Bacillus subtilis self-splitting in stable stage and application thereof

A Bacillus subtilis, stable phase technology, applied in the field of genetic engineering, can solve the problems of target product denaturation, time-consuming and labor-intensive crushing process, etc.

Active Publication Date: 2016-12-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, when Bacillus subtilis is used to overexpress certain enzyme molecules and synthesize some intracellular metabolites, it often occurs that the nature of these products cannot be secreted in large quantities outside the cell, an...

Method used

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  • Bacillus subtilis self-splitting in stable stage and application thereof
  • Bacillus subtilis self-splitting in stable stage and application thereof
  • Bacillus subtilis self-splitting in stable stage and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1 Integration vector construction

[0041] Using Bacillus subtilis 168 genomic template, #1 and #2 as primers, P mmgA Promoter (as shown in SEQID NO.1) DNA fragment; with Bacillus subtilis 168 genome template, #3 and #4 are primers, amplified by PCR P sigW Promoter (as shown in SEQ ID NO.2) DNA fragment; with Bacillus subtilis 168 genome template, #5 and #6 are primers, amplified by PCR P yqfD Promoter (as shown in SEQ ID NO.3) DNA fragment; With Bacillussubtilis 168 genome template, #7 and #8 are primers, by PCR amplification Bacillus subtilis168skfA gene (as shown in SEQ ID NO.4) DNA fragment; With Bacillus subtilis 168 genome template, #9 and #10 are primers, amplify sdpC gene (as shown in SEQ ID NO.5) DNA fragment by PCR; With Bacillus subtilis168 genome template, #11 and #12 are primers, amplify by PCR Increase xpf gene (as shown in SEQ ID NO.6) DNA fragment; with Bacillussubtilis 168 genome template, #13 and #14 are primers, amplify lytC gene (as show...

Embodiment 2

[0042] Example 2 Expression of Fluorescent Protein Using Bacillus subtilis Logarithmic Phase Expression System

[0043] Using the plasmid pMD-19T simple-gfp as a template, #17 and #18 as primers, clone the gfp fluorescent protein gene (as shown in SEQID NO.29), add an XhoI restriction site and RBS to the front, and add a PstI restriction to the rear site. The PCR product obtained by digestion with XhoI and PstI was ligated to pP43 treated with the same enzyme (sequence shown in SEQ ID NO.28) to form a new plasmid pP43-gfp.

[0044] Add 10 μL of pP43-gfp plasmid with a concentration of 100 μg / mL to 500 μL of Bacillus subtilis 168, B. subtilis MSA(pP43-gfp), B. subtilis MSC(pP43-gfp), B. subtilis MXF(pP43-gfp) respectively , B. subtilis MLC (pP43-gfp), B. subtilis MBG (pP43-gfp), B. subtilis SSA (pP43-gfp), B. subtilis SSC (pP43-gfp), B. subtilis SXF (pP43-gfp), B. subtilis SLC (pP43-gfp), B. subtilis SBG (pP43-gfp), B. subtilis YSA (pP43-gfp), B. subtilis YSC (pP43-gfp), B. s...

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Abstract

The invention discloses Bacillus subtilis self-splitting in stable stage and application thereof and belongs to the field of genetic engineering. An expression host herein is Bacillus subtilis that self-degrades when entering the stable stage; after the Bacillus subtilis is cultured in the stable stage, protein molecules capable of promoting cell splitting may be overexpressed under promotion of a stable-stage promoter so that the host is quickly split and matters in cells are released. Green fluorescent protein gene (gfp) is expressed by using the expression host; through fermentation culture for the host as compared with a host targeted with traditional Bacillus subtilis, final bacterial concentration is decreased by at least 63.2%, with extracellular fluorescent protein content increased by at least by 78.8-141.5%.

Description

technical field [0001] The invention relates to a bacillus subtilis which is self-cleaved in a stable period and an application thereof, belonging to the field of genetic engineering. Background technique [0002] Bacillus subtilis (Bacillus subtilis) is playing an important role in applied biology because of its fast growth rate, short fermentation period, and strong ability to secrete extracellular proteins. At the same time, as a well-studied model strain, it is generally recognized as safe (GRAS), and has been widely used in the research and production of food and medicine. [0003] However, when Bacillus subtilis is used to overexpress certain enzyme molecules and synthesize some intracellular metabolites, it often occurs that these products cannot be secreted in large quantities outside the cell due to the nature of these products, and Bacillus subtilis is due to its Gram-positive bacteria It has the characteristics of a thick peptidoglycan layer outside the cell, and...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N1/21C07K14/435C12R1/125
CPCC07K14/32C07K14/43595C12N15/75C12N2800/101
Inventor 康振陈坚堵国成杨森
Owner JIANGNAN UNIV
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