iCatch:a new strategy for capturing large DNA fragments using homing endonucleases  

作　　者：Jingman Wang Anrui Lu Jiakun Liu Weiren Huang Jin Wang Zhiming Cai Guoping Zhao 

机构地区：[1]Carson International Cancer Center,Shenzhen University School of Medicine,Shenzhen Second People's Hospital,The First Affiliated Hospital of Shenzhen University,Shenzhen 518039,China [2]Institute of Synthetic Biology,Shenzhen Institutes of Advanced Technology,Chinese Academy of Sciences,Shenzhen 518055,China [3]Key Laboratory of Synthetic Biology,Institute of Plant Physiology and Ecology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai 200032,China [4]State Key Laboratory of Oncology in South China,Sun Yat-Sen University Cancer Center,Guangzhou 510060,China [5]Shanghai-M0ST Key Laboratory of Health and Disease Genomics,Chinese National Human Genome Center at Shanghai,Shanghai 200000,China [6]Guangdong Key Laboratory of Systems Biology and Synthetic Biology,Urogenital Tumors,Shenzhen 518035,China

出　　处：《Acta Biochimica et Biophysica Sinica》2019年第1期97-103,共7页生物化学与生物物理学报（英文版）

基　　金：the grants from the National Key Basic Research Program of China (973 Program)(No.2014CB745201);the National Natural Science Foundation of China (No.81772737);the Strategic Priority Research Program of the Chinese Academy of Sciences (No.XDB19040200);the International Cooperation Program of the Chinese Academy of Sciences (No.153D31KYSB20170121);the National Science Foundation Projects of Guangdong Province (No.2017B030301015);the Shenzhen Municipal Government of China (Nos.JCYJ20170413161749433 and JSGG20160301161836370);the Sanming Project of Shenzhen Health and Family Planning Commission (Nos.SZSM201412018 and SZSM201512037);and the High Level University Medical Discipline Construction (No.2016031638).

摘　　要：Natural genetic materials contain many biosynthetic gene clusters encoding potentially valuable natural products,many of which can be used directly without codon optimization or other manipulations.With the development of synthetic biology,several DNA assembly standards have been proposed,conveniently facilitating the reuse of natural materials.Among these standards,the iBrick assembly standard was developed by our laboratory to manipulate large DNA fragments,employing two homing endonucleases.Considering the difficulty of cloning large iBrick parts using conventional endonuclease-mediated restriction and ligation methods,we herein present a new method,known as iCatch,which readily captures biosynthetic gene clusters.As the clusters cloned by iCatch have the prefix and suffix of the iBrick standard,they serve as new iBrick parts and are therefore conducive to further editing and assembly with the iBrick standard.iCatch employs the natural homologous recombination system to flank the region of interest with I-Scel and PI-Pspl recognition sites,after which the genome is digested with I-Scel or PI-Pspl and the fragments are then self-ligated to clone the target DNA fragments.We used this method to successfully capture the actinorhodin biosynthetic cluster from Streptomyces coelicolor and then heterologously expressed this cluster in a thermophilic Streptomyces strain.We propose that iCatch can be used for the cloning of DNA sequences that are dozens of kilobases in length,facilitating the heterologous expression of microbial natural products.Moreover,this cloning methodology can be a complementary tool for the iBrick standard,especially in applications requiring the manipulation of large DNA fragments.

关 键 词：GENOMIC CAPTURE HOMING ENDONUCLEASE BIOLOGICAL part HETEROLOGOUS expression 

分 类 号：Q[生物学]