CN108865963B - Genetic operation method for artificially controlling spontaneous mutation rate of bacillus subtilis and application thereof - Google Patents
Genetic operation method for artificially controlling spontaneous mutation rate of bacillus subtilis and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a genetic operation method for artificially controlling spontaneous mutation rate of bacillus subtilis and application thereof. By combining the operon responsible for the mismatch repair system of Bacillus subtilisMutSLUnder the regulation of xylose inducing promoter and repressor protein, and constructing corresponding recombinant plasmid for inducing expression, and its applicationSpizizenIntegrating the recombinant plasmid into the genome of the strain by double exchange, obtaining a target engineering strain by positive and negative screening, and adjusting the operon of a mismatch repair system by different xylose addition concentrationsMutSLDifferent expression levels，Further realizing the artificial control of the spontaneous mutation rate of the bacillus subtilis.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a genetic operation method for artificially controlling spontaneous mutation rate of bacillus subtilis and application thereof.
Background
Bacillus subtilis (A), (B) and (C)Bacillussubtilis) Is a dominant microorganism species existing in soil and plant micro-ecosystem, has no pathogenicity, no harm to people and livestock, no environmental pollution, stronger stress resistance and antibacterial and disease-preventing functions, and is widely used as a molecular biology research model organism and a host for industrially producing high-yield strains. In recent years, the following areThe wide application of second-generation and third-generation high-throughput sequencing technologies, and the researches such as adaptive laboratory evolution and the like increasingly become research hotspots. Adaptive laboratory evolution is a method for artificially simulating variation and selection processes in natural evolution under laboratory conditions, realizing directed evolution of organisms by means of artificial selection pressure, and screening individuals with excellent characters from an evolved population, and is currently applied to the aspects of improving the tolerance of strains to substrates (or products), the utilization rate of the substrates, the growth rate of the strains and the like. For adaptive laboratory evolution, genetic mutation is the main driving force of the evolution, but in the evolution process, in order to maintain the integrity of the genome of the microorganism, a strict mismatch repair mechanism exists in the cell and is used for eliminating mismatch regions generated in replication and recombination events, thereby reducing the natural mutation rate of the cell (1)<10-6) Resulting in a long evolution period. The mismatch repair mechanism is a repair mechanism after the cells are replicated, and the existing DNA mismatch repair system can maintain the high fidelity of DNA replication and inhibit gene mutation. The invention is prepared by mixing Bacillus subtilisBacillus subtilis) Mismatch repair mechanism (from the genomeMutSLOperon responsible for completing functions) are placed under the control of xylose-induced promoter and repressor protein from plasmid pAX01, and the operon controlled by xylose addition concentration is constructedMutSLThe different expression levels of the engineering strains are further obtained, so that excellent starting strains are provided for adaptive laboratory evolution, and the evolution period is shortened.
Disclosure of Invention
The invention provides a genetic operation method for artificially controlling the spontaneous mutation rate of bacillus subtilis and application thereof, which reduce the expression level of an operon corresponding to a strict mismatch repair mechanism in a cell, thereby achieving the purpose of artificially controlling the mutation rate of the cell.
The technical scheme of the invention is realized as follows:
a genetic operation method for artificially controlling spontaneous mutation rate of bacillus subtilis comprises the following steps: mismatch repair system of bacillus subtilisMutSLUnder the control of xylose-inducible promoter and repressor protein from plasmid pAX01Operon for establishing xylose addition concentration induced expressionMutSLAnd then using the recombinant plasmids of (4)SpizizenDouble exchange is integrated into the strain, and the target engineering strain is obtained through screening, so that the spontaneous mutation rate of the bacillus subtilis is artificially controlled.
The artificially controlled spontaneous mutation rate of the bacillus subtilis is applied to preparation of the bacillus subtilis engineering strain with high mutation rate.
The method comprises the following steps:
(1) plasmid pAX01-mutSConstructing;
(2) plasmid pSS-mutS is constructed;
(3) plasmid pSS-mutConstructing an SL;
(4) plasmid pSS-mutSL utilizationSpizizenIntegration of double crossover into the StrainB. subtilis 168 ΔuppA genome, wherein positive transformants with double crossover gene recombination are screened by positive screening marker chloramphenicol resistance;
(5) inoculating the positive transformant which is subjected to double crossover gene recombination in the step (4) on an LB liquid culture medium, deleting all cells with the screening markers between two DR areas (Direct repeat) by generating intramolecular homologous recombination, coating the cells with the screening markers subjected to intramolecular homologous recombination on a 5FU negative screening plate, and screening out a positive strainB. subtilisMutSL, namely the bacillus subtilis engineering strain with high mutation rate.
The plasmid pAX01-mutSContaining xylose promoterP xylA And encoding the XylR repressor proteinxylRA gene.
The plasmid pAX01 of step (1)mutSThe construction method comprises the following specific steps: to be provided withB. subtilis168 genome as template and upstream primer pAX01-mutThe sequence of S-F is shown as SEQ ID NO: 1 and the downstream primer pAX01-mutThe sequence of S-B is shown as SEQ ID NO: 2 is a primer, and a downstream homologous region is obtainedmutSThe target gene fragment of the-B is subjected to enzyme digestion, connection, transformation and verification with the pAX01 plasmid to obtain the plasmid pAX01-mutS。
The plasmid pSS-mutS contains an operonMutSLUpstream homologous region ofmutS-F。
The step (2) plasmid pSS-mutThe S construction comprises the following specific steps: to be provided withB. subtilis168 genome as a template, and using an upstream primer pSS-mutThe sequence of S-F is shown as SEQ ID NO: 3 and the downstream primer pSS-mutThe sequence of S-B is shown as SEQ ID NO: 4, will contain an operonMutSLUpstream homologous regionmutSThe target fragment of the-F and the plasmid pSS are subjected to enzyme digestion, enzyme connection, transformation and verification to obtain the plasmid pSS-mutS。
The plasmid pSS-mutSL promoter containing xyloseP xylA Encoding the XylR repressor proteinxylRGene and operonMutSLUpstream homologous region ofmutS-F and downstream homologous regionmutS-B。
The step (3) plasmid pSS-mutThe SL construction method comprises the following specific steps: plasmid pAX01-mutS as template using upstream primer pSS-xylThe sequence of R-F is shown as SEQ ID NO: 5 and the downstream primer pSS-xylThe sequence of R-F is shown as SEQ ID NO: 6 amplification of a promoter containing xylose inducible promoterP xylA Genes encoding repressor protein XlyRxlyRAnd an operatorMutSLDownstream homologous region ofmutSA target gene fragment of-B, the target gene fragment and the plasmid pSS-mutAfter the S is subjected to enzyme digestion, enzyme ligation, transformation and verification, the plasmid pSS-mutSL。
The invention has the beneficial effects that:
first, the mutation rate determination experiments of the present invention demonstrate that xylose inducer is not addedB. subtilisCompared with the original strain, the mutation rate of the MutSL strain is improved by 58.4 times, and when the concentrations of xylose inducers are respectively 0.5 percent and 1.0 percent, the relative mutation rates of the strain are respectively improved by 27.1 times and 4.2 times;
secondly, prepared by the inventionB. subtilisThe MutSL strain has different spontaneous mutation rates under the conditions of different xylose induction concentrations, and the strain can reach the expected mutation rate level by adding a proper concentration of inducer xylose into a culture medium.
Thirdly, the operon gene which is responsible for the mismatch repair system on the bacillus subtilis genome is treated by recombinant plasmidsmutSL placed on xylose inducible promoterP xylA Under the control of the regulation, the operon gene is regulated by adding different xylose concentrationsmutSL expression level, and the mismatch repair mechanism of the strain is maintained at a desired level, and the strain is subjected to a desired mutation rate level.
Drawings
FIG. 1 plasmid map of plasmid pSS
FIG. 2 plasmid pAX01-mutSThe plasmid map of (1).
FIG. 3 plasmid pSS-mutPlasmid map of SL.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
Raw materials and using method
The original strain used in the present inventionB. subtilis168. Plasmid pAX01 is derived from BGSC (BacillusGenetic Stock Center, http:// www.bgsc.org /). Bacillus subtilis starting strain used in the inventionB. subtilis 168 ΔuppDerived fromB. subtilis168, the basic operation plasmid pSS (plasmid map is shown in FIG. 1) is derived from pUC18, and the detailed construction process of the two can be referred to Shi, T.et al. (2013). "plasmid of a marker delivery system inBacillus subtilis stimulated by a double-strand break in the chromosome." PloS One 8(11): e81370。
Molecular biological reagents used in the present invention, such as restriction enzymes, dephosphorylating enzymes, DNA ligase, etc., were purchased from Thermo corporation (http:// www.thermoscientificbio.com/fermentas). Other biochemicals are purchased from Biotechnology engineering (Shanghai) Inc. (http:// www.sangon.com /). The operations of enzyme digestion, connection, transformation, verification and the like involved in the implementation process of the invention are all conventional molecular biology operations, and are not described in detail in the invention for saving space.
Second, preparation of culture medium
The LB liquid culture medium formula is: 10 g/L peptone, 5 g/L yeast extract, 10 g/L NaCl, pH adjusted to 7.5. Sterilizing under 0.1 Mpa for 20 min. The LB solid culture medium formula is: adding agar powder (final concentration 20 g/L) into LB liquid culture medium, and sterilizing at 0.1 Mpa for 20 min.
The preparation method of the M9 liquid culture medium comprises the following steps: preparation of 1 mol/L MgSO4、1 mol/L CaCl2200 mL of distilled water was dissolved with Na2PO4·7H2O 12.8g、KH2PO4 3.0g、NaCl 0.5g、NH4Cl 1.0g, 5 XM 9 salt solution was prepared and sterilized at 121 ℃ for 15 minutes for use. Preparing a 1L M9 liquid culture medium according to the following mixture ratio: 200 ml of 5 XM 9 salt solution, 2 ml of 1 mol/L MgSO420 ml of 20% glucose solution, 0.1 ml of 1 mol/L CaCl2And adding sterile double distilled water to 1000 ml under aseptic condition to obtain M9 liquid culture medium. M9 solid medium is prepared by adding agar powder (final concentration 20 g/L) into M9 liquid medium, and sterilizing at 0.1 MPa for 20 min.
The formula of the 5FU negative screening culture medium is shown in Table 1:
TABLE 15 FU negative screening culture medium preparation table
The percentage concentrations of the components in table 1 are mass volume percentages.
The basic salt composition of 10 × Spizizen in Table 1 is: 2.0 g/L (NH)4）2SO4，18.3 g/L K2HPO4，6.0 g/L KH2PO4，12.0 g/L C6H5Na3O7.2H2O, pH 7.2, sterilizing at 0.1 MPa for 20 min.
The 1000 x trace element composition in table 1 is: 27.0 g/L FeCl3•6H2O，2.0 g/L ZnCl2•4H2O，2.0 g/L CaCl2•2H2O，2.0 g/L Na2MoO4•2H2O，1.9 g/L CuSO4•5H2O，0.5 g/L H3BO3Sterilizing at pH 7.2 and pressure of 0.1 MPa for 20 min.
Wherein the antibiotic screening plate is LB solid culture medium added with antibiotic, and the chloramphenicol screening plate is added with 5 mug/mL (final concentration) of chloramphenicol. For basal salt medium, 1 mL of 0.5% tryptophan per 100 mL of medium was added to meet the strain growth requirements.
The genetic operation method for artificially controlling the spontaneous mutation rate of the bacillus subtilis comprises the following steps:
1. plasmid pAX01-mutSConstruction of
Using a PCR reaction toB. subtilis168 genome as template, using upstream and downstream primers pAX01-mutThe sequence of S-F is shown as SEQ ID NO: 1 and pAX01-mutThe sequence of S-B is shown as SEQ ID NO: 2 obtaining operon-containingMutSLDownstream homologous regionmutSThe target fragment of-B, the gene fragment and pAX01 plasmidBamHI single enzyme digestion, connection, transformation, verification and the like to obtain a plasmid pAX01-mutS, the plasmid map is shown in FIG. 2.
2. Plasmid pSS-mutConstruction of S
Using a PCR reaction toB. subtilis168 genome as template and pSS as upstream and downstream primersmutThe sequence of S-F is shown as SEQ ID NO: 3 and pSS-mutThe sequence of S-B is shown as SEQ ID NO: 4 obtaining a gene containing an operonMutSLUpstream homologous regionmutSThe target fragment of F, the gene fragment and the plasmid pSS (the plasmid map is shown in figure 1)BglII andXhoafter the operations of double enzyme digestion, enzyme ligation, transformation, verification and the like, the plasmid pSS-mutS。
3. Plasmid pSS-mutConstruction of SL
Using PCR reaction with plasmid pAX01-mutS as template using upstream and downstream primers pSS-xylR-F sequences such asSEQ ID NO: 5 and pSS-xylThe sequence of R-B is shown as SEQ ID NO: 6, obtaining the gene fragment pSS-xylR, the gene segment comprises a xylose promoterP xylA Encoding the XylR repressor proteinxylRGene and operonMutSLDownstream homologous region ofmutS-B. The gene fragment pSS-xylR and pSS-mutS plasmidKpnI obtaining the plasmid pSS-mutSL, plasmid map is shown in FIG. 3.
4. Spizisen transformation
Plasmid pSS-mutSL utilizationSpizizenDouble crossover integration into strains by chemical transformationB. subtilis 168 ΔuppAnd (4) genome, and screening positive transformants with double crossover gene recombination by positive screening marker chloramphenicol resistance.
5. Obtaining engineering strainB. subtilis MutSL
And inoculating the positive transformant subjected to double crossover gene recombination into an LB liquid medium (without adding antibiotics), and selecting a bacterial liquid with an appropriate concentration and coating the bacterial liquid on a 5FU negative screening plate after 6 hours of culture. Due to the fact thatuppThe gene coding UPRTase can catalyze 5-fluorouracil (5 FU) to generate 5-F-dUMP, and the 5-F-dUMP is a strong inhibitor of thymidylate synthase of bacillus subtilis and can cause cell death, so that all screened marked cells without intramolecular homologous recombination deletion between two DR regions (Direct repeat) containuppThe encoded gene, could not be grown on 5FU negative selection plates. All recombinant cells grown on 5FU plates underwent intramolecular homologous recombination and were deleted for all selectable markers. Followed by the use of the primer pSS-mutS-F and pSS-xylPerforming PCR and sequencing verification on R-B to obtain the target engineering strainB. subtilis MutSL。
6. Mutation rate validation
The obtained engineering strainB. subtilisMutSL and control strainsB. subtilis 168 ΔuppAll were inoculated in M9 minimal salt medium and cultured overnight at 37 ℃. The overnight culture solution is transferred to a fresh M9 basic salt culture medium according to the inoculation amount of 1 percent, and after 6 hours of culture, the culture solution is respectively coated onAnd (3) adding an M9 culture medium solid plate with 25 mu g/mL rifamycin and an M9 culture medium solid plate without rifamycin, culturing at 37 ℃ for 12-16 h, counting the number of grown colonies, and calculating the spontaneous mutation rate of the strain. Mutation rate was defined as the number of colonies grown on plates supplemented with rifamycin (Rif 25)R) Total colonies grown on plates without rifamycin addition. Relative mutation rate is measured asB. subtilis 168 ΔuppSpontaneous mutation rates were used as a reference and set at 1.0, and mutation rates of other strains were calculated. The results are summarized in Table 2.
TABLE 2 relationship between spontaneous mutation rate of engineered strain and xylose induction concentration
Mutation Rate determination experiments demonstrated that xylose inducer was not addedB. subtilisCompared with the original strain, the mutation rate of the MutSL strain is improved by 58.4 times, and when the concentrations of xylose inducer are respectively 0.5% and 1.0%, the relative mutation rate of the strain is respectively improved by 27.1 times and 4.2 times.B. subtilisThe MutSL strains have different spontaneous mutation rates under different xylose induction concentration conditions. The strain can be brought to the desired mutation rate level by adding an appropriate concentration of inducer xylose to the medium.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
<110> Zhengzhou light industry academy
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Claims (4)
1. A method for preparing a bacillus subtilis engineering strain with high mutation rate by artificially controlling the spontaneous mutation rate of bacillus subtilis is characterized by comprising the following steps:
(1) plasmid pAX01-mutSConstructing;
(2) plasmid pSS-mutS is constructed;
(3) plasmid pSS-mutConstructing an SL;
(4) plasmid pSS-mutSL utilizationSpizizenIntegration of double crossover into the StrainB. subtilis 168 ΔuppA genome, wherein positive transformants with double crossover gene recombination are screened by positive screening marker chloramphenicol resistance;
(5) inoculating the positive transformant subjected to double crossover gene recombination in the step (4) on an LB liquid culture medium, deleting all cells with the screening markers between the two DR areas by intramolecular homologous recombination, coating the cells with the screening markers deleted on a 5FU negative screening plate, and screening out the target positive strainB. subtilisMutSL, namely the artificially controlled high mutation rate bacillus subtilis engineering strain;
the plasmid pAX01-mutSContaining xylose inducible promoterP xylA Encoding the XylR repressor proteinxylRGenes and operonsMutSLDownstream homologous region ofmutS-B；
The plasmid pSS-mutS contains an operonMutSLUpstream homologous region ofmutS-F；
The plasmid pSS-mutSL promoter containing xyloseP xylA Encoding the XylR repressor proteinxylRGene and operonMutSLUpstream homologous region ofmutS-F and downstream homologous regionmutS-B。
2. The method as claimed in claim 1, wherein the plasmid pAX01 of step (1)mutSThe construction method comprises the following specific steps: to be provided withB. subtilis168 genome as template and upstream primer pAX01-mutThe sequence of S-F is shown as SEQ ID NO: 1 and the downstream primer pAX01-mutThe sequence of S-B is shown as SEQ ID NO: 2 is a primer, and a downstream homologous region is obtainedmutSThe target gene fragment of the-B is subjected to enzyme digestion, connection, transformation and verification with the pAX01 plasmid to obtain the plasmid pAX01-mutS。
3. The method as set forth in claim 1, wherein the plasmid pSS-mutThe S construction comprises the following specific steps: to be provided withB. subtilis168 genome as a template, and using an upstream primer pSS-mutThe sequence of S-F is shown as SEQ ID NO: 3 and the downstream primer pSS-mutThe sequence of S-B is shown as SEQ ID NO: 4, will contain an operonMutSLUpstream homologous regionmutSThe target fragment of the-F and the plasmid pSS are subjected to enzyme digestion, enzyme connection, transformation and verification to obtain the plasmid pSS-mutS。
4. The method as set forth in claim 1, wherein the plasmid pSS-mutThe SL construction method comprises the following specific steps: plasmid pAX01-mutS as template using upstream primer pSS-xylThe sequence of R-F is shown as SEQ ID NO: 5 and the downstream primer pSS-xylThe sequence of R-F is shown as SEQ ID NO: 6 amplification of a promoter containing xylose inducible promoterP xylA Genes encoding repressor protein XlyRxlyRAnd an operatorMutSLDownstream homologous region ofmutSA target gene fragment of-B, a target geneFragments and plasmids pSS-mutAfter the S is subjected to enzyme digestion, enzyme ligation, transformation and verification, the plasmid pSS-mutSL。
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1351660A (en) * | 1999-05-19 | 2002-05-29 | 金克克国际有限公司 | directed evolution of microorganisms |
| CN1989255A (en) * | 2004-02-26 | 2007-06-27 | 麦克西斯法国股份有限公司 | Reduction of spontaneous mutation rates in cells |
| CN101203608A (en) * | 2004-07-06 | 2008-06-18 | 麦克西斯法国股份有限公司 | Generation of recombinant genes in bacteriophages |
| WO2010074562A1 (en) * | 2008-12-22 | 2010-07-01 | Keygene N.V. | Use of double stranded rna to increase the efficiency of targeted gene alteration in plant protoplasts |
| CN102382855A (en) * | 2011-11-14 | 2012-03-21 | 福州大学 | Bacillus subtilis xylose induced exocrine expression vector |
| CN104212830A (en) * | 2014-09-03 | 2014-12-17 | 江南大学 | Self-regulation expression system of bacillus subtilis and building method and application of self-regulation expression system |
-
2018
- 2018-07-10 CN CN201810752050.3A patent/CN108865963B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1351660A (en) * | 1999-05-19 | 2002-05-29 | 金克克国际有限公司 | directed evolution of microorganisms |
| CN1989255A (en) * | 2004-02-26 | 2007-06-27 | 麦克西斯法国股份有限公司 | Reduction of spontaneous mutation rates in cells |
| CN101203608A (en) * | 2004-07-06 | 2008-06-18 | 麦克西斯法国股份有限公司 | Generation of recombinant genes in bacteriophages |
| WO2010074562A1 (en) * | 2008-12-22 | 2010-07-01 | Keygene N.V. | Use of double stranded rna to increase the efficiency of targeted gene alteration in plant protoplasts |
| CN102382855A (en) * | 2011-11-14 | 2012-03-21 | 福州大学 | Bacillus subtilis xylose induced exocrine expression vector |
| CN104212830A (en) * | 2014-09-03 | 2014-12-17 | 江南大学 | Self-regulation expression system of bacillus subtilis and building method and application of self-regulation expression system |
Non-Patent Citations (2)
| Title |
|---|
| 枯草芽孢杆菌木糖诱导型穿梭质粒的构建;王珊瑛;《中国优秀硕士学位论文全文数据库（电子期刊）》;20170228;全文 * |
| 枯草芽孢杆菌表达系统及其启动子的研究进展;熊海涛等;《广西科学》;20180630;第25卷(第3期);233-241 * |
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| CN108865963A (en) | 2018-11-23 |
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