MEGA (Multiple Essential Genes Assembling) Deletion and Replacement Method for Genome Reduction in Escherichia coli

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• 作者：Xiaoli Xue ; Tao Wang ; Peng Jiang ; Yangyang Shao ; Min Zhou ; Li Zhong ; Ronghai Wu ; Jianting Zhou ; Haiyang Xia ; Guoping Zhao ; Zhongjun Qin
• 刊名：ACS Synthetic Biology
• 出版年：2015
• 出版时间：June 19, 2015
• 年：2015
• 卷：4
• 期：6
• 页码：700-706
• 全文大小：313K
• ISSN：2161-5063

文摘

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 鈥淢EGA (Multiple Essential Genes Assembling) deletion and replacement鈥?that functions by assembling multiple essential genes in an E. coli鈥揝. 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鈥?05 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.