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Method for improving bacillus subtilis strain

A Bacillus subtilis and bacterial strain technology, applied in the field of bioengineering, can solve problems such as the production limit of Bacillus subtilis extracellular enzymes, and achieve the effect of avoiding cannibalism and improving yield

Active Publication Date: 2015-12-23
河南新仰韶生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It is precisely because of this phenomenon that the production of Bacillus subtilis extracellular enzymes is fundamentally limited, and there is no better way to improve it at present

Method used

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  • Method for improving bacillus subtilis strain
  • Method for improving bacillus subtilis strain
  • Method for improving bacillus subtilis strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] The method for transforming the Bacillus subtilis bacterial strain provided by the present invention, at first artificially constructing does not contain (knockout) sporulation lethal factor gene ( skf ) gene sequence, and then the constructed gene sequence without (knockout) sporulation lethal factor ( skf ) gene sequence to perform homologous recombination replacement in Bacillus subtilis to obtain a new knockout (excluding) sporulation lethal factor gene ( skf ) of the Bacillus subtilis strain, specifically comprising the following steps.

[0061] (1) The artificial construction does not contain (knockout) the sporulation lethal factor gene ( skf ) gene sequence

[0062] The gene without (knockout) sporulation lethal factor ( skf ) gene sequence, which consists of 3 parts of DNA fragments, which are skf Upstream partial sequence (fragment A), tetracycline resistance gene expression sequence (fragment B), skf The downstream partial sequence of the gene (...

Embodiment 2

[0173] This example is mainly to test the applicability of the new Bacillus subtilis strain constructed in Example 1 (i.e. the No. 5 transformant strain), mainly including the number of viable bacteria and the determination of amylase activity after fermentation. The introduction is as follows.

[0174] Determination of Viable Bacteria

[0175] Transformant No. 5 obtained in Example 1 (the new constructed Bacillus subtilis strain) and the starting strain (Bacillus subtilis WB800) were cultured in shake flasks at 37°C and 220rpm, and 0.1 mL, were added to centrifuge tubes containing 0.9mL sterile water, vortexed, and serially diluted 10 times (10, 10 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 ).

[0176] Draw 0.1mL of the dilution, add it to the LB solid plate, and then spread the bacterial solution evenly with a coating rod, make 3 plates for each dilution, let it stand for 30 minutes, then place it upside down in a constant temperature incubator at 37°C, an...

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Abstract

The invention belongs to the field of biology engineering technologies, and particularly relates to a method for improving bacillus subtilis strains. The method includes the steps of manually constructing a gene sequence not containing spore production lethal factor genes, electric shock conversion, sieving verifying and others. In the prior art, although part of basic research is already implemented on the spore production lethal factor genes (skf), whether the yield of endogenous enzymes can be increased through deleting the spore production lethal factor genes is still not determined, and meanwhile for different microorganism strains, after the spore production lethal factor genes are deleted, whether improvements on the fermentation capacity of the strains are the same lacks more research. With the detailed bacillus subtilis strains being examples, new strains lacking spore production lethal factor genes (skf) are successfully constructed, when the strains are concretely used for fermenting and producing amylase, determination results show that the enzyme activity of the constructed new strains is improved well, and good application prospects are achieved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for transforming bacillus subtilis strains. Background technique [0002] Bacillus subtilis ( Bacillus subtilis ) has the characteristics of non-pathogenicity and strong protein secretion ability, and is widely used in fields such as agriculture, medicine and industrial enzyme preparations. The enzyme preparations produced by Bacillus subtilis include amylase, protease, lipase, cellulase and nattokinase, etc. The commonly used methods to improve the activity of its fermentation enzymes are physical and chemical mutagenesis to screen new strains or use genetic engineering to increase related purposes gene expression. Therefore, an in-depth understanding of the regulation mechanism of Bacillus subtilis extracellular enzyme synthesis will be of great benefit to the breeding of Bacillus subtilis high-producing enzyme strains. [0003] It has been sugges...

Claims

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Application Information

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IPC IPC(8): C12N15/87C12N1/21C12N9/26C12R1/125
Inventor 焦国宝邱立友田芳王明道孙利鹏宋安东
Owner 河南新仰韶生物科技有限公司
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