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A kind of Bacillus subtilis recombinant strain and its preparation method and application

A technology of Bacillus subtilis and recombinant strains, which is applied in the biological field and can solve the problems of high secreted protease and unstable expression plasmids.

Active Publication Date: 2019-07-16
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The application of B. subtilis is limited to a certain extent due to the influence of many factors such as self-secretion of proteases and the instability of the constructed expression plasmid.

Method used

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  • A kind of Bacillus subtilis recombinant strain and its preparation method and application
  • A kind of Bacillus subtilis recombinant strain and its preparation method and application
  • A kind of Bacillus subtilis recombinant strain and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0075] Embodiment 1 Construction of ten gene knockout vectors of Bacillus subtilis ATCC6051

[0076] Using the genomic DNA of Bacillus subtilis ATCC6051 (purchased from NBRC, product number NBRC 13719) as a template, use the primers in Table 1 to amplify F-X-L, R-X-L and F-X-R, R-X-R (X is the gene to be knocked out, the same below), respectively, Amplified products X-L (homologous arm upstream of the gene to be knocked out) and X-R (homologous arm downstream of the gene to be knocked out) were obtained.

[0077] Using the X-L fragment and the X-R fragment as templates, and using F-X–L and R-X–R as primers, fusion PCR was performed to obtain ten corresponding fusion products (L+R fragments). The ten fusion products were respectively cloned into the pMD18-T vector, and after sequencing, the ten fusion fragments were the nucleotides shown in sequence 1 in the sequence table (the DNA molecule of the homology arm of the nprE encoding gene), and sequence 2. Nucleotide shown in seq...

Embodiment 2

[0087] (1) Construction of recombinant bacteria 1 (Bacillus subtilis ATCC6051ΔnprE)

[0088] ①Transform the recombinant plasmid pKS2-nprE into Escherichia coli TG1 (purchased from Yubao Bioengineering (Dalian) Co., Ltd.), extract the plasmid, and perform the same operation for other recombinant plasmids. Then, the plasmid pKS2-nprE was transferred into Bacillus subtilis ATCC6051 by electroporation method (for specific methods, refer to the non-patent literature record Natalia P, Zakataeva, Oksana V et al. A simple method to introduce marker-free genetic modification into chromosome of naturally nontransformable Bacillus amyloliquefaciens strains [J].Appl Microbiol Biotechnol.2010,85:1201-1209.), cultivated upside down at 30°C, and screened positive clones with 5 μg / mL erythromycin (erm) resistant LB plates.

[0089] ②Pick the positive clones on the resistance plate, inoculate them in a 50mL Erlenmeyer flask with 10mL LB liquid medium (5μg / mL erm), culture at 30°C and 200rpm fo...

Embodiment 3

[0111] Example 3 Expression of transglutaminase in Bacillus subtilis ATCC6051Δ10 (nprE, nprB, aprE, mpr, bpf, epr, vpr, wprA, spollAC, srfAC)

[0112] Using the plasmid pBEp43-proMTG (constructed according to CN201210052578.2, a recombinant Bacillus subtilis and its method for producing transglutaminase) as the expression plasmid, Bacillus subtilis ATCC6051 and Bacillus subtilis ATCC6051Δ10 were transferred into Bacillus subtilis ATCC6051Δ10 by electroporation, and two Transformation strains Bacillus subtilis ATCC6051 (pBEp43-proMTG) and Bacillus subtilis ATCC6051Δ10 (pBEp43-proMTG).

[0113] Pick single colonies of Bacillus subtilis ATCC6051(pBEp43-proMTG) and Bacillus subtilisATCC6051Δ10(pBEp43-proMTG) and inoculate them in 10mL LB medium (kanamycin 20μg / mL) at 37°C and 200rpm for 12h, and the activated seeds solution was inoculated in 50mL LB medium (kanamycin 20μg / mL), the inoculation amount was 1% (volume ratio), and fermented at 37°C and 200rpm for 48h; meanwhile, the st...

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Abstract

The invention discloses a recombinant strain of Bacillus subtilis, a preparation method and application thereof, and belongs to the field of biotechnology. The recombinant strain provided by the present invention is obtained by knocking out or inactivating ten protease genes in Bacillus subtilis, these ten protease genes are: nprE, nprB, aprE, mpr, bpf, epr, vpr, wprA, spollAC and srfAC. In the present invention, the aforementioned 10 protease genes are knocked out through the method of homologous recombination, and the obtained Bacillus subtilis recombinant strain grows fast and has high expression of foreign protein. Therefore, the recombinant strain can be used as a host for high-efficiency expression of foreign proteins.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant strain of Bacillus subtilis and a preparation method and application thereof. Background technique [0002] Bacillus subtilis (Bacillus subtilis) belongs to the genus Bacillus, is a Gram-positive bacterium, has no pathogenicity, and is recognized as a safe strain (GRAS). B. subtilis has only a single layer of outer cell membrane, fast growth, simple nutritional requirements, easy to survive, colonize and reproduce; has a clear heritage background, good protein secretion ability and a series of efficient signal peptides, and has been used in food for a long time Fermentation and production of important enzyme preparations; ideal host for expressing and secreting foreign proteins in prokaryotic expression systems. [0003] The application of B. subtilis is limited to a certain extent due to the influence of many factors such as self-secretion of proteases and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/75C12R1/125
CPCC12N9/54C12N15/75C12N1/205C12R2001/125
Inventor 潘力刘欣王斌
Owner SOUTH CHINA UNIV OF TECH
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