Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A self-regulated expression system of Bacillus subtilis and its construction method and application

A Bacillus subtilis, self-regulating technology, applied in the field of genetic engineering, can solve problems such as low copy number, loss of function, unstable plasmid, etc.

Active Publication Date: 2016-09-07
JIANGNAN UNIV
View PDF3 Cites 1 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A self-regulated expression system of Bacillus subtilis and its construction method and application
  • A self-regulated expression system of Bacillus subtilis and its construction method and application
  • A self-regulated expression system of Bacillus subtilis and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 In vitro recombinant fragment construction

[0044] Using the Bacillus subtilis168 genome template, #1 (as shown in SEQ ID NO.1) and #2 (as shown in SEQ ID NO.2) as primers, the 452bp fragment upstream of the endB-ndoA of the Bacillus subtilis168 genome was amplified by PCR and named as F ; With plasmid P7Z6 (as shown in SEQ ID NO.27) as template, #3 (as shown in SEQ ID NO.3) and #4 (as shown in SEQ ID NO.4) as primers, by PCR amplification primer Bo The lyomycin resistance gene fragment is designated as Z; with the template of plasmid PAX01 (as shown in SEQ ID NO.28), #5 (as shown in SEQ ID NO.5) and #6 (as shown in SEQ ID NO.6) Shown) as primers, amplified by PCR T 0 Terminator (as shown in SEQ ID NO.7) and xylose-inducible promoter Pxyl (as shown in SEQ ID NO.8) fragments, designated as T 0 -Pxyl; with Bacillus subtilis168 genome template, #7 (as shown in SEQ ID NO.9) and #8 (as shown in SEQ ID NO.10) are primers, by PCR amplification Bacillus subtilis168...

Embodiment 2

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

[0047] Combine the fused F-Z-T 0 - After the Pxyl-N-R PCR product is purified, dilute it with water, adjust the DNA concentration to 100 μg / mL, and then add 40 μL of the diluent to 500 μL of Bacillus subtilis168-transformed competent cells (for the competent preparation method, refer to Anagnostopoulos, C., and Spizizen , J.: Requirements for transformation in Bacillus subtilis.J.Bacterial.81 (1961) 741-746.), heat shock at 45°C for 3min, put it in a shaker at 37°C, and cultivate it at 220rpm for 1h, and then spread it on the surface containing 50ug / ml bleomycin LB plate, dark culture 10h, select the grown colony, #11 (as shown in SEQ ID NO.14) and #12 (as shown in SEQ ID NO.15) are primers, carry out After PCR verification, the correct bacterial strain of the verification result is extracted genome, and with this genome as template, #11 (as shown in SEQ ID NO.14) and #12 (as shown in SEQ ID NO.15...

Embodiment 3

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

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a Bacillus subtilis self-regulating expression system and its construction method and application, belonging to the field of genetic engineering. The expression system of the present invention comprises a bacillus subtilis that expresses an endonuclease gene induced by xylose and a plasmid expression vector that constitutively expresses an endonuclease gene. In the culture medium containing the inducer xylose, only the Bacillus subtilis with the stable expression vector can grow normally. Fluorescent protein gene (gfp), hyaluronic acid synthase gene (hasA) and keratinase gene (ker) were respectively expressed by the expression system of the present invention, and by fermentation and cultivation, the fluorescence intensity, hyaluronic acid content and keratinase activity were compared with Plasmids maintained by antibiotics increased by 21.8%, 45% and 30.2%.

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 because of its fast growth rate, short fermentation cycle, and strong ability to secrete proteins. Among them, Bacillus subtilis, as a well-researched model strain, is also generally recognized as safe (GRAS), and has been widely used in the research and production of food and medicine. [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 p...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/75C12N1/21
Inventor 陈坚堵国成康振杨森
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com