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

Bacillus licheniformis industrial production strain genetic transformation method

A technology of Bacillus licheniformis and genetic transformation method, which is applied in the field of microbial genetic engineering and fermentation engineering, can solve the problems of unstable and difficult genetic information, and the lack of obvious advantages of Bacillus licheniformis, and achieve the effect of ensuring genetic stability

Inactive Publication Date: 2008-07-30
JIANGNAN UNIV
View PDF0 Cites 9 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But there is no obvious advantage (Waschkau B et al, Appl MicrobiolBiotechnol, 2007) from the plasmid obtained from the Bacillus licheniformis R-M system deletion mutant that is used for the genetic transformation of Bacillus licheniformis; And isolate and purify plasmid DNA (microbial molecule Improved important molecular material, which is essentially extrachromosomal genetic material capable of self-replicating independently) is also very difficult
In addition, it is impossible for us to carry out deletion mutations on the R-M system of industrial production strains of Bacillus licheniformis, because the genetic information of the obtained genetically improved industrial strains will be very unstable

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
  • Bacillus licheniformis industrial production strain genetic transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Identification and Cloning of the Bacillus licheniformis Methyltransferase Encoding Gene

[0045] Analysis of the genome sequence of B. licheniformis ATCC 14580 (GenBank accession number: AE017333, Veith B et al, J Mol Biotechnol, 2004), determined that there is an open reading frame in the region 4169271 to 4170800 of the genome, and its encoded gene product may be a B. licheniformis-specific type I DNA methyltransferase. Chromosomal DNA was extracted with Chromosomal DNA Extraction Kit (Qiagen Company), using this DNA as a template, chemically synthesized oligonucleotides

[0046] metP1 5′-TC AGAATTC TGAGGAACACAACCATGGCTGAATT-3′, the underlined part is the EcoRI site, and

[0047] metP2 5′-TAC GAATTC TCCTAACTTACGCTCTTCCCA-3', the underlined part is the EcoRI site, which is a gene amplification primer, and under the action of PfuDNA polymerase, the coding gene of B. licheniformis methyltransferase ( Bli-met). The PCR product was digested with EcoRI, an...

Embodiment 2

[0050] Example 2: Methylation of Exogenous DNA Molecules

[0051] Competent cells of the recombinant strain Ec(met) were prepared by the method introduced by Sambrook (Sambrook et al, Molecular Cloning: ALab Manual, 1989) and stored at -70°C. The Escherichia coli-Bacillus licheniformis shuttle plasmids pHY 300PLK, pDG148-Stu, and pHY-WZX were prepared with a plasmid purification kit (Qiagen). Using the competent method, the above plasmids were transformed into recombinant bacteria Ec(met), and the transformants were selected on LB plates with a final concentration of 100 μg / mL ampicillin, 50 μg / mL tetracycline or 5 μg / mL kanamycin. After the positive transformants are isolated and purified, they are cultured in LB medium at 37°C for 24-48 hours, and the plasmid DNA transformed into it is methylated by the expression of the met gene. The plasmid DNA molecules in the above transformants were isolated and purified using a plasmid purification kit (Qiagen Company). That is, a pl...

Embodiment 3

[0052]Example 3: Specific methylated DNA molecules are used for genetic transformation of Bacillus licheniformis

[0053] Inoculate industrial production of Bacillus licheniformis strain B2004 or B0204 in 2.5mL LB medium containing 0.5mol / L sorbitol, after overnight culture, transfer to 50mL LB medium containing 0.5mol / L sorbitol, at 37℃ Grow to A 600 0.85-0.95. The bacteria were placed in an ice-water bath for 10 minutes, and the bacteria were collected by centrifugation at 10,000 r / min at 4°C for 5 minutes. The cells were repeatedly washed 4 times with pre-cooled EP buffer (0.5mol / L sorbitol, 0.5mol / L mannitol and 10% glycerol). Suspend the cells in about 1 mL of pre-cooled EP buffer. Aliquot 60 μL of bacteria into 1.5 mL Eppendorf tubes. Take 1 μL of the methylated or unmethylated plasmid DNA (50ng / L) prepared by the above method, mix it with the cells, transfer it into a pre-cooled electroporation cell, and add 1mL recovery medium (containing 0.5mol / L sorbitol and 0.3...

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 provides a bacillus licheniformis industrially producing bacterial strain genetic transformation method, which belongs to microbial gene engineering and fermentation technology field. The invention provides a bacillus licheniformis industrially producing bacterial strain genetic transformation method; firstly DNA molecule which needs to be genetically transformed is methylated by the bacillus licheniformis specificity methyltransferase medium; specifically methylated DNA molecule adopts optimized electroporation method to realize the genetic transformation of bacillus licheniformis industrially producing bacterial strain. By the method, the genetic transformation rate can reach 25-1250 strains / microgram DNA. In particular, the invention can be used in the genetic transformation of bacillus licheniformis bacterial strain that the prior genetic transformation cannot be realized.

Description

technical field [0001] The invention discloses a method for genetic transformation of bacillus licheniformis industrial production strains, which belongs to the field of microbial genetic engineering and fermentation engineering. Background technique [0002] Bacillus licheniformis is the main strain producing high-temperature α-amylase, alkaline protease, new antimicrobial peptides, and new biodegradable polymers (such as polyglutamic acid, polylysine, etc.). Realizing its efficient genetic improvement will be an important guarantee for realizing the industrialized production and industrialization of the above-mentioned important industrial products. However, due to the lack of effective genetic transformation technology, it is extremely difficult and seldom successful to carry out target-directed genetic improvement on both experimental strains and industrial strains of Bacillus licheniformis. Therefore, to study and solve the genetic transformation system of Bacillus lic...

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): C12N1/21C12N15/11C12N15/54C12N15/70C12N15/75C12R1/10
Inventor 王正祥牛丹丹
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