High-efficiency scarless genetic modification in Escherichia coli by using lambda red recombination and I-SceI cleavage.

Abstract

Genetic modifications of bacterial chromosomes are important for both fundamental and applied research. In this study, we developed an efficient, easy-to-use system for genetic modification of the Escherichia coli chromosome, a two-plasmid method involving lambda Red (λ-Red) recombination and I-SceI cleavage. An intermediate strain is generated by integration of a resistance marker gene(s) and I-SceI recognition sites in or near the target gene locus, using λ-Red PCR targeting. The intermediate strain is transformed with a donor plasmid carrying the target gene fragment with the desired modification flanked by I-SceI recognition sites, together with a bifunctional helper plasmid for λ-Red recombination and I-SceI endonuclease. I-SceI cleavage of the chromosome and the donor plasmid allows λ-Red recombination between chromosomal breaks and linear double-stranded DNA from the donor plasmid. Genetic modifications are introduced into the chromosome, and the placement of the I-SceI sites determines the nature of the recombination and the modification. This method was successfully used for cadA knockout, gdhA knock-in, seamless deletion of pepD, site-directed mutagenesis of the essential metK gene, and replacement of metK with the Rickettsia S-adenosylmethionine transporter gene. This effective method can be used with both essential and nonessential gene modifications and will benefit basic and applied genetic research.

• Authors: Junjie Yang, Bingbing Sun, He Huang, Yu Jiang, Liuyang Diao, Biao Chen, Chongmao Xu, Xin Wang, Jinle Liu, Weihong Jiang, Sheng Yang
• Journal: Applied and environmental microbiology (Appl Environ Microbiol) Vol. 80 Issue 13 Pg. 3826-34 (Jul 2014) ISSN: 1098-5336 [Electronic] United States
• PMID: 24747889 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Chemical References

• Recombinases
• Endonucleases

Topics

• Endonucleases
• Escherichia coli (genetics)
• Genetics, Microbial (methods)
• Molecular Biology (methods)
• Plasmids
• Recombinases
• Recombination, Genetic