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A Bacillus subtilis expression system and genetically engineered bacteria producing recombinant glutamic acid decarboxylase

A technology of glutamic acid decarboxylase and genetically engineered strains, applied in the field of Bacillus subtilis expression system and genetically engineered bacteria producing recombinant glutamic acid decarboxylase, can solve the lack of exogenous gene promoter and expression efficiency in B.subtilis expression system low development

Inactive Publication Date: 2015-10-21
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

Exogenous genes from various sources have been expressed in the B. subtilis expression system, but there is still a big gap compared with Escherichia coli, and the low expression efficiency is still a bottleneck restricting its development
[0003] An important reason for this problem is that the B. subtilis expression system lacks a promoter capable of efficiently transcribing foreign genes
However their problem is that a binary vector must be used for induction and two inducers are required

Method used

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  • A Bacillus subtilis expression system and genetically engineered bacteria producing recombinant glutamic acid decarboxylase
  • A Bacillus subtilis expression system and genetically engineered bacteria producing recombinant glutamic acid decarboxylase
  • A Bacillus subtilis expression system and genetically engineered bacteria producing recombinant glutamic acid decarboxylase

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Embodiment 1

[0029] (1) Construction of T7 RNA polymerase integrated expression vector

[0030] build route see figure 2 .

[0031] 1. Escherichia coli BL21(DE3)pLysS (purchased from Novagen) genomic DNA was used as a template to amplify the T7 RNA polymerase gene T7plo by PCR with P1 (SEQ ID NO.1) and P2 (SEQ ID NO.2) as primers;

[0032] T7plo is obtained through the following reaction system and reaction procedure:

[0033]

[0034] The PCR program is 94°C 2min; 30×(94°C 45s; 58°C 50s; 72°C 4min); 72°C 10min.

[0035] 2. With P3 (SEQ ID NO.3) and P4 (SEQ ID NO.4) as primers, with pHT01 plasmid (purchased from Mo Bi Tec company) as template, PCR amplification promoter Pgrac gene (Pgrac is induced by lactose expression promoter, including Bacillus subtilis groE promoter sequence, lacO operating region, and gsiB SD sequence);

[0036] The Pgrac gene was obtained through the following reaction system and reaction procedure:

[0037]

[0038] The PCR program is 94°C 2min; 30×(94°...

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Abstract

The invention relates to a novel bacillus subtilis expression system and genetic engineering bacteria producing recombined glutamic acid decarboxylase. The novel bacillus subtilis expression system comprises a genetic engineering strain BS-T710 and an expression carrier pBT-23a. According to genetic engineering bacteria BS-T710-gadB producing recombined glutamic acid decarboxylase, a gene gadB is inserted into the expression carrier pBT-23a so as to obtain the recombined expression carrier pBT-23a-gad, the pBT-23a-gad is converted into the genetic engineering strain BS-T710 through a chemical conversion method, so as to obtain the genetic engineering bacteria BS-T710-gadB producing the recombined glutamic acid decarboxylase. The system can be used for expressing foreign genes efficiently and only needs one inducer. MSG (monosodium glutamate) can be converted into gamma-GABA (aminobutyric acid) efficiently by the recombined engineering bacteria BS-T710-gadB cell.

Description

technical field [0001] The invention belongs to the field of food biotechnology, and relates to a Bacillus subtilis expression system and a genetically engineered bacterium producing recombinant glutamic acid decarboxylase. Background technique [0002] Bacillus subtilis (Busillus.subtilis) is recognized as a safe strain (GRAS), and has a set of efficient secretion signals and molecular chaperone systems, which can endow it with the ability to efficiently secrete target proteins; at the same time, B.subilis grows rapidly and has simple culture conditions , The genetic background is clearer. B. subtilis has the potential to be developed as a food-grade high-efficiency expression system. Exogenous genes from various sources have been expressed in the B. subtilis expression system, but there is still a big gap compared with Escherichia coli, and the low expression efficiency is still the bottleneck restricting its development. [0003] An important reason for this problem is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/75C12N1/21C12N9/88C12P13/00C12R1/125
Inventor 陆兆新张充吕凤霞别小妹陈琳
Owner NANJING AGRICULTURAL UNIVERSITY
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