Mutant of bacillus subtilis transcriptional regulation protein PadR, gene of mutant, expression vector of gene, genetically engineered bacterium and application thereof

Abstract

The invention relates to the technical field of gene engineering, in particular to a mutant of a bacillus subtilis transcriptional regulatory protein PadR, a gene of the mutant, an expression vector of the gene, a genetically engineered bacterium and application of the genetically engineered bacterium, the mutant is PadR-M, the nucleotide sequence is shown as SEQ ID NO: 3, the amino acid sequence is shown as SEQ ID NO: 4, a parent of the mutant is wild type bacillus subtilis transcriptional regulatory protein PadR, and the mutant has a nucleotide sequence shown as SEQ ID NO: 5 and an amino acid sequence shown as SEQ ID NO: 6. The nucleotide sequence of the parent is as shown in SEQ ID NO: 1, and the amino acid sequence of the parent is as shown in SEQ ID NO: 2. According to the present invention, the mutant of the Bacillus subtilis transcriptional regulation protein PadR, the gene of the mutant, the expression vector of the gene, and the related gene engineering bacteria are used for constructing the special biosensor, and the rapid, sensitive and low-cost detection of the alternariol content is achieved by using the highly specific response of the biosensor to the alternariol.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and is a mutant of Bacillus subtilis transcriptional regulatory protein PadR, a gene of the mutant, an expression vector of the gene, a genetic engineering bacterium and an application thereof. Background Art

[0002] Alternariol is a common mycotoxin produced by Alternaria spp. and widely found in food and agricultural products. This toxin is potentially toxic and carcinogenic, so its detection and control are of great importance to public health.

[0003] Traditionally, the detection of spore phenol mainly relies on chemical analysis techniques such as high performance liquid chromatography (HPLC) and mass spectrometry. Although these methods can provide accurate measurement results, they usually require expensive equipment, complex sample preparation and professional operators, which limits their application in rapid field detection and large-scale screening.

[0004] Bacillus subtilis is a model Gram-positive bacterium widely used in genetic engineering. Its transcriptional regulatory protein PadR plays an important role in bacterial antitoxin response and drug resistance mechanisms. By engineering this protein, specific biosensors can be developed to detect specific chemicals in the environment. Therefore, it is necessary to develop a new biosensor based on the Bacillus subtilis transcriptional regulatory protein PadR to achieve rapid and sensitive detection of cross-linked spore phenol content. Summary of the invention

[0005] The present invention provides a mutant of Bacillus subtilis transcriptional regulatory protein PadR, a gene of the mutant, an expression vector of the gene, a genetically engineered bacterium and applications thereof, which overcome the deficiencies of the above-mentioned prior art and can effectively solve the problems of complex sample processing procedures, expensive detection equipment and excessively high professional requirements for operators in the existing cross-linked spore phenol detection.

[0006] One of the technical solutions of the present invention is achieved through the following measures: a mutant of the Bacillus subtilis transcriptional regulatory protein PadR, the mutant is PadR-M, the nucleotide sequence of PadR-M is shown in SEQ ID NO: 3, and the amino acid sequence of PadR-M is shown in SEQ ID NO: 4.

[0007] The following is a further optimization and / or improvement of one of the above-mentioned technical solutions: The mutants described above are alanine mutations at position 77, glutamate mutations at position 78, arginine mutations at position 79, and alanine mutations at position 80 of the parent of the Bacillus subtilis transcriptional regulatory protein PadR, or lysine mutations at position 77 to glutamine, glutamine mutations at position 78 to lysine, glutamate mutations at position 79 to lysine, and leucine mutations at position 80 to methionine of the parent of the Bacillus subtilis transcriptional regulatory protein PadR.

[0008] The parent of the Bacillus subtilis transcription regulatory protein PadR is a wild-type Bacillus subtilis transcription regulatory protein PadR, the nucleotide sequence of the parent is shown in SEQ ID NO: 1, and the amino acid sequence of the parent is shown in SEQ ID NO: 2.

[0009] The second technical solution of the present invention is achieved through the following measures: a gene encoding a mutant of the Bacillus subtilis transcriptional regulatory protein PadR.

[0010] The third technical solution of the present invention is achieved by the following measures: an expression vector carrying the gene of the mutant of the Bacillus subtilis transcriptional regulatory protein PadR, the expression vector comprising a pBAD / HisA vector.

[0011] The fourth technical solution of the present invention is achieved by the following measures: a genetically engineered bacterium expressing a mutant of the Bacillus subtilis transcriptional regulatory protein PadR, wherein the genetically engineered bacterium uses Escherichia coli as a starting strain, and the starting strains include E. coli MC1061 and BW25113.

[0012] The fifth technical solution of the present invention is achieved by the following measures: a genetically engineered bacterium containing the gene of the mutant of the Bacillus subtilis transcriptional regulatory protein PadR, wherein the genetically engineered bacterium uses Escherichia coli as a starting strain, and the starting strains include E. coli MC1061 and BW25113.

[0013] The sixth technical solution of the present invention is achieved by the following measures: a genetically engineered bacterium transformed with an expression vector of a gene carrying a mutant of the Bacillus subtilis transcriptional regulatory protein PadR, wherein the genetically engineered bacterium uses Escherichia coli as a starting strain, and the starting strains include E. coli MC1061 and BW25113.

[0014] The seventh technical solution of the present invention is achieved through the following measures: a genetically engineered bacterium expressing a mutant of the Bacillus subtilis transcriptional regulatory protein PadR, or a genetically engineered bacterium containing a gene of the mutant of the Bacillus subtilis transcriptional regulatory protein PadR, or a genetically engineered bacterium transformed with an expression vector carrying a gene of the mutant of the Bacillus subtilis transcriptional regulatory protein PadR in the detection of cross-linked spore phenol.

[0015] The eighth technical solution of the present invention is achieved by the following measures: use of a mutant of Bacillus subtilis transcriptional regulatory protein PadR, or a gene encoding a mutant of Bacillus subtilis transcriptional regulatory protein PadR, or an expression vector of a gene carrying a mutant of Bacillus subtilis transcriptional regulatory protein PadR, or a genetically engineered bacterium expressing a mutant of Bacillus subtilis transcriptional regulatory protein PadR, or a genetically engineered bacterium containing a gene of a mutant of Bacillus subtilis transcriptional regulatory protein PadR, or a genetically engineered bacterium transformed with an expression vector of a gene carrying a mutant of Bacillus subtilis transcriptional regulatory protein PadR in the construction of a biosensor that responds to cross-linked spore phenol.

[0016] The present invention applies mutants of Bacillus subtilis transcriptional regulatory protein PadR, genes of the mutants, gene expression vectors, and related genetically engineered bacteria to construct a special biosensor, and utilizes its highly specific response to cross-sporin to achieve rapid, sensitive and low-cost detection of cross-sporin content. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Attached Figure 1 This is the pPadR expression plasmid map obtained by extracting the plasmid of the recombinant bacteria in Example 10 of the present invention.

[0018] Attached Figure 2 This is a comparison diagram of the induction specificity of the whole-cell biosensor that can specifically respond to the induction of cross-linked spore phenol obtained in Example 10 of the present invention. DETAILED DESCRIPTION

[0019] The present invention is not limited by the following embodiments, and specific implementation methods can be determined according to the technical scheme of the present invention and actual conditions. Unless otherwise specified, the various chemical reagents and chemicals mentioned in the present invention are all known and commonly used chemical reagents and chemicals in the prior art.

[0020] The present invention will be further described below in conjunction with embodiments: Example 1: A mutant of the Bacillus subtilis transcriptional regulatory protein PadR, wherein the mutant is PadR-M, the nucleotide sequence of PadR-M is shown in SEQ ID NO: 3, and the amino acid sequence of PadR-M is shown in SEQ ID NO: 4.

[0021] Example 2: As an optimization of the above example, the mutant is a parent of the Bacillus subtilis transcriptional regulatory protein PadR with alanine mutation at position 77, glutamate mutation at position 78, arginine mutation at position 79, and alanine mutation at position 80; or, the mutant is a parent of the Bacillus subtilis transcriptional regulatory protein PadR with lysine (Lys) mutation at position 77 to glutamine (Gln), glutamine (Gln) mutation at position 78 to lysine (Lys), glutamate (Glu) mutation at position 79 to lysine (Lys), and leucine (Leu) mutation at position 80 to methionine (Met).

[0022] Example 3: As an optimization of the above example, the parent of the Bacillus subtilis transcriptional regulatory protein PadR is a wild-type Bacillus subtilis transcriptional regulatory protein PadR, the nucleotide sequence of the parent is shown in SEQ ID NO: 1, and the amino acid sequence of the parent is shown in SEQ ID NO: 2.

[0023] Example 4: The gene encoding the mutant of the Bacillus subtilis transcriptional regulatory protein PadR.

[0024] Example 5: The expression vector carrying the gene of the mutant of the Bacillus subtilis transcriptional regulatory protein PadR, the expression vector comprises a pBAD / HisA vector.

[0025] Example 6: The genetically engineered bacteria expressing the mutant of the Bacillus subtilis transcriptional regulatory protein PadR, the genetically engineered bacteria uses Escherichia coli as the starting strain, and the starting strains include E. coli MC1061 and BW25113.

[0026] Example 7: The genetically engineered bacteria containing the gene of the mutant of the Bacillus subtilis transcriptional regulatory protein PadR, the genetically engineered bacteria uses Escherichia coli as the starting strain, and the starting strains include E. coli MC1061 and BW25113.

[0027] Example 8: The genetically engineered bacteria transformed with an expression vector carrying a gene of a mutant of the Bacillus subtilis transcriptional regulatory protein PadR are derived from Escherichia coli as starting strains, including E. coli MC1061 and BW25113.

[0028] Example 9: Application of the genetically engineered bacteria expressing a mutant of Bacillus subtilis transcriptional regulatory protein PadR, or the genetically engineered bacteria containing a gene of a mutant of Bacillus subtilis transcriptional regulatory protein PadR, or the genetically engineered bacteria transformed with an expression vector carrying a gene of a mutant of Bacillus subtilis transcriptional regulatory protein PadR in detecting cross-linked spore phenol.

[0029] The signal intensity of the genetically engineered bacteria can be used to characterize the content level of the cross-linked spore phenol in the sample to be detected. The signal intensity of the genetically engineered bacteria is the fluorescence signal intensity of the fluorescent protein. The higher the signal intensity, the higher the content of the cross-linked spore phenol in the sample.

[0030] The signal strength testing method of the genetically engineered bacteria described in Example 9 of the present invention.

[0031] (1) Flow cytometry: Electroporate the plasmid of the PadR mutant library into MC1061 competent cells (about 10 7 After shaking for 45 minutes at 37°C, the resuscitation solution was added to 50 mL of LB medium containing Amp (ampicillin resistance), and 1 nM cross-linked spores were added, and cultured at 37°C for 12 hours. Take 1 mL of cell suspension, centrifuge at 3,000 × g for 10 minutes, discard the supernatant and resuspend in 1 mL PBS buffer. Take 200 μL of the suspension, mix with 1.8 mL PBS, and add to a 5 mL round-bottom test tube. Use flow cytometry for sorting (excitation wavelength 561 nm, emission wavelength 610 nm), and select cells with higher fluorescence intensity. The sorted cells were cultured in 3 mL LB medium for 6 hours, and the final OD 600 =0.2, and transferred again to 3 mL LB medium containing 1 nM spore spore for the second round of screening. After eight rounds of screening, 10,000 cells were collected for the final round of screening, 300 μL of the cell suspension was spread on the AmpR plate, single clones were picked, and retested in test tubes containing 0 nM, 0.01 nM, 0.1 nM, 1 nM and 10 nM spore spore spore.

[0032] (2) Multifunctional microplate reader: Select a single clone and add it to 3 mL LB medium containing 100 μg / mL Amp. Culture it in a shaking incubator at 37°C overnight as seed solution. The next day, transfer the clone at a 1% inoculation volume (final OD6 00 After culturing at 37 °C for 12 h, 200 μL of the cell suspension was taken and the OD was measured using an ELISA reader. 600 and RFP fluorescence intensity (test conditions: excitation wavelength 556 nm, emission wavelength 586 nm).

[0033] Example 10: Use of the mutant of Bacillus subtilis transcriptional regulatory protein PadR, or the gene encoding the mutant of Bacillus subtilis transcriptional regulatory protein PadR, or the expression vector of the gene carrying the mutant of Bacillus subtilis transcriptional regulatory protein PadR, or the genetically engineered bacteria expressing the mutant of Bacillus subtilis transcriptional regulatory protein PadR, or the genetically engineered bacteria containing the gene of the mutant of Bacillus subtilis transcriptional regulatory protein PadR, or the genetically engineered bacteria transformed with the expression vector of the gene carrying the mutant of Bacillus subtilis transcriptional regulatory protein PadR in the construction of a biosensor responsive to crosslinked spore phenol.

[0034] Example 11: Screening of PadR mutants and construction of biosensor.

[0035] The raw materials used in Example 11 are: (1) The pBAD / HisA vector in the expression vector was from Invitrogen.

[0036] (2) Escherichia coli MC1061 and BW25113 were purchased from Quanshijin Company.

[0037] Using pBAD / HisA vector as a template, PCR amplification was performed using 5'-AAGCTTGGCTGTTTTGGCGG-3' (upstream primer sequence as shown in SEQ ID NO: 5) and 5'-CTCGAGCTCGGATCCCCATC-3' (downstream primer sequence as shown in SEQ ID NO: 6) as primers. The genome of Bacillus subtilis was extracted as a template, and the parent gene of padR (the nucleotide sequence of the parent is shown in SEQ ID NO: 1) was amplified using 5'-GATGGGGATCCGAGCTCGAGATGAGAGTATTAAAATACGC-3' (upstream primer sequence as shown in SEQ ID NO: 7) and 5'-CCGCCAAAACAGCCAAGCTTTTAATCCTTATCTATCATAG-3' (downstream primer sequence as shown in SEQ ID NO: 8) as primers. PCR system: 0.2μM of each primer, 0.2mM of dNTPs, 10ng of template, 5μL of 2×Buffer, 0.5μL of Hieff, and deionized water to make up the volume to 100μL. PCR reaction conditions are: step 1, 94℃ 20s; step 2, 94℃ 30s; step 3, 55℃ 30s; step 4, 72℃ 2min; step 5, 4℃ 5min, 30 cycles of steps 2 to 5. The obtained PCR products were connected using the Gibson Assembly Kit (Anhui General Biotechnology), and the connected products were transferred into Escherichia coli MC1061 to obtain recombinant bacteria. The plasmid of the recombinant bacteria was extracted to obtain the pPadR expression plasmid. The plasmid map is shown in the figure. Figure 1 .

[0038] Error-prone PCR was performed on the parental gene of padR (nucleotide sequence shown in SEQ ID NO: 1) using primers 5'-ATGAGAGTATTAAAATACGC-3' (upstream primer sequence as shown in SEQ ID NO: 9), 5'-TTAATCCTTATCTATCATAG-3' (downstream primer sequence as shown in SEQ ID NO: 10). The error-prone PCR system was 0.2mM dATP, 1mM dCTP, 1mMdTTP, 0.025mM MnCl2, 10×PCR Buffer 10μL, rTaq 0.5μL, deionized water to make up the volume to 100μL, and then evenly distributed into 10 PCR tubes. Error-prone PCR reaction conditions were: 94℃ 2min, 94℃ 30s, 50℃ 30s, 72℃ 1min, 4℃ 5min, and the above program was repeated for 30 cycles. The obtained PCR product was subjected to MEGAWHOPE PCR using the pPadR expression plasmid (nucleotide sequence as shown in SEQID NO: 11) as a template. The reaction system was: 0.2 μg of the PCR product obtained by the above error-prone PCR, 1 mM dNTPs, 30 ng of template pPadR, 5 μL of 10×Pyrobest Buffer, 0.3 μL of Pyrobest, and the volume was supplemented to 100 μL with deionized water. The PCR reaction conditions were: step 1, 94°C 20 s; step 2, 94°C 30 s; step 3, 50°C 30 s; step 4, 72°C 2 min; step 5, 4°C 5 min, and 30 cycles of the above steps 2 to 5. The obtained PCR product was added with DpnI (20 U) and digested at 37°C for 2 h, and DpnI was inactivated at 80°C for 20 min. The padR error-prone mutant library (mutant plasmid mixture) was obtained. The error-prone mutant library was screened by flow cytometry sorter to obtain a mutant PadR-M (amino acid sequence as shown in SEQ ID NO: 4) of the transcriptional regulatory protein gene padR that can specifically respond to cross-sporin induction. Sequencing showed that the 77th amino acid of the mutant was mutated from lysine (Lys) to glutamine (Gln), the 78th amino acid was mutated from glutamine (Gln) to lysine (Lys), the 79th amino acid was mutated from glutamic acid (Glu) to lysine (Lys), and the 80th amino acid was mutated from leucine (Leu) to methionine (Met). The pPadR-M plasmid containing the PadR-M coding gene (nucleotide sequence as shown in SEQ ID NO: 3) was transferred into the Escherichia coli BW25113 strain to obtain the recombinant strain BW25113-PadR-M, that is, a whole-cell biosensor that can specifically respond to cross-sporin induction was obtained, and its induction specificity was as shown in FIG. Figure 2 shown. Figure 2 It can be seen that the biosensor can specifically respond to the induction of spore phenol, but not to the induction of analogs spore phenol monomethyl ether, spore phenol, spore phenol, spore phenol acid, and tengtoxin.

[0039] In summary, the present invention uses mutants of the Bacillus subtilis transcriptional regulatory protein PadR, genes of the mutants, expression vectors of the genes, and related genetically engineered bacteria to construct a special biosensor, and utilizes its highly specific response to cross-sporin to achieve rapid, sensitive and low-cost detection of cross-sporin content.

[0040] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Non-essential technical features can be added or reduced according to actual needs to meet the requirements of different situations.

Claims

1. A mutant of the transcriptional regulatory protein PadR of Bacillus subtilis, characterized in that The mutant is PadR-M, the nucleotide sequence of PadR-M is shown in SEQ ID NO: 3, and the amino acid sequence of PadR-M is shown in SEQ ID NO:

4.

2. The mutant of the Bacillus subtilis transcriptional regulatory protein PadR according to claim 1, characterized in that The mutant is a parent of the Bacillus subtilis transcriptional regulatory protein PadR in which the alanine at position 77, the glutamic acid at position 78, the arginine at position 79, and the alanine at position 80 are mutated; or the mutant is a parent of the Bacillus subtilis transcriptional regulatory protein PadR in which the lysine at position 77 is mutated to glutamine, the glutamine at position 78 is mutated to lysine, the glutamic acid at position 79 is mutated to lysine, and the leucine at position 80 is mutated to methionine.

3. The mutant of the Bacillus subtilis transcriptional regulatory protein PadR according to claim 2, characterized in that The parent of the Bacillus subtilis transcription regulatory protein PadR is a wild-type Bacillus subtilis transcription regulatory protein PadR, the nucleotide sequence of the parent is shown in SEQ ID NO: 1, and the amino acid sequence of the parent is shown in SEQ ID NO:

2.

4. A gene encoding a mutant of the Bacillus subtilis transcriptional regulatory protein PadR according to any one of claims 1 to 3.

5. An expression vector carrying a gene of a mutant of the Bacillus subtilis transcriptional regulatory protein PadR according to claim 4, characterized in that The expression vector includes pBAD / HisA vector.

6. A genetically engineered bacterium expressing a mutant of the Bacillus subtilis transcriptional regulatory protein PadR according to any one of claims 1 to 3, characterized in that The genetically engineered bacteria use Escherichia coli as the starting strain, and the starting strains include E. coli MC1061 and BW25113.

7. A genetically engineered bacterium containing the gene of the mutant of the Bacillus subtilis transcriptional regulatory protein PadR according to claim 4, characterized in that The genetically engineered bacteria use Escherichia coli as the starting strain, and the starting strains include E. coli MC1061 and BW25113.

8. A genetically engineered bacterium transformed with the expression vector carrying the gene of the mutant of the Bacillus subtilis transcriptional regulatory protein PadR according to claim 5, characterized in that The genetically engineered bacteria use Escherichia coli as the starting strain, and the starting strains include E. coli MC1061 and BW25113.

9. Use of the genetically engineered bacterium expressing a mutant of Bacillus subtilis transcriptional regulatory protein PadR according to claim 6, or the genetically engineered bacterium containing a gene of a mutant of Bacillus subtilis transcriptional regulatory protein PadR according to claim 7, or the genetically engineered bacterium transformed with an expression vector carrying a gene of a mutant of Bacillus subtilis transcriptional regulatory protein PadR according to claim 8 in detecting crosslinked spore phenol.

10. Use of the mutant of Bacillus subtilis transcriptional regulatory protein PadR according to any one of claims 1 to 3, or the gene encoding the mutant of Bacillus subtilis transcriptional regulatory protein PadR according to claim 4, or the expression vector of the gene carrying the mutant of Bacillus subtilis transcriptional regulatory protein PadR according to claim 5, or the genetically engineered bacteria expressing the mutant of Bacillus subtilis transcriptional regulatory protein PadR according to claim 6, or the genetically engineered bacteria containing the gene of Bacillus subtilis transcriptional regulatory protein PadR according to claim 7, or the genetically engineered bacteria transformed with the expression vector of the gene carrying the mutant of Bacillus subtilis transcriptional regulatory protein PadR according to claim 8 in the construction of a biosensor responsive to crosslinked spore phenol.

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