Method for efficiently and specifically removing plasmids in bacteria by utilizing integrated suicide vector

Abstract

The invention provides a method for efficiently and specifically removing plasmids in bacteria by utilizing an integrated suicide vector. The method comprises the following steps: (1) constructing theintegrated suicide vector targeting to-be-knocked-out plasmids, (2) screening and converting the integrated suicide vector, (3) carrying out first-round screening on the bacteria, and (4) carrying out second-round screening on the bacteria. According to the method for efficiently and specifically removing the plasmids in the bacteria by utilizing the integrated suicide vector, the stability of genomes of the bacteria is not influenced, the method is verified in various strains, the plasmids with the sizes of 24kbp to 340kbp can be efficiently knocked out, the method is simple and effective, and the removal rate is as high as 100 percent.

Description

technical field [0001] The invention belongs to the field of gene editing, and in particular relates to a method for efficiently and specifically removing plasmids in bacteria by using integrated suicide vectors. Background technique [0002] Plasmids are widely found in the biological world, ranging from bacteria, actinomycetes, filamentous fungi, macrofungi, yeast to plants, and even human organisms. Plasmids contained in bacteria are DNA molecules that self-replicate independently of the bacterial extrachromosomal. Plasmids are not essential for host survival, however, plasmids often confer environmental resistance to the host. Plasmids carry genes with many functions, including resistance to antibiotics and heavy metals, sensitivity to mutagens, susceptibility or resistance to bacteriophages, production of restriction enzymes, production of rare amino acids and toxins, determinant toxicity forces, degrade complex organic molecules, and the ability to form symbiotic rel...

AI Technical Summary

Problems solved by technology

[0007] However, there are often multiple copies of plasmids inside the bacteria. CRISPR / Cas9 enzyme digestion is difficult to remove each plasmid, and the bacteria itself contains DNA ligase, which will reconnect the severed plasmids, which cannot guarantee the efficiency of plasmid elimination.

Plasmid incompatibility methods require precise cloning of origins of replication and are often ineffective for large plasmids containing multiple origins of replication

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