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Self-regulation expression system of bacillus subtilis and building method and application of self-regulation expression system

A Bacillus subtilis, expression system technology, applied in bacteria, introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve the problems of low expression level, low copy number, loss of function, etc.

Active Publication Date: 2014-12-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the following problems commonly exist in the expression of exogenous genes in Bacillus subtilis: when exogenous genes are recombined into the genome for expression, the expression level is often low due to the low copy number; , the phenomenon of plasmid instability often occurs, and the stability of the plasmid is mainly maintained by adding an excessive amount of antibiotics during the fermentation process
However, the expression of the antitoxin gene can release the dormancy effect caused by the toxin protein, because the unstable antitoxin protein can bind to the stable toxin protein and make it lose its function

Method used

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  • Self-regulation expression system of bacillus subtilis and building method and application of self-regulation expression system
  • Self-regulation expression system of bacillus subtilis and building method and application of self-regulation expression system
  • Self-regulation expression system of bacillus subtilis and building method and application of self-regulation expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of in vitro recombinant fragments

[0044] Using Bacillus subtilis168 genome template, #1 (shown in SEQ ID NO.1) and #2 (shown in SEQ ID NO.2) as primers, PCR was used to amplify the 452bp upstream fragment of endB-ndoA of the Bacillus subtilis168 genome and named F; Using plasmid P7Z6 (shown in SEQ ID NO.27) as a template, #3 (shown in SEQ ID NO.3) and #4 (shown in SEQ ID NO.4) as primers, amplified by PCR Primer bleomycin resistance gene fragment, named Z; with plasmid PAX01 (shown in SEQ ID NO. 28) template, #5 (shown in SEQ ID NO. 5) and #6 (shown in SEQ ID NO. 6) are primers, and T is amplified by PCR 0 Terminator (shown in SEQ ID NO. 7) and xylose-inducible promoter Pxyl (shown in SEQ ID NO. 8) fragments, named T 0 -Pxyl; Using Bacillus subtilis168 genome template, #7 (shown in SEQ ID NO.9) and #8 (shown in SEQ ID NO.10) as primers, the Bacillus subtilis168 genome ndoA gene fragment (nucleoside The acid sequence is shown in SEQ ID NO.11), name...

Embodiment 2

[0046] Example 2 F-Z-T 0 -Pxyl-N-R fragment transforms Bacillus subtilis168

[0047] F-Z-T after fusion 0 -After the Pxyl-NR PCR product is purified, it is diluted with water, the DNA concentration is adjusted to 100μg / mL, and then 40μL of dilution is added to 500μL of Bacillus subtilis168 transformed competent cells (refer to Anagnostopoulos, C., and Spizizen for the preparation method of competent ,J.:Requirements for transformation in Bacillus subtilis.J.Bacterial.81(1961)741-746.), heat shock at 45℃ for 3min, put it in a shaker at 37℃, incubate at 220rpm for 1h, then apply 50ug / ml bleomycin LB plate, dark culture for 10h, select the grown colonies, #11 (shown in SEQ ID NO.14) and #12 (shown in SEQ ID NO.15) as primers, Perform PCR verification, then extract the genome of the strain with the correct verification result, and use the genome as a template, #11 (as shown in SEQ ID NO.14) and #12 (as shown in SEQ ID NO.15) as primers, PCR The obtained fragments were verified by se...

Embodiment 3

[0048] Example 3 Determination of the lethal concentration of xylose

[0049] The modified strain Bacillus subtilis169 with endB gene deletion and xylose-induced expression of ndoA gene was inoculated onto LB plates containing different concentrations of xylose, and the lethal concentration of xylose was determined to be 1.5 g / L.

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Abstract

The invention discloses a self-regulation expression system of bacillus subtilis, and a building method and an application of the self-regulation expression system, and belongs to the field of genetic engineering. The expression system disclosed by the system comprises bacillus subtilis for xylose-induced expression of an endonuclease gene and a plasmid expression vector for constitutive expression of an anti-endonuclease gene. In a culture medium containing an inducer xylose, only the bacillus subtilis containing stably existing expression vector can normally grow. A fluorescent protein gene (gfp), a hyaluronic acid synthase gene (hasA) and a keratinase gene (ker) are respectively expressed by using the expression system disclosed by the invention; and by fermented cultivation, the fluorescence intensity, the hyaluronic acid content and the keratinase activity are respectively improved by 21.8%, 45% and 30.2% in comparison with those of the plasmid maintained by using antibiotic.

Description

Technical field [0001] The invention relates to a Bacillus subtilis self-regulating expression system and its construction and application, belonging to the field of genetic engineering. Background technique [0002] Bacillus is playing an important role in applied biology due to its fast growth, short fermentation cycle, and strong ability to secrete extracellular proteins. Among them, Bacillus subtilis, as a well-researched model strain, is also generally considered safe (GRAS), and has been widely used in food and drug research and production. [0003] At present, the following problems generally exist when using Bacillus subtilis to express foreign genes: When the foreign genes are recombined into the genome for expression, the expression level is often low due to the low copy number; when using plasmids for expression , Plasmid instability often occurs. The most important thing in plasmid stability is to maintain it by adding excessive antibiotics during the fermentation proc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N1/21
Inventor 陈坚堵国成康振杨森
Owner JIANGNAN UNIV
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