Method for increasing yield of Bacillus subtilis antimicrobial peptide by overexpression comA gene

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

Inactive Publication Date: 2011-10-19
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 yield of Bacillus subtilis antimicrobial peptide by overexpression comA gene
  • Method for increasing yield of Bacillus subtilis antimicrobial peptide by overexpression comA gene
  • Method for increasing yield of Bacillus subtilis antimicrobial peptide by overexpression comA gene

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specific Embodiment approach

[0041] The PCR amplification system is as follows:

[0042] 10x Pfu PCR buffer 10μl

[0043] 10 μM upstream primer 10 μl

[0044] 10μl of 10μM downstream primer

[0045] 2.5mM dNTPs 8μl

[0046] Bacillus subtilis genomic DNA

[0047] Or pMUTIN4 plasmid DNA 1μl

[0048] Pfu DNA polymerase 1μl

[0049] wxya 2 O 60μl

[0050] 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.

[0051] pDK (purchased from Bacilluse Genetic Stock Center ID: ECE143) snaBI / SalI The large fragment obtained after double digestion, and the fragment including the Pspac promoter and multiple cloning site (MCS) obtained after the same double digestion treatment of the pMUTIN4 vector, use T 4 DNA ligase ligation to construct the pDK-Pspac vector, after the enzyme digestion was verified to be correct, it was stored at -20°C for later use;

[0052] For pDK-Pspac SnaBI / sacI The large fragment obtained after double enzyme digestion is the sam...

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Abstract

The invention relates to a method increasing yield of Bacillus subtilis antimicrobial peptide by overexpression comA gene, belonging to the field of biotechnology. In the method, Bacillus subtilis ATCC9943 is used as an initial strain, and integrated vector pDK is used as a frame to construct a gene overexpression vector pDK-ComA; and by utilizing the homologous recombination principle, the comA gene is integrated into Bacillus subtilis ATCC9943 genome through a double exchange process, thereby constructing a high-yield antimicrobial peptide strain FMB27. The high efficiency liquid chromatography analysis proves that the surfactin / fengycin yield of the improved strain is greatly increased.

Description

1. Technical field [0001] The present invention relates to the overexpression of comA 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. 2. Background technology [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. [0004] At present, in the world, the me...

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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