title: MEGA (Multiple Essential Genes Assembling) deletion and replacement method for genome reduction in Escherichia coli.
abstract: Top-down reduction of the bacterial genome to construct desired chassis cells is important for synthetic biology. However, the current progress in the field of genome reduction is greatly hindered by indispensable life-essential genes that are interspersed throughout the chromosomal loci. Here, we described a new method designated as "MEGA (Multiple Essential Genes Assembling) deletion and replacement" that functions by assembling multiple essential genes in an E. coli-S. cerevisiae shuttle vector, removing targeted chromosomal regions containing essential and nonessential genes using a one-round deletion, and then integrating the cloned essential genes into the in situ chromosomal loci via I-SceI endonuclease cleavage. As a proof of concept, we separately generated three large deletions (80-205 kbp) in the E. coli MDS42 chromosome. We believe that the MEGA deletion and replacement method has potential to become widely used in large-scale genome reductions in other sequenced organisms in addition to E. coli.
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Xue
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Tao
authors.initials: T
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 200032, China.
authors.forename: Peng
authors.initials: P
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 200032, China.
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Shao
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Min
authors.initials: M
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhong
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Ronghai
authors.initials: R
authors.lastname: Wu
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Jianting
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Haiyang
authors.initials: H
authors.lastname: Xia
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Zhongjun
authors.initials: Z
authors.lastname: Qin
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D017353:Gene Deletion; D020043:Genes, Essential / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D016680:Genome, Bacterial; D012441:Saccharomyces cerevisiae / Q000235:genetics
keywords: essential gene; gene assembly; genome engineering; genome reduction