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Method for increasing output of antibacterial peptides of bacillus subtilis through knockout (i)abrB(/i) genes

A technology of Bacillus subtilis and antimicrobial peptides, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc.

Inactive Publication Date: 2011-09-07
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0004] At present, in the world, the methods to increase its production are mainly to screen excellent strains and optimize fermentation conditions, but few use molecular biology techniques to improve strains method to increase the production of antimicrobial substances

Method used

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  • Method for increasing output of antibacterial peptides of bacillus subtilis through knockout (i)abrB(/i) genes
  • Method for increasing output of antibacterial peptides of bacillus subtilis through knockout (i)abrB(/i) genes
  • Method for increasing output of antibacterial peptides of bacillus subtilis through knockout (i)abrB(/i) genes

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specific Embodiment approach

[0042] The PCR amplification system is as follows:

[0043] 10x Pfu PCR buffer 10μl

[0044] 10 μM upstream primer 10 μl

[0045] 10μl of 10μM downstream primer

[0046] 2.5mM dNTPs 8μl

[0047] Bacillus subtilis genomic DNA

[0048] Or pHCMC04 plasmid DNA 1μl

[0049] Pfu DNA polymerase 1μl

[0050] wxya 2 O 60μl

[0051] The PCR program was 94°C for 2min; 34×(94°C for 45s; 58°C for 50s; 72°C for 4min; 72°C for 10min.

[0052] p abrB5' -T for EcoRI / wxya Obtained after double digestion abrB5' Fragment, and the pGEM-T vector that has been treated with the same double enzyme digestion, are connected with T4 DNA ligase to construct pGEM- abrB5' Carriers, after enzyme digestion and verification are correct, store them at -20°C for later use;

[0053] p abrB3' -T for wxya / SphI Obtained after double digestion abrB3' Fragment, with pGEM- abrB5' Vector, ligated with T4 DNA ligase to construct pGEM- abrB Carriers, after enzyme digestion and verif...

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Abstract

The invention relates to a method for increasing the output of antibacterial peptides of bacillus subtilis through knockout (i)abrB( / i) genes, and belongs to the field of biotechnology. The method comprises the following steps of: firstly establishing a gene knockout vector pAbrB-Cm by taking bacillus subtilis ATCC9943 as a starting strain and taking a cloning vector pGEM-T of escherichia coli asa skeleton; and establishing a high-yield antibacterial peptide strain FMB29 through a knockout method of genes of a genome abrB of the bacillus subtilis ATCC9943 by applying a double exchange process based on a homologous recombination principle. Through high performance liquid chromatograph, the capacity of producing surfactins and fengycins of improved strains is greatly improved, and the existence of the surfactins and fengycins in fermentation products of improved strains FMB29 is identified through mass spectrum.

Description

technical field [0001] The present invention involves knocking out abrB The invention relates to a method for improving the production of antimicrobial peptides of Bacillus subtilis by gene, which belongs to the field of biotechnology. Background technique [0002] Bacillus subtilis ( Bacillus subtilis ) is currently recognized as one of the safe microorganisms, and its antibacterial metabolites are rich and diverse, especially lipopeptide antibacterial substances, which not only have a wide antibacterial spectrum but also have the function of biosurfactant, so they can not only be used for biological control in agriculture , and also has a wide range of applications in industry and environmental protection. [0003] Bacillus subtilis ( Bacillus subtilis ) ATCC 9943 is a Bacillus subtilis that can secrete surfactin, fengycin and other antimicrobial peptide substances (Valérie Leclère, Romain Marti, Max Béchet. The lipopeptides mycosubtilin and surfactin enhance spreadin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N1/21C12P21/02C12R1/125
Inventor 陆兆新曹国强吕凤霞别小妹张充钟蕾
Owner NANJING AGRICULTURAL UNIVERSITY
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