Direct cloning and transplanting of large DNA fragments from Escherichia coli chromosome  

作　　者：Ying Zhu Yan Yang Pingping Den Yong Huang Mengxiang Ni Hongqing Fang 

机构地区：[1]Institute of Biotechnology, Academy of Military Medical Sciences [2]Institute of Life Science and technology, China Pharmaceutical University [3]Institute of Health Sciences, Anhui University [4]Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences

出　　处：《Science China(Life Sciences)》2016年第10期1034-1041,共8页中国科学（生命科学英文版）

基　　金：supported by the National Natural Science Foundation of China(81373286);National Basic Research Program of China(2011CBA00800)

摘　　要：We applied a resistance split-fusion strategy to increase the in vivo direct cloning efficiency mediated by Red recombination. The cat cassette was divided into two parts： cma （which has a homologous sequence with cmb） and cmb, each of which has no resistance separately unless the two parts are fused together. The crab sequence was integrated into one flank of a target clon- ing region in the chromosome, and a linear vector containing the cma sequence was electroporated into the cells to directly capture the target region. Based on this strategy, we successfully cloned an approximately 48 kb DNA fragment from the E. coli DH1-Z chromosome with a positive frequency of approximately 80%. Combined with double-strand breakage-stimulated homologous recombination, we applied this strategy to successfully replace the corresponding region of the E. coli DH36 chromosome and knock out four non-essential genomic regions in one step. This strategy could provide a powerful tool for the heterologous expression of microbial natural product biosynthetic pathways for genome assembly and for the functional study of DNA sequences dozens of kilobases in length.We applied a resistance split-fusion strategy to increase the in vivo direct cloning efficiency mediated by Red recombination.The cat cassette was divided into two parts: cma(which has a homologous sequence with cmb) and cmb, each of which has no resistance separately unless the two parts are fused together. The cmb sequence was integrated into one flank of a target cloning region in the chromosome, and a linear vector containing the cma sequence was electroporated into the cells to directly capture the target region. Based on this strategy, we successfully cloned an approximately 48 kb DNA fragment from the E.coli DH1-Z chromosome with a positive frequency of approximately 80%. Combined with double-strand breakage-stimulated homologous recombination, we applied this strategy to successfully replace the corresponding region of the E. coli DH36 chromosome and knock out four non-essential genomic regions in one step. This strategy could provide a powerful tool for the heterologous expression of microbial natural product biosynthetic pathways for genome assembly and for the functional study of DNA sequences dozens of kilobases in length.

关 键 词：Red homologous recombination resistance split-fusion target cloning transferring 

分 类 号：Q78[生物学—分子生物学]