Junjie Yang1,2,3, Bingbing Sun4,5, He Huang6,7, Biao Chen8,9, Chongmao Xu10,11, Xin Wang12,13, Jinle Liu14,15, Liuyang Diao16,17. 1. 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. 2. Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, 201201, China. yangjunjie@sibs.ac.cn. 3. Shanghai Collaborative Innovation Center for Biomanufacturing Technology, Shanghai, 200237, China. yangjunjie@sibs.ac.cn. 4. 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. 5. Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, 201201, China. bbsun@sibs.ac.cn. 6. 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. 7. Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, 201201, China. huanghe@sibs.ac.cn. 8. 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. 9. Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, 201201, China. bchen@cibt.ac.cn. 10. 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. 11. Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, 201201, China. cmxu@cibt.ac.cn. 12. 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. 13. University of the Chinese Academy of Sciences, Beijing, 100080, China. wangxin@sibs.ac.cn. 14. 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. 15. University of the Chinese Academy of Sciences, Beijing, 100080, China. liujinle@sibs.ac.cn. 16. 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. 17. Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, 201201, China. lydiao@sibs.ac.cn.
Abstract
OBJECTIVES: 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.
OBJECTIVES: 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.