A method for rapid gene knockout of Bacillus licheniformis

Abstract

The invention relates to a method for achieving bacillus licheniformis gene knockout rapidly. The method comprises the following steps of 1, obtaining a gene fragment in a bacillus licheniformis to-be-knocked-out gene coding frame as a homologous arm sequence; 2, obtaining a resistance label gene fragment; 3, conducting an overlapped and extended PCR on the homologous arm sequence and the resistance label gene fragment, so that a fused gene sequence of the homologous arm sequence and the resistance label gene fragment is obtained, and treating the fused gene sequence as a gene knockout fragment for use; 4, conducting single digestion on the fused gene sequence, converting bacillus licheniformis competent cells, and conducting recovery culture and screening culture, so that bacillus licheniformis with a gene knocked out is obtained. According to the method, the bacillus licheniformis gene is knocked out based on the single exchange principle, in the knockout process, homologous recombination only needs to be generated once, and the integration process is high in efficiency; whole knockout and screening work can be completed within three or four days, the gene knockout cycle is short, and knockout cost is effectively lowered.

Description

technical field [0001] The invention relates to a method for quickly realizing gene knockout of Bacillus licheniformis, belonging to the technical field of biotechnology. Background technique [0002] Bacillus licheniformis (Bacillus licheniformis), a Gram-positive thermophilic bacterium that exists widely in nature and can resist harsh environments, is one of the important production strains in the field of biotechnology industry. At present, it is mainly used in medicine, biological washing powder, nanotechnology and other fields, and has emerged in the degradation of biomass materials. It has been reported to regulate the metabolism of Bacillus licheniformis by molecular modification to achieve the accumulation of target products. [0003] Gene knockout (gene knockout), also known as gene targeting, is a new type of molecular biology technology. It uses DNA transformation technology to introduce the constructed targeting vector into the target cell, and then recombines t...

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Problems solved by technology

Although the method of gene knockout based on the above principles has been widely used, the overall construction process is complicated and the operation cycle is long

[0004] For example, the serpin gene knockout vector constructed in Chinese patent document CN103146739A (application number: CN201310070885) is composed of upstream homology arm (Up-serpin), resistance gene, downstream homology arm (Down-serpin) and pUC57 and other elements connected end-to-end. Therefore, only the construction of the vector requires several enzyme digestion ligation and sequencing operations, and it takes several weeks or even more than a month to complete the entire operation cycle. At the same time, due to the complex structure of the vector and the long sequence, the transformation efficiency is generally low.

Although the above method can achieve special needs such as gene knockout without trace, but in the gene knockout operation for the purpose of gene function verification, this kind of method often becomes the speed-limiting link of the whole experimental process

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