple operation, high sensitivity, and can linearly detect pollutant molecules in a low concentration range, so it has a good application prospect.

Description

A microbial fluorescence sensor for detecting p-nitrophenol and its preparation method and application

Technical Field

The invention belongs to the technical field of genetic engineering and molecular biology, and specifically relates to a microbial fluorescence sensor for detecting p-nitrophenol and a preparation method and application thereof.

Background Art

p-Nitrophenol (PNP) is mainly derived from pesticides, dyes, medicines, rubber and other fields. It has "three-hazard" effects and is identified as an environmental endocrine disruptor and is listed as a priority controlled pollutant in my country. Therefore, the effective detection of p-nitrophenol is of great significance to people's health and environmental protection.

At present, the common methods for the determination of p-nitrophenol are: spectroscopy, electrochemical method, gas chromatography, liquid chromatography, chromatography-mass spectrometry, etc. Most of these chemical analysis methods have the problems of cumbersome operation, expensive instruments, and high analysis cost. In recent years, microbial sensors have become a hot topic of research as a new technology for environmental monitoring due to their good selectivity, high sensitivity, ease of use, low cost, and continuous online monitoring in complex systems. Microorganisms are genetically modified by genetic engineering methods, so that they respond to certain special toxic substances or physical stresses in the environment and produce measurable signals, ultimately realizing the detection and quantification of specific chemicals in the air, soil or water.

Microbial sensors are mainly composed of sensing elements and reporter genes. The sensing elements are usually composed of specific recognition protein genes and promoter elements related to the detected compounds, which determine the specificity of the system; while the reporter gene does not require specific recognition, and its expression level and intensity reflect the sensitivity and detection threshold of the system. At present, although there are many studies on the degradation characteristics, metabolic pathways and regulatory mechanisms of p-nitrophenol-degrading strains, the discovery of p-nitrophenol-specific response elements has limited the development of p-nitrophenol microbial sensors.

Summary of the invention

In order to solve the above technical problems, the present invention provides a preparation method and application of a microbial fluorescent sensor synthesized by using the specific response element of an efficient degradation strain. The microbial fluorescent sensor contains a regulatory protein and its corresponding promoter and green fluorescent protein, which can realize specific linear detection of p-nitrophenol with high sensitivity, is suitable for environmental monitoring, and has broad application prospects.

The present invention is implemented by the following technical solutions:

A recombinant plasmid is obtained by using pBBR-MCS2 as a starting vector and inserting a p-nitrophenol sensing element _PR -pnpR- _PC1 and a green fluorescent protein gene gfp between the BamH I and Xho I sites, wherein the nucleotide sequence of the promoter _PR is shown in SEQ ID NO.1, the nucleotide sequence of the regulatory protein PnpR is shown in SEQ ID NO.2, and the nucleotide sequence of the promoter _PC1 is shown in SEQ ID NO.3.

As a preferred embodiment of the present invention, the green fluorescent protein gene gfp sequence is shown as SEQ ID NO.4.

As a preferred embodiment of the present invention, the nucleotide sequence of the p-nitrophenol sensing element _PR -pnpR-P _C1 is shown in SEQ ID NO.5.

As a further preference of the present invention, the recombinant plasmid is mainly constructed by the following method:

(1) Using the total genomic DNA of the p-nitrophenol efficient degradation strain DLL-E4 as a template, the p-nitrophenol response element _PR -pnpR-P _C1 was amplified;

(2) Amplify the green fluorescent protein (GFP) gene by PCR;

(3) using the recovered _PR -pnpR- _PR1 and gfp as templates to amplify the fusion fragment PR- _pnpR - _PR1 -gfp, which was purified and recovered again;

(4) The plasmid vector pBBR-MCS2 was double-digested with BamH I and Xho I, and the plasmid digested fragment and the fusion fragment were seamlessly connected at a molar ratio of 1:2.

As a further preferred embodiment of the present invention, the primer sequences for amplifying _PR -pnpR- _PC1 are SEQ ID NO.7 and SEQ ID NO.8.

A method for preparing a microbial fluorescence sensor for detecting p-nitrophenol comprises the following steps:

(1) The recombinant plasmid of the present invention is transformed into KT2440 competent cells, and positive clones are screened on an LB solid plate containing antibiotics to obtain an engineered bacterium PNPgfp-1 containing the recombinant plasmid pBBR- _PR -pnpR-P _C1 -gfp;

(2) The engineered bacteria PNPgfp-1 was cultured in LB liquid culture medium containing kanamycin and activated to grow to OD ₆₀₀ = 0.2, and finally a microbial fluorescent sensor was obtained.

The microbial fluorescence sensor capable of linearly responding to p-nitrophenol is obtained according to the preparation method of the present invention.

Application of the microbial fluorescence sensor of the present invention in real-time detection of p-nitrophenol.

As a preferred embodiment of the present invention, the microbial fluorescence sensor and the sample to be tested are mixed at a volume ratio of 1:4, reacted for 6 hours, and then the sample is added to a black transparent ELISA plate, and the fluorescence intensity RFU value is monitored by an ELISA instrument.

As a preferred embodiment of the present invention, the concentration of pollutant molecules that can be sensed by the microbial fluorescence sensor is 10 ppm-100 ppm.

Furthermore, the microbial fluorescent biosensor can sense pollutant molecules of different concentrations, thereby generating fluorescence with different RFU values, and coupling the pollutant molecule concentration with the self-luminous RFU value to achieve the purpose of real-time monitoring of pollutant molecules.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

The present invention uses pollutant-tolerant strain KT2440 as chassis cells, screens and obtains the regulatory unit pnpR-P _C1 from the p-nitrophenol efficient degradation strain DLL-E4 as a sensing element, connects GFP protein as a reporter protein, and prepares a microbial fluorescent sensor. The biosensor can specifically respond to p-nitrophenol in a short time, thereby generating fluorescence with different RFU values, and is linearly related within a certain concentration range, thereby realizing the fluorescent detection of pollutant molecules. Compared with the existing p-nitrophenol detection method, the biosensor has a rapid response, high specificity and sensitivity, and strong environmental adaptability, so it has a relatively broad application prospect in the field of p-nitrophenol detection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the growth of Escherichia coli DH5α at different p-nitrophenol concentration levels.

Figure 2 shows the growth of Pseudomonas putida KT2440 at different p-nitrophenol concentration levels.

Fig. 3 is a plasmid map of the recombinant plasmid pBBR- _PR -pnpR-P _C1 -gfp.

Fig. 4 is a plasmid map of the recombinant plasmid pBBR- _PR1 -pnpR1- _PA -gfp.

FIG5 shows the fluorescence detection result data of the constructed sensor strain PNPgfp-1.

FIG6 shows the fluorescence detection result data of the constructed sensor strain PNPgfp-2.

FIG7 shows the fluorescence detection data of the constructed sensor strain PNPgfp-1 under fixed pollutant concentration conditions within 12 h.

FIG8 shows the pollutant-specific fluorescence detection result data of the constructed sensor strain PNPgfp-1.

FIG9 shows the fluorescence detection data of the constructed sensor strain PNPgfp-1 under different pH conditions.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited by the embodiments.

If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased.

Example 1: Screening of chassis cells

Prepare p-nitrophenol mother solutions of different concentrations (120000ppm, 60000ppm, 30000ppm); use anhydrous ethanol as the solvent.

Preparation of p-nitrophenol culture medium of different concentrations: 4 mL LB medium + 1 mL bacterial solution + 5 μL p-nitrophenol stock solution of corresponding concentration.

DH5α and KT2440 strains (ATCC47057) with OD ₆₀₀ = 1 were inoculated at a 1% inoculum amount and cultured in a constant temperature shaker (37° C. and 30° C., 180 rpm), respectively.

200 μL of samples were taken at regular intervals and added to a transparent ELISA plate. Three parallel plates were made to measure the OD ₆₀₀ at different times to detect the growth of different strains.

The results are shown in Figures 1 and 2. Compared with strain DH5α, strain KT2400 can tolerate high concentrations of p-nitrophenol, so KT2440 was selected as the chassis cell to construct the biosensor.

Example 2: Gene acquisition and vector construction

1. Gene acquisition

PR, pnpR and PC1 genes were amplified from the nitrophenol efficient degrading strain DLL-E4 (Liu Zhi, Hong Qing et al. Degradation characteristics of p-nitrophenol by methyl parathion-degrading bacteria DLL-E4 [J]. China Environmental Science, Issue ₀₀₄ , 2003, 435-439), and their nucleotide sequences are shown in SEQ _{ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. The nucleotide sequences of PR1, pnpR1 and PA genes are} shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8. The primers are RF and RR, _PC1 -F and _PC1 -R, R1-F and R1-R, _PA -F and _PA -R, respectively. The template is DLL-E4 total DNA, and polymerase chain reaction (PCR) is performed. The PCR amplification system is as follows:

The PCR program was: 95°C for 5 min; 32 cycles × (94°C for 30 s, 55°C for 30 s, 72°C for 45 s); 72°C for 5 min; 4°C∞.

The primer sequences are as follows:

R-F:

5’-cgctctagaactagtggatccGGAAAGTGGCCGCTTCAAG-3’;

R-R:

5’-TCAGACGACCCAGCCACG-3’.

_PC1 -F:

5’-CATTGAGCAGTGTTCGCGTTT-3’;

_PC1 -R:

5’-CGCATTCACCGCACTTGTT-3’.

R1-F:

5’-cgctctagaactagtggatccCGGGATTTGTGGGCCATC-3’;

R1-R:

5’-TCAGCAGTTTTGCAAGGGCA-3’.

_PA -F:

5’-GTGAATTCCTGAATACGTCATTGTTAG-3’;

P _A -R:

5’-GTAGGCGACTGCCCGCGG-3’.

The PCR product was purified by gel recovery using a gel recovery kit (Vazyme).

Then, the recovered _PR -pnpR and _PC1 genes were used as templates, and RF and _PC1 -R were used as primers to perform polymerase chain reaction (PCR) to amplify the fusion fragment PR- _pnpR - _PC1 , whose nucleotide sequence is shown in SEQ ID NO.5. The PCR amplification system is as follows:

The PCR program was: 95°C for 5 min; 32 cycles × (94°C for 30 s, 55°C for 30 s, 72°C for 70 s); 72°C for 5 min; 4°C∞.

The above method was then used to amplify the fusion fragment _PR1- pnpR1- _PA using _PR1 -pnpR1 and _PA as templates and R1-F and _PA -R as primers. The nucleotide sequence of the fragment is shown in SEQ ID NO.9.

The PCR product was purified by gel recovery using a gel recovery kit (Vazyme).

The gfp gene was amplified from the plasmid pMD19-T-gfp containing green fluorescent protein GFP. The plasmid was purchased from Wuhan Miaoling Biotechnology Co., Ltd. The nucleotide sequence is shown in SEQ ID NO.4. The primers are gfp-F and gfp-R for polymerase chain reaction (PCR). The PCR amplification system is as follows:

The PCR program was: 95°C for 5 min; 35 cycles × (94°C for 30 s, 55°C for 30 s, 72°C for 30 s); 72°C for 5 min; 4°C∞.

The primer sequences are as follows:

Primer 1 (gfp-F):

5’-ATGAGTAAAGGAGAAGAACTTTTCACTG-3’;

Primer 2 (gfp-R):

5’-ggtaccgggccccccctcgagGTGTATCCAGGAGCTGTTACAACTCA-3’.

Then, the recovered _PR -pnpR-P _C1 and gfp gene were used as templates, RF and gfp-R were used as primers to amplify the fusion fragment _PR- pnpR-P _C1 -gfp, and _PR1 -pnpR1- _PA and gfp were used as templates, R1-F and gfp-R were used as primers to amplify the fusion fragment _PR1 -pnpR1- _PA -gfp, and polymerase chain reaction (PCR) was performed. The PCR amplification system is as follows:

The PCR program was: 95°C for 5 min; 32 cycles × (94°C for 30 s, 55°C for 30 s, 72°C for 70 s); 72°C for 5 min; 4°C∞.

The PCR product was purified by gel recovery using a gel recovery kit (Vazyme).

2. Construction of pBBR- _PR- pnpR-P _C1 -gfp and pBBR- _PR1 -pnpR1-PA _- gfp expression vectors

(1) The pBBR-MCS2 plasmid was double-digested with restriction endonuclease 1BamH I (Takara Bio, Catalog No. 1010S) and restriction endonuclease 2Xho I (Takara Bio, Catalog No. 1094S). The restriction digestion system was as follows:

The enzyme digestion system was incubated at 37°C for 3 h, and then the gel was recovered and purified.

_PR- pnpR-P _C1 -gfp and PR1 _- pnpR1- _PA- gfp fragments were cloned into the pBBR-MCS2 plasmid using seamless cloning. The system is as follows:

The ligation system was incubated at 37°C for 30 min. The ligation product was transformed into E. coli DH5α competent cells and spread on LB solid plates containing 50 mg/L kanamycin to screen positive clones. Recombinant plasmids pBBR- _PR -pnpR-P _C1 -gfp (Figure 3) and pBBR- _PR1- pnpR1-P _A -gfp (Figure 4) were extracted from the positive clones and then identified by restriction enzyme digestion 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-voltage electroporation, plated on LB solid plates containing 100 mg/L kanamycin, and positive clones were obtained by PCR screening, thereby obtaining the engineered strain PNPgfp-1 containing the vector pBBR- _PR- pnpR-P _C1- gfp and the engineered strain PNPgfp-2 containing the vector pBBR- _PR1 -pnpR1-P _A -gfp.

Example 4: Application of microbial fluorescence sensor to detect p-nitrophenol

1. Strain activation and cultivation

The correctly sequenced engineered strains PNPgfp-1 and PNPgfp-2 were respectively transferred to LB liquid culture medium containing 100 mg/L kanamycin and cultured at 30° C. overnight to obtain bacterial suspension.

2. Dose effect detection of sensors

Prepare p-nitrophenol mother solutions of different concentrations (100000ppm, 80000ppm, 60000ppm, 50000ppm, 40000ppm, 30000ppm, 20000ppm, 10000ppm); use anhydrous ethanol as the solvent.

Preparation of p-nitrophenol culture medium with different concentrations: 4 mL LB medium + 1 mL bacterial solution + 5 μL p-nitrophenol mother solution of corresponding concentration to obtain culture medium with p-nitrophenol concentrations of 100 ppm, 80 ppm, 60 ppm, 50 ppm, 40 ppm, 30 ppm, 20 ppm, and 10 ppm, respectively.

The cells were cultured in a constant temperature shaker (30°C, 180 rpm) for 6 h.

Take 200 μL of the sample and add it to a transparent black ELISA plate. Make three parallels for each experimental group.

3. Specificity detection of sensors

Prepare 40000ppm of mother liquor of pollutants with similar structures (benzene, hydroquinone, catechol, p-nitrophenol, p-nitrobenzene); use anhydrous ethanol as solvent.

Preparation of culture medium for different pollutants: 4 mL LB medium + 1 mL bacterial solution + 5 μL corresponding pollutant mother solution to obtain culture medium for pollutants with different structures and similar structures with a final concentration of 40 ppm.

The cells were cultured in a constant temperature shaker (30°C, 180 rpm) for 6 h.

Take 200 μL of the sample and add it to a clear black ELISA plate. Make 3 parallels for each concentration.

3. Sensor response time detection

Configuration of pollutant culture medium: 4 mL LB medium + 1 mL bacterial solution + 5 μL p-nitrophenol stock solution (40000 ppm).

The culture was incubated in a constant temperature shaker (30°C, 180 rpm) for 12 h, and samples were taken every two hours for testing.

Take 200 μL of the sample and add it to a transparent black ELISA plate. Make three parallels for each experimental group.

4. pH adaptability test of the sensor

LB medium with different pH values was prepared (pH was adjusted using NaOH and HCl).

Different pollutant culture medium configuration: 4mL LB medium with different pH values + 1mL bacterial solution + 5μL 40000ppm p-nitrophenol pollutant stock solution

The cells were cultured in a constant temperature shaker (30°C, 180 rpm) for 6 h.

Take 200 μL of the sample and add it to a transparent black ELISA plate. Make three parallels for each experimental group.

5. Fluorescence detection

The concentration of bacterial solution OD ₆₀₀ was detected by using a microplate reader (Biotek) at a constant temperature of 30°C.

The fluorescence intensity (FU) of PNPgfp-1 strain was detected by microplate reader at 30℃, and RFU was calculated as FU/OD ₆₀₀ - FU _CK /OD' ₆₀₀

As shown in Figures 5 and 6, compared with the constructed PNPgfp-2 recombinant strain, the PNPgfp-1 recombinant strain constructed by the present invention, as a microbial fluorescent sensor for sensing pollutant molecules, shows different changes in its RFU value under the action of different concentrations of p-nitrophenol. The higher the concentration of p-nitrophenol, the higher the RFU value, the more obvious the detection effect, and a linear relationship is shown at 10-100ppm. As shown in Figure 7, the PNPgfp-1 recombinant strain can respond quickly in 2 hours, and the fluorescence intensity increases with time. Based on the response time and fluorescence intensity, the cultivation time of the microbial fluorescence sensor is selected to be 6 hours. As shown in Figures 8 and 9, the PNPgfp-1 recombinant strain can specifically respond to p-nitrophenol and express a strong fluorescence signal, and can maintain the pollutant response ability of the strain in a relatively wide pH range.

The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

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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