Plasmid for improving spontaneous mutation frequency of bacillus subtilis

A Bacillus subtilis, spontaneous mutation technology, applied in the field of biological high, to achieve the effect of increasing diversity and improving stability

Pending Publication Date: 2021-12-17
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there is no such ideal type of mutation method in Bacillus subtilis

Method used

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  • Plasmid for improving spontaneous mutation frequency of bacillus subtilis
  • Plasmid for improving spontaneous mutation frequency of bacillus subtilis
  • Plasmid for improving spontaneous mutation frequency of bacillus subtilis

Examples

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Effect test

Embodiment 1

[0024]Example 1 Plasmid pUS20D9M is used to improve the spontaneous mutation rate of Bacillus subtilis

[0025] Escherichia coli and Bacillus subtilis were cultured in LB medium (1% peptone, 0.5% yeast powder, 1% sodium chloride, pH 7.0) at 37°C. like figure 1 As shown in a, the xylose-induced dCas9 expression cassette and the sgRNA expression cassette interfering with mutSL were constructed in the plasmid pUS20 by homologous recombination (Ye et al.. 2018. J Biotechnol. 284:57-62.doi: 10.1016 / j.jbiotec.2018.08.001). The construction process is as follows: First, the plasmid pJMP1 (Peterset al..2016.Cell. 165:1493-506.doi:10.1016 / j.cell.2016.05.003) was used as a template, and primers P1 / P2 were used to amplify the xylose-induced dCas9 expression cassette xylR-P xyl -dcas9 part; the sequence of the sgRNA expression cassette interfering with mutSL was entrusted to biocompany to synthesize (8310bp-8480bp in SEQ ID NO.1), and then amplified by primers P3 / P4 to obtain fragment...

Embodiment 2

[0027] Example 2 Plasmid pUS20XP1 is used to improve the spontaneous mutation rate of Bacillus subtilis

[0028] The chromosomally integrated plasmid pJMP1 (Peters et al.. 2016. Cell. 165:1493-506.doi:10.1016 / j.cell.2016.05.003) containing the xylose-inducible dCas9 expression cassette was transformed into Bacillus subtilis 168, in Strain 168D was obtained by screening for erythromycin resistance on the chromosome containing the xylose-induced dCas9 expression cassette after double crossover at the gene lacA.

[0029] like image 3 As shown, the DNA polymerase PolC, which has lost its proofreading function, is induced by xylose by homologous recombination. * The expression cassette and the sgRNA expression cassette interfering with polC were constructed on plasmid pUS20 to generate plasmid pUS20XP1. The construction process is as follows: First, using the Bacillus subtilis 168 genome as a template, the DNA polymerase polC was amplified with primers P7 / P8, P9 / P10 and P11 / P12,...

Embodiment 3

[0031] Example 3 Plasmid pUS20XP2 is used to improve the spontaneous mutation rate of Bacillus subtilis

[0032] In order to further improve the spontaneous mutation rate in the plasmid-type Bacillus subtilis spontaneous mutation system, we tried to combine the interference mutSL system and the expression polC* system to construct the plasmid pUS20XP2 ( Figure 5 ). The construction process is as follows: First, using the plasmid pUS20XP1 as a template, the entire sequence of the plasmid pUS20XP1 was amplified with primers P17 / P18; the plasmid pUS20D9M was used as a template, and the fragment P was amplified with primers P19 / P20 to obtain fragment P veg -sgmutSL. Then, the above-mentioned two fragments were fused into a circular plasmid by homologous recombination. Finally, the above fusion product was transformed into Escherichia coli DH5α, and the plasmid pUS20XP2 was obtained after screening with 100 ppm ampicillin.

[0033] like Figure 5 As shown, the plasmid contains...

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Abstract

The invention discloses a plasmid for improving spontaneous mutation frequency of bacillus subtilis. The plasmid is selected from any one of a PUS20D9M, a PUS20XP1 and a PUS20XP2; wherein the plasmid pUS20D9M has a sequence as shown in SEQ ID NO. 1, the plasmid pUS20XP1 has a sequence as shown in SEQ ID NO. 2, and the plasmid pUS20XP2 has a sequence as shown in SEQ ID NO. 3. Bacillus subtilis is an important microorganism. Many breeding works need to improve gene mutation frequency of bacillus subtilis. However, an ideal method for controlling spontaneous mutation frequency of bacillus subtilis does not exist at present. According to the invention, by virtue of a CRISPRi technology and a method for overexpressing error-prone DNA polymerase, the spontaneous mutation frequency of a host is improved (as high as 5128 times). The whole operation can be realized through transformation and elimination of plasmids, and gene knockout of a host is not needed.

Description

technical field [0001] The invention belongs to the technical field of biology and discloses a plasmid used for improving the spontaneous mutation rate of Bacillus subtilis. Background technique [0002] Bacillus subtilis plays an important role in the fields of fermentation industry, agriculture and environmental protection. For example, Bacillus subtilis is used for recombinant protein expression, compound production, plant growth promotion, biological control and pollutant degradation. Before practical application, strains are usually domesticated and modified, that is, bred. Physical or chemical mutagenesis is a commonly used breeding method, that is, to increase the gene mutation frequency of strains by physical or chemical means, and then screen the target mutant strains. The disadvantages of this method are low mutation efficiency and narrow mutation spectrum, and there are hazards and contamination problems. Therefore, researchers try to increase the frequency of ...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N1/21C12N15/66C12N15/90C12R1/125
CPCC12N15/75C12N15/902C07K14/32Y02E50/10
Inventor 闫新叶斌
Owner NANJING AGRICULTURAL UNIVERSITY
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