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

Antibiotic resistance maker-free bacillus subtilis constructing method and method for screening bacillus subtilis with inactivated target gene

A technology of Bacillus subtilis and resistance markers, applied in biochemical equipment and methods, microbial measurement/testing, DNA preparation, etc., can solve the problems of cumbersome construction of knockout vectors and low efficiency of recombination mutation, and achieve recombination mutation High efficiency, simple construction of integrated mutation vector, and simple process

Inactive Publication Date: 2013-09-11
NANJING AGRICULTURAL UNIVERSITY
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above method can achieve the purpose of screening without antibiotic resistance markers, the process of constructing a knockout vector is cumbersome and the efficiency of recombination mutation is low

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
  • Antibiotic resistance maker-free bacillus subtilis constructing method and method for screening bacillus subtilis with inactivated target gene
  • Antibiotic resistance maker-free bacillus subtilis constructing method and method for screening bacillus subtilis with inactivated target gene
  • Antibiotic resistance maker-free bacillus subtilis constructing method and method for screening bacillus subtilis with inactivated target gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0048] Construction of embodiment 1BS-PS mutant strain

[0049] (1) Construction of SpovAF-CM-LysA homologous exchange fragment

[0050] build route see figure 2 . With spoVAF-F (SEQ ID NO.1) and spoVAF-R (SEQ ID NO.2) as primers, with Bacillus subtilis 168 (BS168) genomic DNA as template PCR amplification spoVAF gene; with LysA-F (SEQ ID NO. NO.3) and LysA-R (SEQ ID NO.4) were primers, and the Lys gene was amplified by PCR using BS168 genomic DNA as a template; CM-F (SEQ ID NO.5) and CM-R (SEQ ID NO. 6) was used as a primer, and the CM (chloramphenicol resistance gene) including the promoter and the terminator was amplified by PCR using the pJC164.3 plasmid (BGSC, containing the CM gene) as the template. The three amplified genes were respectively cloned into the pMD19-T (purchased from Takara Company) vector, and after verification and correct sequencing, they were named pMD19-T-spoVAF, pMD19-T-LysA, and pMD19-T-CM respectively. Store at -20°C for later use.

[0051] T...

Embodiment 2

[0064] Example 2 Utilize the non-antibiotic resistance marker gene screening technology to screen the Bacillus subtilis with target gene inactivation

[0065] (1) Construction of BS-PS strain protease nprE and aprE inactivation integration vector

[0066] In the present invention, two protease genes aprE and nprE secreted by Bacillus subtilis are selected, and an insertion inactivation strategy is adopted to conduct mutation inactivation research. Using E. coli pMD19-T as the backbone, a general integration vector PLC-T was constructed. The PLC-IaprE-T integration vector for insertion inactivation of aprE gene and the PLC-InprE-T integration vector for insertion inactivation of nprE gene were constructed on the basis of the universal vector. Build routes like Figure 5 shown.

[0067] The specific construction process of the PLC-T integration vector is as follows:

[0068]Using P43-F (SEQ ID NO.9) / P43-R (SEQ ID NO.10) as primers, using BS168 genomic DNA as a template for P...

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 an antibiotic resistance maker-free bacillus subtilis constructing method and a method for screening bacillus subtilis with an inactivated target gene. The method comprises the steps of: constructing two homologous exchange segments by using bacillus subtilis 168 as a starting strain; constructing a controllable lysine auxotroph strain BS-PS by two dual exchange processes on the basis of a homologous recombination principle; constructing a universal integrated mutation carrier PLC by using a colibacillus clone carrier pMD19-T as a framework; and constructing an integrated carrier PLC-InprE of the insertional inactivated nprE by using the neutral protease nprE of the bacillus subtilis as the inactivated target gene and by adopting an insertional inactivation strategy based on a programmable logic controller (PLC), and converting the PLC-nprE into BS-PS, controlling lysine to synthesize a screening marker by a single exchange process and a spontaneous single exchange process and screening protease insertional inactivation mutant strains by combining the result of polymerase chain reaction (PCR) verification.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for constructing Bacillus subtilis without antibiotic resistance markers and a method for screening Bacillus subtilis with inactivated target genes. Background technique [0002] Bacillus subtilis is recognized as a safe strain (GRAS), which can secrete and express active products from exogenous genes. In addition, Bacillus subtilis has a clear genetic background, rapid growth, simple culture conditions, good fermentation foundation and production technology, making it widely used in scientific research and industrial and agricultural production. As an important industrial microorganism, B. subtilis has the advantage of being developed as a food-grade expression host, reducing the cost of downstream isolation and purification of recombinant biological products used in the food industry. [0003] At present, the biggest problem of B. subtilis as a host is the protease secreted...

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): C12N15/09C12N15/10C12N15/63C12Q1/68C12Q1/04
Inventor 陆兆新张充别小妹吕凤霞王煜赵海珍
Owner NANJING AGRICULTURAL UNIVERSITY
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