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

A suicide vector, specific technology, applied in the field of gene editing, can solve the problem that the efficiency of plasmid elimination cannot be guaranteed

Inactive Publication Date: 2020-10-30
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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

Method used

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

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Embodiment 1

[0038] A method for efficient and specific removal of plasmids in bacteria using an integrated suicide vector, the vector pCs-ΔpESA3 displays a pESA3-specific fragment and the ampicillin resistance gene bla, replication initiator oriR6K, sucrose-sensitive marker sacB and broad host range mobility Region localization of mobRP4. pCVD442 was linearized by PCR and ligated with pESA3-specific DNA fragment to obtain plasmid pCS-ΔpESA3.

[0039] The plasmid pCS-ΔpESA3 was extracted from S17-1λpir Escherichia coli and transformed into Enterobacter sakazakii BAA-894. Through genetic recombination of the pCS-ΔpESA3 plasmid and the integration of the endogenous plasmid pESA3 of BAA-894, the Enterobacter sakazakii BAA-894 containing the integrated plasmid was obtained through ampicillin selection, and cultured in a non-resistant medium caused the spontaneous loss of the integrated plasmid of some bacteria , sucrose exposure selectively kills bacteria harboring integrated plasmids for the...

Embodiment 2

[0061] In order to verify the generality of the method, another integrated suicide vector was constructed in an attempt to knock out the plasmids leading to multi-drug resistance in a variety of bacteria.

[0062] Cronobacter sakazakii GZcsf-1, Shigellaflexneri strain M2901, Salmonella enterica subsp.enterica serovarTyphi strain, Citrobacter freundii strain Iona 4 and pneumonia Klebsiella pneumoniae strain555 all have antibiotic resistance, and their drug resistance is considered to be related to the plasmids they carry, which are pGW1 plasmid (340kbp) of Enterobacter sakazakii, pB4878 plasmid (55kbp) of Salmonella, pM2901 plasmid (81 kbp) for Shigella flexneri, pCFI-2 plasmid (24 kbp) for Citrobacter flexneri and pSCKLB555-1 plasmid (247 kbp) for Klebsiella pneumoniae. There are homologous segments in these 5 plasmids, and these 5 plasmids may have a common origin. Utilizing the homologous segments in these five plasmids, the homologous segments were cloned into the suicide ...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74A61K48/00A61P31/04C12R1/22C12R1/01
CPCA61K48/005A61P31/04C12N15/74
Inventor 季学猛王硕路平
Owner NANKAI UNIV
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