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Construction and application of bacillus subtilis linear plasmid system

A plasmid and nature technology, applied in the field of Bacillus subtilis genetic engineering, can solve the problems of easy loss, difficult industrial production, unstable tool plasmids, etc.

Active Publication Date: 2020-06-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the tool plasmids used to increase protein expression are very unstable and prone to loss, requiring antibiotic maintenance (Zhang XZ, Cui ZL, Hong Q, Li SP. High-level expression and secretion of methylparathion hydrolase in Bacillus subtilis WB800.Applied and environmentalmicrobiology.2005; 71(7):4101-3), so the Bacillus subtilis strain with tool plasmid is difficult to be used in industrial production and is not suitable for food and pharmaceutical production
[0003] For the realization of the development of a new generation of tool vectors for Bacillus subtilis, there is no report yet. Currently, circular plasmids and genome expression are mainly used for gene expression. The development of new and effective linear plasmid systems may be useful for subsequent enzyme engineering, laying the groundwork for metabolic engineering and other fields

Method used

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  • Construction and application of bacillus subtilis linear plasmid system

Examples

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Effect test

Embodiment 1

[0029] Construction of embodiment 1 recombinant plasmid pP43NMK-phi29

[0030] The recombinant plasmid is the P of the Pp43NMK plasmid 43 The promoter was inserted into the Bacillus subtilis phi29 phage replication-related gene cluster sequence to be transformed into Bacillus subtilis for expression. To introduce the Bacillus subtilis phi29 phage replication-associated gene cluster sequence into P 43 After the promoter, design primers rh_p43NMK-Phi29_F: 5'-GGTACCATTATAGATGGCAAAAATGATGCAGAGAGAAATCAC-3', rh_p43NMK-Phi29_R: 5'-ACGCCAAGCTTTCATCATACAAACATCTCCTTTTAAACAAACGTTTTATTTGATTG-3', using phage containing B. PCR, obtain the subtilis phi29 phage replication-related gene cluster sequence fragment (as shown in SEQ ID NO.1); design primers

[0031] fx_p43NMK-Phi29_F: 5’-GGAGATGTTTGTATGATGAAAGCTTGGCGTAATCATGGTC’, fx_p43NMK-Phi29_R: 5’-CATTTTTGCCATCTATAATGGTACCGCTATCACTTTATATTTTACATAATCGC-3’,

[0032] Using the plasmid pP43NMK as a template, the plasmid fragment was obtained by ...

Embodiment 2

[0033] Example 2 Construction of linear plasmid LP3

[0034] The linear plasmid LP3 is composed of four parts, from left to right: left origin of replication, P 43 Promoter and GFP coding gene, chloramphenicol resistance gene, right origin of replication. Design primers to amplify the left replicon:

[0035] rh1_LP3_F: 5'-AAAGTAAGCCCCCCACCCTCACATGATACCA-3',

[0036] rh1_LP3_R: 5'-CAGCATCTTTCCTCTGCGACACAGACGAAGCGCTA-3';

[0037] Design primers to amplify the green fluorescent protein gene:

[0038]rh2_LP3_F: 5'-CGTCTGTGTCGCAGAGGAAAGATGCTGTTCTTGTAAATGAGTTGCTAG-3',

[0039] rh2_LP3_R: 5'-GGGAAAACCCTGGCGTTAACACTTTATGCTTCCGGCTCGTATGT-3';

[0040] Primers designed to amplify the chloramphenicol resistance gene:

[0041] rh3_LP3_F: 5'-GGAAGCATAAAGTGTTAACGCCAGGGTTTTCCCAGTCACGAC-3',

[0042] rh3_LP3_R: 5'-CCAATCATAGGAGGAATTCGAGCTCGGTACCCGGGGAT-3';

[0043] Design primers to amplify the right replicon:

[0044] rh4_LP3_F: 5'-GGGTACCGAGCTCGAATTCCTCCTATGATTGGTTGTCTTATTACCTTACTTC-...

Embodiment 3

[0047] Example 3 Recombinant Bacillus subtilis P43NMK-P lytr - Construction of Phi29+LP3

[0048] The specific implementation method is the same as that of Examples 1-2, the difference is that P 43 promoter replaced by P lytr , constructed to obtain Bacillus subtilis P43NMK-P lytr -Phi29+LP3.

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Abstract

The invention discloses construction and application of a bacillus subtilis linear plasmid system, and belongs to the field of bacillus subtilis gene engineering. According to the invention, bacillussubtilis is used as an expression host, and a genome replication-related gene cluster derived from bacillus subtilis phage phi29 is expressed in the bacillus subtilis, so that replication and expression of linear plasmids in cells are realized. Through change of different promoters, the copy number and expression level of the linear plasmids can be controlled. According to verification, when the linear plasmid system is used for expressing the green fluorescent protein, a condition of plasmid loss does not occur after shaking-flask culture for 70 hours, and thus the novel expression tool laysa foundation for realizing efficient and stable gene expression, gene editing, metabolic engineering transformation and the like of bacillus subtilis.

Description

technical field [0001] The invention relates to the construction and application of a Bacillus subtilis linear plasmid system, belonging to the field of Bacillus subtilis genetic engineering. Background technique [0002] Bacillus subtilis is widely distributed in nature, including soil surface, water environment and animal stomach. As a model microorganism of Gram-positive bacteria, it is used in laboratory studies of sporulation mechanism and metabolic regulation. It is a non-pathogenic microorganism, does not contain endotoxins, and is generally recognized as safe (GRAS) food-grade microorganisms by the U.S. Food and Drug Administration (FDA). In addition, Bacillus subtilis has many advantages, including: fast growth, short culture time, and low culture requirements; the system for secreting signal peptide is very efficient, which makes it have a strong ability to secrete proteins, which provides great convenience for the subsequent protein extraction process; Its molec...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N15/65C12N1/21C12R1/125
CPCC12N15/75C12N15/65
Inventor 刘延峰堵国成田荣臻陈坚
Owner JIANGNAN UNIV
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