title: 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.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, China.
authors.forename: Junjie
authors.initials: J
authors.lastname: Yang
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, China.
authors.forename: Bingbing
authors.initials: B
authors.lastname: Sun
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, China.
authors.forename: He
authors.initials: H
authors.lastname: Huang
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, China.
authors.forename: Liuyang
authors.initials: L
authors.lastname: Diao
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, China.
authors.forename: Biao
authors.initials: B
authors.lastname: Chen
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, China.
authors.forename: Chongmao
authors.initials: C
authors.lastname: Xu
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China University of the Chinese Academy of Sciences, Beijing, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China University of the Chinese Academy of Sciences, Beijing, China.
authors.forename: Jinle
authors.initials: J
authors.lastname: Liu
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Weihong
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, China syang@sibs.ac.cn.
authors.forename: Sheng
authors.initials: S
authors.lastname: Yang
mesh_terms: D004720:Endonucleases; D004926:Escherichia coli / Q000235:genetics*; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D010957:Plasmids; D045522:Recombinases; D011995:Recombination, Genetic
chemical_list: D045522:Recombinases; D004720:Endonucleases
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