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Antibiotic lysobacter spp gene knockout system as well as construction method and application thereof

An antibiotic lysobacteria, gene knockout technology, applied in microorganism-based methods, biochemical equipment and methods, chemical instruments and methods, etc. And other issues

Inactive Publication Date: 2017-02-15
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because lysobacteria are resistant to many antibiotics, and the types of antibiotics that are sensitive to them are different, it is not easy to construct a gene knockout system for lysobacteria, and the effective gene knockout system for different lysobacteria or even different strains of the same species is also different. same, cannot share

Method used

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  • Antibiotic lysobacter spp gene knockout system as well as construction method and application thereof
  • Antibiotic lysobacter spp gene knockout system as well as construction method and application thereof
  • Antibiotic lysobacter spp gene knockout system as well as construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Knockout of LaPhzD gene in the antibiotic Lysobacterium OH13 of embodiment 1

[0044] Follow these steps:

[0045] (1) According to the complete genome information of the antibiotic Lysobacterium OH13, design the target gene LaPhzD upstream arm primer LaPhzD-F1 / LaPhzD-R1 and downstream arm primer LaPhzD-F2 / LaPhzD-R2 and mutant verification primer LaPhzD-deletion-MF / LaPhzD- deletion-MR.

[0046] LaPhzD-F1: CGGGATCCCG ACGAAGCCCTGCTGCTACCC

[0047] LaPhzD-R1: CCCAAGCTTGGG CGAAGCGGCATTCTTGACC

[0048] LaPhzD-F2: CCCAAGCTTGGG ACGCCGTCGCCGATTTCT

[0049] LaPhzD-R2: GCTCTAGAGC CGTGCGCCCTTCGATGAA

[0050] LaPhzD-deletion-MF: AAGATCCAGCCTTATGCG

[0051] LaPhzD-deletion-MR: CAGCAACGGGGTAGTCAT

[0052] (2) The 1100-bp upstream arm was amplified by PCR, and the restriction site was BamHI / HindIII; the downstream arm was 940-bp, and the restriction site was HindIII / XbaI, and the plasmid pJQ200SK was digested with BamHI / XbaI.

[0053] (3) The upper and lower arms after ...

Embodiment 2

[0057] Knockout of LaPhzB gene in the antibiotic Lysobacterium OH13 of embodiment 2

[0058] Follow these steps:

[0059] (1) According to the complete genome information of the antibiotic Lysobacterium OH13, design the target gene LaPhzB upstream arm primer LaPhzB-F1 / LaPhzB-R1 and downstream arm primer LaPhzB-F2 / LaPhzB-R2 and mutant verification primer LaPhzB-deletion-MF / LaPhzB- deletion-MR.

[0060] LaPhzB-F1: CGGGATCCCG CGGAGTCGGACTTGCTGTGG

[0061] LaPhzB-R1: CCCAAGCTTGGG CGCCCTTGCGGCTCATAT

[0062] LaPhzB-F2: CCCAAGCTTGGG GATTCCGGCGATCAAGCG

[0063] LaPhzB-R2: GCTCTAGAGC CAGCAATCCAACCAGGTCTCG

[0064] LaPhzB-deletion-MF: CGGTGAAACCCGACCTGC

[0065] LaPhzB-deletion-MR: GGCGTCTGCCCACTTCTT

[0066] (2) The 1318-bp upstream arm was amplified by PCR, and the restriction site was BamHI / HindIII; the downstream arm was 1102-bp, and the restriction site was HindIII / XbaI, and the plasmid pJQ200SK was digested with BamHI / XbaI.

[0067] (3) The upper and lower arms aft...

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Abstract

The invention discloses an antibiotic lysobacter spp gene knockout system as well as a construction method and an application thereof, and belongs to the technical field of microbiological genetic engineering. A gene knockout mutant is prepared by the following steps: constructing a recombinant vector for gene knockout by virtue of a suicide plasmid; transferring the recombinant vector into competent cells; conducting primary recon screening and secondary recon screening on a gentamicin containing resistance medium and a sucrose containing medium; and conducting mutant screening by virtue of PCR (polymerase chain reaction). With the application of the knockout system, two genes in antibiotic lysobacter spp OH13 can be successfully knocked out, proving that the system is applicable to gene function researches of the antibiotic lysobacter spp; therefore, a foundation is laid for researches on small-molecule metabolite biosynthesis, genetic regulation and action mechanism.

Description

technical field [0001] The invention relates to an antibiotic Lysobacterium OH13 gene knockout system based on homologous recombination and its construction method and application, belonging to the field of microbial genetic engineering. Background technique [0002] Lysobacter spp are a class of Gram-negative bacteria belonging to the family Xanthomonadaceae, genus Lysobacter. In 1978, Christensen and Cook established the genus Lysobacterium and named four species: L.enzymogenes, L.antibioticus, L.brunescens and L.brunescens. Lysobacterium colloidus (L. gummosus). Lysobacteria are ubiquitous in the environment and can survive in soil, fresh water or decaying organic matter. The main feature of Lysobacterium is that it has no flagella but has the ability to slide. The G+C% content of the genome reaches 65-70%. It can produce a large number of lyases and small molecular secondary metabolites. It is effective for many microorganisms, such as fungi, oomycetes, and yeast Bact...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12R1/01
CPCC07K14/195C12N15/74C12N2800/101
Inventor 赵杨扬刘凤权徐会永钱国良赵延存
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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