A highly efficient and stable dual-plasmid system for Bacillus subtilis

A Bacillus subtilis and plasmid technology, applied in the field of genetic engineering, can solve the problems of insolubility, easy formation of inclusion body of target protein, low expression level, etc., and achieve stable expression effect

Active Publication Date: 2022-01-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, most of the host bacteria used for construction are Escherichia coli, but Escherichia coli is a well-known pathogenic bacteria in the field, which affects the safety of the protein, and if the recombinantly expressed foreign protein contains a large number of continuous rare codons, it often causes expression. Low amount, or premature termination of translation, the target protein tends to form inclusion bodies and is insoluble

Method used

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  • A highly efficient and stable dual-plasmid system for Bacillus subtilis
  • A highly efficient and stable dual-plasmid system for Bacillus subtilis
  • A highly efficient and stable dual-plasmid system for Bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction of simplified pHTmin-sfGFP and pBmin-mCherry recombinant bacteria

[0054] (1) Construction of pHTOriP43-sfGFP strain

[0055] After the sfGFP fragment (nucleotide sequence shown in SEQ ID NO.5) and the constitutive promoter P43 (nucleotide sequence shown in SEQ ID NO.7) were connected to the pHT01 vector by the Infusion method for homology arm assembly , into the Escherichia coli JM109 host, spread it on a 100 μg / mL ampicillin-resistant plate, culture overnight at 37°C, pick a single colony and inoculate it into a 100 μg / mL ampicillin-resistant liquid medium and send it for sequencing; After the plasmid was correct, the plasmid was extracted and transformed into a Bacillus subtilis B.S168 host, and it was verified that a recombinant strain containing the plasmid pHTOriP43-sfGFP was obtained.

[0056] (2) Construction of pBOriP43-mCherry strain

[0057] The mCherry fragment (nucleotide sequence shown in SEQ ID NO.6) and the constitutive promote...

Embodiment 2

[0062] Example 2: Recombinant bacteria containing double plasmids and verification of their protein expression

[0063] (1) Construction of double-plasmid recombinant bacteria containing shuttle version

[0064] The pHTOriP43-sfGFP recombinant bacteria constructed in step (1) of Example 1 were prepared into competent cells, and pBOriP43-mCherry was transformed into the pHTOriP43-sfGFP recombinant bacteria, and placed on a plate containing 5 μg / mL kanamycin resistance After screening and sequencing verification, a Bacillus subtilis containing double plasmids (pHTOriP43-sfGFP+pBOriP43-mCherry) was obtained.

[0065] (2) Construction of a streamlined double-plasmid recombinant bacterium

[0066] Prepare pHTminP43-sfGFP recombinant bacteria into competent cells, transform pBminP43-mCherry into pHTminP43-sfGFP recombinant bacteria, screen on a plate containing 5 μg / mL kanamycin resistance, and sequence verification to obtain a simplified version containing Bacillus subtilis with ...

Embodiment 3

[0071] Example 3: Gene Shakers Based on Dual Plasmids and Inducible Promoters

[0072] (1) Construction of pHTminPgrac-sfGFP-XylR plasmid

[0073] The promoter of pHTminP43-sfGFP is replaced by a lactose-inducible promoter: the lactose-inducible promoter Pgrac-i (nucleotide sequence is shown in SEQ ID NO.1), and then it is combined with the primer pHTPgrac-sfGFP-v1 / The pHTminPgrac-sfGFP-v amplified from v2 (using pHTminP43-sfGFP as a template) was ligated through homology arms, and the two fragments were assembled using Infusion enzyme to obtain a plasmid pHTminPgrac-sfGFP containing a lactose-inducible promoter.

[0074] Integrative expression of xylose-inducible promoter repressor protein in plasmid pHTminPgrac-sfGFP: xylose repressor protein xylR-i fragment (nucleotide sequence shown in SEQ ID NO.2), and then combine it with primer pHTPgrac-xylR-v1 The pHTminPgrac-sfGFP-v amplified from pHTminPgrac-sfGFP / v2 (with pHTminPgrac-sfGFP as a template) was ligated by homology ar...

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Abstract

The invention discloses a high-efficiency and stable dual-plasmid system for Bacillus subtilis, belonging to the field of genetic engineering. The present invention constructs a vector suitable for double plasmid transformation. The multi-copy replication in Escherichia coli is omitted, the Bacillus subtilis can be directly transformed, and the expression of the target protein can be induced by adding a corresponding inducer from an external source. The expression effect of this system is stable, and Bacillus subtilis is used as the host. The obtained product is safe and non-pathogenic, and is suitable for various production scenarios.

Description

technical field [0001] The invention relates to a high-efficiency and stable double-plasmid system of Bacillus subtilis, which belongs to the field of genetic engineering. Background technique [0002] Plasmid is a covalently closed circular DNA with the function of self-replication and expression of foreign genes. Because of its good self-replication ability in different hosts, it is often used as a vector for expressing target genes in the fields of genetic engineering and molecular biology. In the transformation and research of genetic engineering, usually only one kind of plasmid is transformed in a host bacterium, because the plasmid is incompatible, that is, two kinds of plasmids of the same species or close relatives cannot be stably maintained in one cell at the same time Inside. At present, the same plasmid is also transformed into E. coli to construct a dual-plasmid system for the expression of dual enzymes. The double plasmid was kept stable by adding antibioti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/75C12P21/02C07K14/435C12N9/24C12N9/38C12R1/125
CPCC12N15/75C12P21/02C07K14/43595C12N9/2471C12Y302/01023C12N9/2402C12Y302/01031C12N2800/40
Inventor 周哲敏崔文璟陈巧青刘中美周丽郭军玲
Owner JIANGNAN UNIV
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