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

CRISPR Cas9 system system for Bacillus subtilis genome edition and establishment method thereof

A Bacillus subtilis genome editing technology, applied in the field of genetic engineering, can solve problems such as bacterial infection and difficulty in controlling the fermentation process

Active Publication Date: 2016-06-15
JIANGNAN UNIV +1
View PDF4 Cites 48 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But B.subtilisWSH10-03 will produce a lot of foam during the fermentation process, which makes the fermentation process difficult to control and may cause bacterial contamination

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
  • CRISPR Cas9 system system for Bacillus subtilis genome edition and establishment method thereof
  • CRISPR Cas9 system system for Bacillus subtilis genome edition and establishment method thereof
  • CRISPR Cas9 system system for Bacillus subtilis genome edition and establishment method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction of CRISPR / Cas9 gene editing system to knock out plasmid PHY300dsrf

[0036] (1) According to the gene sequence of Bacillus subtilis, the sgRNA for specifically targeting the srfA-C gene is designed, and then the sgRNA double-stranded oligonucleotide sequence (such as SEQ ID NO: 2) is designed on the basis of the sgRNA of the srfA-C gene , look up the gene sequence of the PE194 temperature-sensitive replication origin (such as SEQ ID NO: 3) from NCBI and then synthesize sgRNA and PE194 sequences and connect them to the PMD18-T vector, using the following primers 5'-GGAACGTACAGACGCATTTTACATTTTTAGAAATGGGC-3' (such as SEQ ID NO: 5) Perform PCR amplification with 5'-CGTTTGTTGAACTACGCAGTCGGCTTAAACCAG-3' (such as SEQ ID NO: 6) to obtain Frag1. The reaction system is shown in Table 1.

[0037] Table 1 reaction system

[0038] 5xPhusion HF Reaction Buffer

[0039] The reaction procedure was as follows: pre-denaturation at 94°C for 4 min; 30 cy...

Embodiment 2

[0056] Embodiment 2: Bacillus subtilis transformation method

[0057] Dip the frozen Bacillus subtilis with an inoculation loop, then streak on the LB plate, and cultivate overnight at 37°C for activation. Pick a single colony and inoculate in 5mL LB liquid medium, and culture overnight at 37°C for 18h. Take a certain amount of overnight culture into 4.5mL of GMI, so that the OD600 value reaches 0.1-0.2, leaving 4.5mL of mixed bacterial liquid. Shake culture at 200 rpm at 37°C, measure OD600 every 20 minutes, when OD600 reaches 0.4-0.6 (about 60-90 minutes); continue shaking culture for 90 minutes, draw 0.05ml of bacterial liquid into a sterile test tube containing 0.45mL of preheated GMⅡ medium; shaking at 37°C for 90 minutes, at this time many competent cells formed in the culture; add 1 μg of plasmid (15-20uL), shake and culture at 37°C for 30 minutes; centrifuge to remove most of the supernatant, resuspend the cells, and spread on Incubate overnight at 37°C on a screenin...

Embodiment 3

[0062] Embodiment 3: use plasmid PHY300dsrf to knock out the srfA-C gene in B.subtilis168 genome

[0063]Using the method in Example 2, the knockout plasmid PHY300dsrf was transformed into competent cells of Bacillus subtilis, spread on LB solid medium (containing 20ug / mL tetracycline), and cultured overnight at 37°C. Pick positive clones, extract the genome, use the genome as a template, use the following primers 5'-CTCGAGGCTAGGGGCAGCGAGCAAACAGC-3' (such as SEQ ID NO: 17) and 5'-GAGCAGCTCTTTCGGCTCATAG-3' (such as SEQ ID NO: 18) to carry out PCR amplification, and then in Perform XbaI digestion verification at 37°C. When the srfA-C gene knockout strain performs genome homology repair, an XbaI restriction site is inserted at the srfA-C gene break, so it can be cut by the XbaI endonuclease, while the wild type will not be cut ( figure 2 , image 3 ).

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 CRISPR Cas9 system for Bacillus subtilis genome edition and an establishment method thereof, belonging to the technical field of gene engineering. A knock-out plasmid PHY300dsrf is established; and the plasmid comprises sgRNA and cas9 genes of the specific targeted target gene, a homologous restoration arm, and replication initial points and tolerance screening markers of Escherichia coli and Bacillus subtilis. The sgRNA of the specific targeted srfA-C gene can guide the cas9 protein to cut double strands at the specific site of the srfA-C gene, and can be used for accurate homologous recombination on the genome under the guide of the homologous restoration arm, thereby introducing the XhoI enzyme digestion site at the rupture. When the tank feed fermentation culture is carried out on the Bacillus subtilis of which the srfA-C gene is successfully knocked out, the foam is obviously reduced, which proves that the CRISPR / Cas9 gene editing system can effectively perform gene edition on the Bacillus subtilis genome.

Description

technical field [0001] The invention relates to a CRISPR Cas9 system for genome editing of Bacillus subtilis and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] Bacillus subtilis (Bacillus subtilis) is a Gram-positive bacterium, because it is easy to isolate and culture, has a relatively clear genetic background, good secretion, and no pathogenicity, it has become an important industrial strain and is increasingly used More are used to produce antibiotics, pharmaceutical proteins and industrial enzyme preparations. B. subtilis168 is a laboratory type strain containing many mutations. The strains currently used in industry such as B.subtilisWB600 and B.subtilisWB800 are all derived from B.subtilis168. A heat-resistant acidic pullulanase production strain was previously screened in our laboratory, which was named Bacillus (Bacillus. Preservation number CCTCCNO: M2013012, patent publication number CN103255...

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
IPC IPC(8): C12N15/75C12N15/31
CPCC07K14/32C12N15/75C12N2800/80C12N2999/007
Inventor 吴敬段绪果张康
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