An optimized promoter PyqjfC55 and its application in the preparation of a high-temperature resistant microbial sensor

Abstract

The present invention discloses an optimized promoter PyqjfC55 and its application in the preparation of a high-temperature-resistant microbial sensor. In the present invention, the promoter yqjfC55 is mutated by error-prone PCR to obtain the optimized promoter PyqjfC55, and the yellow fluorescent protein EYFP is ligated by a fusion enzyme, and finally a high-temperature-resistant and highly sensitive microbial sensor capable of sensing the explosive molecule 2,4-DNT in a high-temperature environment is obtained. The optimum culture temperature of the Escherichia coli host in the present invention is 37 °C, and the effective working temperature is 42 °C, and it has the ability to maintain metabolic activity in a high-temperature detection environment. The biosensor provided by the present invention realizes the high-sensitivity detection of explosive molecules in a high-temperature environment, improves the sensitivity of the detector for detecting explosive molecules, and expands the application scenarios.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and molecular biology, and particularly relates to an optimized promoter PyqjfC55 and its application in the preparation of high-temperature-resistant microbial sensors. Background Art

[0002] Explosives (such as TNT, RDX, 2,4-DNT, etc.) pose a serious threat to public safety and ecological health due to their high toxicity and widespread environmental residues. Although traditional detection methods (such as gas chromatography and mass spectrometry) have high precision, they rely on complex pretreatment processes and expensive equipment, making it difficult to meet the requirements of on-site rapid detection. Therefore, the development of efficient and portable detection technologies has become an urgent need. A microbial sensor is a biosensor that combines a living microorganism with a signal conversion element. Its core principle is to use the metabolic activities or specific recognition abilities of microorganisms to convert the presence of a target substance into a detectable electrical signal or optical signal. The development of such sensors began in the 1960s. With the progress of immobilization technology, the stability and sensitivity of microbial sensors have been significantly improved, and they have gradually been applied in fields such as environmental monitoring, medical diagnosis, and industrial process control.

[0003] The advantages of microbial sensors lie in their high selectivity and environmental adaptability. For example, certain microorganisms can generate electrochemical signals by metabolizing specific compounds (such as nitroaromatic explosives), or enhance the specific response to target molecules through genetic engineering modification. In addition, microbial sensors have the characteristics of low cost, simple operation, and real-time monitoring, making their application potential in complex environments huge. However, current microbial sensors are limited by the survival environment of microorganisms and thus cannot meet some extreme environments. Summary of the Invention

[0004] The object of the present invention is to provide an optimized promoter PyqjfC55 and its application in the preparation of high-temperature-resistant microbial sensors. The present invention first optimizes the original yqjfC55 promoter to obtain the high-temperature-resistant promoter PyqjfC55, and uses the optimized promoter and the yellow fluorescent protein EYFP to prepare a biosensor capable of detecting explosive molecules under high-temperature conditions, realizing rapid fluorescence detection of explosives under high-temperature conditions.

[0005] To achieve the above object of the invention, the present invention is implemented by the following technical solutions:

[0006] The present invention provides an optimized promoter PyqjfC55, whose nucleotide sequence is shown in SEQ ID NO.3.

[0007] The present invention provides the application of the above-mentioned optimized promoter PyqjfC55 in the preparation of high-temperature-resistant microbial sensors.

[0008] Further, the application includes the following steps:

[0009] (1) Amplify yellow fluorescent protein EYFP and promoter PyqjfC55 by PCR respectively. Connect the two purified and recovered fragments EYFP and PyqjfC55 with plasmid pACYC. Transform the ligation product into competent E. coli cells and screen for positive clones on an LB solid plate containing antibiotics to obtain recombinant plasmid pACYC-PyqjfC55-EYFP.

[0010] (2) Chemically transform recombinant plasmid pACYC-PyqjfC55-EYFP into competent E. coli cells and screen on an LB solid plate at 37 °C with a tetracycline resistance concentration of 12.5 μg / mL. Introduce the obtained positive clones into E. coli cells to obtain a high-temperature resistant microbial sensor containing the optimized promoter PyqjfC55.

[0011] Further, the mass ratio of fragment EYFP, fragment PyqjfC55 to plasmid pACYC is 1:1:5.

[0012] Further, the nucleotide sequence of promoter yqjfC55 is as shown in SEQ ID NO.1.

[0013] Further, the nucleotide sequence of yellow fluorescent protein EYFP is as shown in SEQ ID NO.2.

[0014] Further, the nucleotide sequence of the optimized PyqjfC55 promoter is as shown in SEQ ID NO.3.

[0015] The present invention also provides the application of the high-temperature resistant microbial sensor in detecting explosive molecules in a high-temperature application environment.

[0016] Further, the usage method of the microbial sensor is as follows: Mix the high-temperature resistant biosensor and the sample to be tested at a volume ratio of 8 - 10:1, incubate at 40 - 42 °C, sample once every hour for a total of ten hours, and detect the fluorescence intensity value using an enzyme-linked immunosorbent assay (ELISA) reader. The higher the concentration of the sample to be tested, the higher the fluorescence intensity.

[0017] Further, the sample to be tested is explosive molecule 2,4-DNT, and its concentration is 0.1 mg / L or above.

[0018] Further, the detection concentration range of 2,4-DNT is 0.1 mg / L - 10 mg / L.

[0019] Further, the high-temperature application environment includes deserts, mountains, and grasslands.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] 1. By molecularly modifying the specific promoter of Escherichia coli, the present invention enables it to have high tolerance and high sensitivity under extreme high-temperature environments. The present invention uses Escherichia coli as the host cell, with an optimal growth temperature of 37 °C, and it can stably exist at a high temperature of 42 °C.

[0022] 2. The present invention uses the plasmid containing the mutated PyqjfC55 promoter and the yellow fluorescent protein EYFP as the basic plasmid to prepare a biosensor for detecting explosive molecules under high-temperature conditions. Therefore, compared with common microbial sensors, the biosensor of the present invention can quickly and sensitively detect explosive molecules under high-temperature conditions and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the plasmid map of the constructed vector pACYC-PyqjfC55-EYFP.

[0024] Figure 2 It is the screening result of yellow fluorescence of the mutant strain.

[0025] Figure 3 It is the detection result of yellow fluorescence of the constructed engineering strain.

[0026] Figure 4 It is the schematic diagram of the mutation sites of the optimized promoter PyqjfC55. DETAILED DESCRIPTION OF THE INVENTION

[0027] The following further describes the present invention with reference to specific embodiments, but the present invention is not limited by the embodiments.

[0028] For those not specifying specific techniques or conditions in the embodiments, the techniques described in the literature in this field or according to the product specifications are followed. For reagents or instruments not specifying the manufacturer, they are all conventional products that can be obtained by purchase.

[0029] Example 1: Obtaining of Genes and Construction of Vectors

[0030] 1. Obtaining of Genes

[0031] The yellow fluorescent protein EYFP, whose nucleotide sequence is as shown in SEQ ID NO.2, was synthesized by GenScript onto the general vector pUC-Amp vector to obtain the pUC-EYFP vector.

[0032] 2. Construction of the pACYC-yqjfC55-EYFP Expression Vector

[0033] (1)Using the pUC-yqjfC55 plasmid (the plasmid p-yqjF cited from Patent CN 113005070B) as a template, with primers C55-F and C55-R, perform polymerase chain reaction (PCR) to amplify the yqjfC55 promoter fragment. The PCR amplification system is as follows: template 1 μL; primer C55-F 2 μL; primer C55-R 2 μL; 2 × Phanta Max Master Mix (Dye Plus) (Vazyme) 25 μL; ultra-pure water to make up to 50 μL.

[0034] The PCR amplification program is: 95ºC for 3 min; 30 cycles × (95ºC for 15 s, 61ºC for 15 s, 72ºC for 30 s); 72ºC for 5 min; 16ºC ∞.

[0035] The primer sequences are as follows:

[0036] C55-F:

[0037] 5’-GTTTTTTTACCTCCTGAGCTCGCCACTCAGGCTGCTGATT-3’ (SEQ ID NO.4);

[0038] C55-R:

[0039] 5’-GGGATCCCCGGGTACCCGGTTTTGGCGTAT-3’ (SEQ ID NO.5);

[0040] The PCR product is purified by gel extraction using a gel extraction purification kit (Vazyme).

[0041] (2)Using pUC-EYFP as a template, with primers EYFP-F and EYFP-R, perform polymerase chain reaction (PCR) to amplify the EYFP fragment. The PCR amplification system is as follows: template 1 μL; primer EYFP-F 2 μL; primer EYFP-R 2 μL; 2× Phanta Max Master Mix (Dye Plus) (Vazyme) 25 μL; ultra-pure water to make up to 50 μL.

[0042] The PCR amplification program is: 95ºC for 3 min; 30 cycles × (95ºC for 15 s, 57ºC for 15 s, 72ºC for 30 s); 72ºC for 5 min; 16ºC ∞.

[0043] The primer sequences are as follows:

[0044] EYFP-F:

[0045] 5’-CTCACTATAGGGCGAATTCGAATTACTTGTACAGCTCGTCCATGCC-3’ (SEQ ID NO.6);

[0046] EYFP-R:

[0047] 5’-AGGAGGTAAAAAAACATGGTGAGCAAGGGC-3’ (SEQ ID NO.7).

[0048] The PCR products were purified by gel extraction using a Gel Extraction Purification Kit (Vazyme).

[0049] (3) Cloning the yqjfC55 and EYFP fragments into the pACYC plasmid using seamless cloning: The two fragments EYFP and yqjfC55 and the plasmid pACYC were fused and ligated at a mass ratio of 1:1:5 using the seamless cloning enzyme 2 × ClonExpress Mix (Vazyme).

[0050] The ligation system was incubated at 50 ºC for 30 min. The ligation products were transformed E. coli into DH5α competent cells, spread on LB solid plates containing 12.5 mg / L tetracycline resistance, and positive clones were screened by PCR. The recombinant plasmid pACYC-yqjfC55-EYFP was extracted from the positive clones, and then positive screening and sequencing were performed by PCR for identification.

[0051] Example 2: Obtaining the sensor plasmid

[0052] The recombinant plasmid pACYC-yqjfC55-EYF was transformed Escherichia coli MG1655 into competent cells, spread on LB solid plates containing 12.5 mg / L tetracycline resistance, and positive clones were obtained by PCR screening, thereby obtaining the pACYC-C55-EYFP plasmid and the engineering bacterium G4.

[0053] Example 3: Molecular modification to optimize the promoter yqjfC55 gene to improve the high-temperature tolerance and sensitivity of the microbial sensor

[0054] 1. Obtaining the mutant PyqjfC55 promoter gene fragment by error-prone PCR

[0055] The yqjfC55 promoter gene in plasmid pACYC-yqjfC55-EYFP was amplified using the following primers. By using a low-fidelity enzyme, base pair changes occurred during the amplification of the yqjfC55 promoter gene to achieve the purpose of mutating the yqjfC55 promoter. The PCR amplification system is as follows: Template 1 μL; C55-F 2 μL; C55-R 2 μL; 2 × Rapid Taq Master Mix (Vazyme) 25 μL; Ultra-pure water to make up 50 μL.

[0056] The PCR amplification program was: 95ºC for 3 min; 10 cycles × (95ºC for 15 s, 60ºC for 15 s, 72ºC for 5 min); 72ºC for 5 min; 95ºC for 3 min; 30 cycles × (95ºC for 15 s, 59ºC for 15 s, 72ºC for 2 min); 72ºC for 5 min; 16ºC ∞.

[0057] The primer sequences are as follows:

[0058] C55-F:

[0059] 5’-GTTTTTTTACCTCCTGAGCTCGCCACTCAGGCTGCTGATT-3’ (SEQ ID NO.4);

[0060] C55-R:

[0061] 5’-GGGATCCCCGGGTACCCGGTTTTGGCGTAT-3’ (SEQ ID NO.5); The obtained PCR product was purified using the Novizan kit FastPure Gel DNA Extraction Mini Kit (Vazyme).

[0062] 2. Using the pACYC-yqjfC55-EYFP plasmid as a template, primers C55-RF and C55-RR, polymerase chain reaction (PCR) was carried out to amplify the pACYC-EYFP framework. The PCR amplification system is as follows: Template 1 μL; Primer 1 2 μL; Primer 2 2 μL; 2 × Phanta Max Master Mix (Dye Plus) (Vazyme) 25 μL; Ultra-pure water to make up 50 μL.

[0063] The PCR amplification program was: 95ºC for 3 min; 30 cycles × (95ºC for 15 s, 61ºC for 15 s, 72ºC for 5 min); 72ºC for 5 min; 16ºC ∞.

[0064] C55 - RF: 5’ - GTTTTTTTACCTCCT - 3’ (SEQ ID NO.8);

[0065] C55 - RR: 5’ - GTACCCGGGGATCCCGAT - 3’(SEQ ID NO.9)

[0066] The obtained PCR products were purified using the Novizan kit FastPure Gel DNA Extraction Mini Kit (Vazyme).

[0067] 3. The mutated PyqjfC55 fragment was cloned into the pACYC - EYFP plasmid using seamless cloning. The system is as follows: plasmid digestion fragment 80 ng; PCR product fragment 200 ng; 2 × ClonExpress Mix (Vazyme) 5 μL; made up to 10 μL with ultrapure water. The ligation system was incubated at 50 ºC for 30 min. The ligation product was transformed E. coli MG1655 into competent cells and spread on an LB solid plate containing 12.5 mg / L tetracycline resistance.

[0068] Example 4: Screening of the heat - resistant PyqjfC55 promoter

[0069] 1. Strain activation and culture

[0070] Single colonies on the above - mentioned plate were picked and transferred to a 48 - well plate containing LB liquid medium with 12.5 mg / L tetracycline resistance. One single colony was inoculated into each well and cultured overnight at 37 °C to obtain mutant engineering bacteria.

[0071] 1% of the bacterial solution was transferred to a 48 - well plate containing M9 liquid medium with 12.5 mg / L tetracycline resistance and cultured until the OD reached 0.2; 10 mg / L of 2,4 - DNT was added to the bacterial solution in the 48 - well plate, and then cultured on a shaker at 42 °C.

[0072] 2. Preparation of 2,4 - DNT solution

[0073] Prepare a 2 mg / mL stock solution of 2,4 - DNT (10 mg of 2,4 - DNT dissolved in 5 mL of absolute ethanol);

[0074] Prepare the diluted 2,4 - DNT solution according to the following ratio:

[0075] 10 mg / L: 5 μL of the stock solution + 980 μL of M9 medium + 15 μL of absolute ethanol;

[0076] 3. Luminescence detection

[0077] Each sample was detected for the fluorescence intensity of the strain using a microplate reader (Biotek) at 3 h, and the mutant engineering strains with the largest fluorescence difference, namely strains G1, G2, G3, and G4, were screened out.

[0078] The results were as Figure 2 , indicating that among the screened mutant strains G1, G2, G3, and G4, G1 had the best effect under the induction of a 10 mg / L 2,4-DNT concentration at 42°C and could form an obvious gap with the control group G1. At the same time, an obvious gap could be formed when there was a 10 mg / L 2,4-DNT explosive molecule and when there was no 2,4-DNT explosive molecule. Thus, the high-temperature-resistant microbial sensor G1 was obtained.

[0079] Example 5: Application of a microbial self-luminescent bioreactor for detecting explosive molecules under high-temperature conditions

[0080] 1. Strain activation and culture

[0081] The engineered strain G1 with correct sequencing was transferred to an LB liquid medium containing 12.5 mg / L tetracycline and cultured overnight at 37°C to obtain a bacterial solution.

[0082] 200 μL of the bacterial solution was added to 10 mL of an LB liquid medium containing 12.5 mg / L tetracycline resistance, and the culture was carried out on a shaker at 42°C.

[0083] 2. Preparation of 2,4-DNT solution

[0084] Prepare a 2 mg / mL stock solution of 2,4-DNT (10 mg of 2,4-DNT dissolved in 5 mL of absolute ethanol);

[0085] Prepare the diluted 2,4-DNT solution according to the following ratio:

[0086] 10 mg / L: 5 μL of the stock solution + 980 μL of M9 medium + 15 μL of absolute ethanol;

[0087] 5 mg / L: 2.5 μL of the stock solution + 980 μL of M9 medium + 17.5 μL of absolute ethanol;

[0088] 1 mg / L: 0.5 μL of the stock solution + 980 μL of M9 medium + 19.5 μL of absolute ethanol;

[0089] 0 mg / L: 980 μL of ASYE medium + 20 μL of absolute ethanol.

[0090] Make the ethanol concentration in each DNT solution 2%.

[0091] 3. Sample addition

[0092] Take 90 μL of the bacterial solution + 10 μL of the 2,4-DNT stock solution at concentrations of 0 mg / L, 1 mg / L, 5 mg / L, and 10 mg / L and add them to 1.5 ml EP tubes, so that the final concentrations of 2,4-DNT are 0 mg / L, 0.1 mg / L, 0.5 mg / L, and 1 mg / L respectively. Take 99 μL of the bacterial solution + 1 μL of the 2,4-DNT stock solution at concentrations of 1 mg / L and 5 mg / L and add them to 1.5 ml EP tubes, so that the final concentrations of 2,4-DNT are 0.01 mg / L and 0.05 mg / L respectively. Do 4 replicates for each concentration. Incubate at 42 °C respectively.

[0093] 4. Luminescence detection

[0094] Take samples from each sample once every hour for a total of 10 hours. Detect the fluorescence intensity of strain G1 with a microplate reader (Biotek).

[0095] 5. Sequence the mutated promoter PyqjfC55 contained in strain G1 through a biological company (Sangon Biotech, Shanghai). It is found that there are three base mutations compared with yqjfC55 as Figure 4 . The nucleotide sequence of the mutated promoter PyqjfC55 is shown in SEQ ID NO.3.

[0096] The results are as Figure 3 , indicating that the G1 recombinant strain constructed by the present invention, as a biosensor that can detect explosive molecules under high-temperature conditions, has different fluorescence intensities under the action of different concentrations of DNT. The higher the DNT concentration, the more obvious the detection effect. The fluorescence effect induced by 2,4-DNT explosive molecules at a minimum of 0.1 mg / L can form a difference from that without induction ( Figure 3 ). Moreover, at 42 °C, explosive molecules can be stably and effectively detected.

[0097] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, for those of ordinary skill in the art, it is still possible to modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions required to be protected by the present invention.

Claims

1. An optimized promoter PyqjfC55, characterized in that, Its nucleotide sequence is as shown in SEQ ID NO.

3.

2. Use of the optimized promoter PyqjfC55 according to claim 1 in the preparation of a high-temperature resistant microbial sensor.

3. The application according to claim 2, wherein The said use comprises the following steps: (1) Respectively amplify yellow fluorescent protein EYFP and promoter PyqjfC55 by PCR, ligate the two purified and recovered fragments EYFP and PyqjfC55 with plasmid pACYC, transform the ligation product into Escherichia coli competent cells, and screen for positive clones on an LB solid plate containing antibiotics to obtain the recombinant plasmid pACYC-PyqjfC55-EYFP; (2) Transform the recombinant plasmid pACYC-PyqjfC55-EYFP into Escherichia coli competent cells, screen on an LB solid plate at 37°C with a tetracycline resistance concentration of 12.5 μg / mL, and the obtained positive clone is the pACYC-C55-EYFP plasmid, which is then introduced into Escherichia coli cells to obtain an engineered strain of a high-temperature resistant microbial sensor containing the optimized promoter PyqjfC55.

4. The application according to claim 3, wherein The mass ratio of the fragment EYFP, the fragment PyqjfC55 and the plasmid pACYC is 1:1:

5.

5. The application according to claim 3, wherein The nucleotide sequence of the coding gene of the said EYFP is as shown in SEQID NO.

2.

6. Use of the high-temperature resistant microbial sensor prepared according to claim 3 for detecting explosive molecules in a high-temperature application environment, characterized in that, The method of using the said microbial sensor is: after mixing the engineered strain containing the high-temperature resistant biosensor with the sample to be detected, incubate at 40 - 42°C, sample once every hour for a total of ten hours, detect the fluorescence intensity value using a microplate reader, and detect the concentration of the sample to be detected according to the fluorescence intensity. The said sample to be detected is the explosive molecule 2,4-DNT, and the host of the said engineered strain is Escherichia coli.

7. The application according to claim 6, characterized in that, The detection concentration range of the said 2,4-DNT is 0.1 mg / L to 10 mg / L.

8. The application according to claim 6, wherein The said high-temperature application environment includes deserts, mountains, and grasslands.

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