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Myxococcus flavus large-fragment knockout plasmid and knockout method thereof

A large fragment and plasmid technology, applied in the biological field, can solve the problems of high screening intensity, long mutant purification time, restricting the application of DK1622, etc.

Pending Publication Date: 2022-02-11
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Claims
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Problems solved by technology

However, the existing knockout methods have problems such as high screening intensity and long purification time of mutants in the construction of large genome fragment knockouts, which seriously restrict the application of DK1622 as a chassis engineering strain

Method used

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  • Myxococcus flavus large-fragment knockout plasmid and knockout method thereof
  • Myxococcus flavus large-fragment knockout plasmid and knockout method thereof
  • Myxococcus flavus large-fragment knockout plasmid and knockout method thereof

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Embodiment 1

[0026] The Myxococcus flavum DK1622 strain involved in the present invention is a model strain.

[0027] This example provides a method for constructing a large fragment knockout of Myxococcus xanthus, first of all, transforming the pBJ113 suicide vector. Using the genome of Myxococcus xanthus DK1622 as a template, primers pilA-1 and pilA-2 were used to amplify the promoter fragment P of pilA gene pilA (Its nucleotide sequence is shown in SEQ ID NO.1); With pJQ200SK plasmid as template, utilize primer SacB-1 and SacB-2 to amplify the ORF fragment of sacB gene (its nucleotide sequence is as shown in SEQ ID NO.2 shown); the promoter fragment P of the pilA gene pilA There is a 20bp homologous sequence at the 3' end of the sacB gene and the 5' end of the ORF fragment of the pilA gene, and the promoter fragment P of the pilA gene pilA The ORF fragment of the sacB gene was used as a template, and PCR amplification was performed using pilA-1 and SacB-2 primers to obtain the ORF fus...

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Abstract

The invention discloses a myxococcus flavus large-fragment knockout plasmid and a knockout method thereof. According to the method, a promoter fragment PpilA of a pilA gene and an ORF fragment of a sacB gene are fused to obtain a PpilA-SacB module, a tetracycline resistance gene is amplified to obtain a PpilA-SacB module, and the PpilA-SacB module and the Ptet-TCR module are constructed to BamHI and XbaI sites of a pBJ113 vector to obtain the plasmid pBJ116. According to the invention, the plasmid pBJ116 contains a kanamycin positive screening marker, a galactose negative screening marker, a tetracycline positive screening module and a sucrose negative screening module, and large fragments on a myxococcus flavus genome are knocked out through a positive and negative screening system twice; tests find that the siderophore generation level of the Myxochelin A coding gene delta MXAN_3618 mutant obtained by the method disclosed by the invention is remarkably reduced, and the capability of preying on pseudomonas aeruginosa is remarkably reduced; and the time required by the method is short.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a large fragment knockout plasmid of Myxococcus flavum and a knockout method thereof. Background technique [0002] Myxobacteria can produce biologically active metabolites with novel structures, rich types, and diverse mechanisms of action, mainly including: peptides, macrocycles, aromatics, etc., which have important research and application values ​​in agriculture and medicine. Due to the fact that most myxobacteria have aggregate growth, slow colony growth, and difficulty in forming single colonies, it is difficult to perform genetic manipulation, which seriously hinders the development and utilization of bioactive metabolites in myxobacteria. Myxococcus xanthus DK1622 is an ideal model strain for studying the heterologous expression of bioactive metabolites of myxobacteria due to its good dispersibility and fast growth rate. However, the existing knockout methods ha...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N1/21C12N15/65C12N15/31C12R1/01
CPCC12N15/74C12N15/65C07K14/195
Inventor 董义杰朱红惠董红红冯广达姚青
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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