Method of rapidly eliminating antibiotic-resistant plasmids in E. coli

A technology of Escherichia coli and antibiotics, applied in the field of microbial genetics, to achieve good results, high plasmid elimination rate, and promote breakdown

Inactive Publication Date: 2013-06-19
JIANGNAN UNIV
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Problems solved by technology

Using electric shock combined with special electric shock buffer to quickly eliminate antibiotic-resistant plasmids in E. coli has not been reported at home and abroad

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  • Method of rapidly eliminating antibiotic-resistant plasmids in E. coli

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Embodiment 1

[0047] Escherichia coli SCS110, JM109, and DH5α contain the antibiotic resistance plasmid pK19mobsacB, which belongs to Gram-negative bacteria. Because there is a kanamycin resistance gene on the pK19mobsacB plasmid, Escherichia coli SCS110, JM109, and DH5α containing the pK19mobsacB plasmid can all be used in the Grow on kanamycin-resistant plates.

[0048] A method for obtaining Escherichia coli that eliminates antibiotic resistance plasmids after electric shocking human enterobacteria, the steps are as follows:

[0049] A. Preparation of competent cells:

[0050] (1) Pick the antibiotic-resistant plasmid pk19mobsacB from the LB plate (Schafer A, Tauch A, Jager W, et al. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in The chromosome of Corynebacterium glutamicum.Gene, 1994, 145 (1): 69-73) Escherichia coli single bacterium colony, such as Escherichia coli SCS110 strain single bacteri...

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Abstract

A method of rapidly eliminating antibiotic-resistant plasmids in E. coli is mainly used for rapidly and effectively eliminating antibiotic-resistant plasmids from the E. coli. A method of electric shock for the E. coli is used to eliminate the antibiotic-resistant plasmids in the E. coli, and a special electric shock buffer is used to promote elimination of antibiotic-resistant plasmids, thereby improving elimination rate of the plasmids. When cells are in an electric field, a voltage reaches a critical value, localized cell membranes are punctured to form instantaneous pores, wherein pore size is suitable for discharging of the plasmids from the cells. After the electric shock, damaged cell membranes can restore by themselves. Alkali metals in the electric shock buffer can combine with the cell membranes to promote breakdown of the cell membranes, so that plasmids in the cells are easy to be discharged. An elimination rate of the plasmid reaches 93 %.

Description

technical field [0001] The invention belongs to the related field of microbial genetics. technical background [0002] Plasmids are small covalently closed circular double-stranded DNA molecules that are independent of chromosomes and have independent replication capabilities and are not necessary for bacterial survival. They are ubiquitous in bacterial cells. Since the genome is easily disturbed by the plasmid DNA when the genome is extracted, a genome with higher purity can be obtained after eliminating the plasmid, which is of great significance for the study of bacterial genetics and molecular biology; at the same time, when people study bacterial strains with plasmids , I always hope to get the corresponding plasmid-eliminating bacteria. Compared with the original strain of the plasmid, the bacteria that completely eliminate the plasmid can observe and study the genetic function of the plasmid. The bacteria that completely eliminate the plasmid can also be used as genet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/01C12N13/00C12N1/20C12R1/19
Inventor 张震宇刘合栋
Owner JIANGNAN UNIV
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