title: Multiple-site genetic modifications in Escherichia coli using lambda-Red recombination and I-SceI cleavage.
abstract: OBJECTIVE
  Genetic modifications to bacterial chromosomes are important for research; recently we reported a two-plasmid system for single locus modification in Escherichia coli and an improved method for simultaneous multiple-loci modification is needed.
  
  
  RESULTS
  An intermediate bacterial strain was generated with different resistance marker genes flanked by I-SceI recognition sites at multiple target loci. Then a donor plasmid carrying several alleles with desired modifications was transformed into the intermediate strain together with a bifunctional helper plasmid encoding λ-Red recombinase and I-SceI endonuclease. I-SceI would induce double-strand breaks (DSBs) in the chromosome and λ-Red would induce recombination between chromosome DSBs and allele fragments from the donor plasmid, resulting in genomic modifications.
  
  
  CONCLUSIONS
  This method has been used to successfully perform three different loci modifications simultaneously.
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, China. yangjunjie@sibs.ac.cn.|Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, 201201, China. yangjunjie@sibs.ac.cn.|Shanghai Collaborative Innovation Center for Biomanufacturing Technology, Shanghai, 200237, China. yangjunjie@sibs.ac.cn.
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, 300 Fenglin Road, Shanghai, 200032, China. bbsun@sibs.ac.cn.|Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, 201201, China. bbsun@sibs.ac.cn.
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, 300 Fenglin Road, Shanghai, 200032, China. huanghe@sibs.ac.cn.|Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, 201201, China. huanghe@sibs.ac.cn.
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, 300 Fenglin Road, Shanghai, 200032, China. bchen@cibt.ac.cn.|Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, 201201, China. bchen@cibt.ac.cn.
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, 300 Fenglin Road, Shanghai, 200032, China. cmxu@cibt.ac.cn.|Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, 201201, China. cmxu@cibt.ac.cn.
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, 300 Fenglin Road, Shanghai, 200032, China. wangxin@sibs.ac.cn.|University of the Chinese Academy of Sciences, Beijing, 100080, China. wangxin@sibs.ac.cn.
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, 300 Fenglin Road, Shanghai, 200032, China. liujinle@sibs.ac.cn.|University of the Chinese Academy of Sciences, Beijing, 100080, China. liujinle@sibs.ac.cn.
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, 300 Fenglin Road, Shanghai, 200032, China. lydiao@sibs.ac.cn.|Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, 201201, China. lydiao@sibs.ac.cn.
authors.forename: Liuyang
authors.initials: L
authors.lastname: Diao
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D045522:Recombinases / Q000235:genetics* / Q000378:metabolism*; D011995:Recombination, Genetic; D014169:Transformation, Bacterial
chemical_list: D011994:Recombinant Proteins; D045522:Recombinases
keywords: Escherichia coli; Genetic modification; I-SceI endonuclease; Lambda Red recombination; Markerless