Construction of Highly Efficient and Rigorous Targetron System in Escherichia coli

doi:10.13560/j.cnki.biotech.bull.1985.2018-0883

Abstract

Abstract: The objective is to construct a highly efficient and rigorous Targetron gene targeting system in Escherichia coli. Firstly,the T7-lac operon from pET28a and the group II intron from pSY6 were assembled to construct an IPTG-inducible Targetron plasmid system in E. coli. Then,taking the lacZ gene as an example,the efficiency and stringency of the IPTG-inducible Targetron targeting system were verified by analyzing the insertion efficiency of the group II introns before and after induction. Finally,a highly efficient and rigorous inducible Targetron gene targeting system in E. coli was obtained after optimizing the IPTG concentration and induction time. As results,group II introns were unable to be inserted into 2 target sites at all in the absence of IPTG induction,the targeting efficiency was 0. When induced for 45 min after adding 0.5 mmol/L IPTG,the targeting efficiency at lacZ-635s site increased to 90.8±5.5%,and the targeting efficiency at lacZ-1063a site increased to 92.6±2.4%. In conclusion,the IPTG-inducible Targetron gene targeting system in E. coli is successfully established,laying a foundation for the research and application of the group II intron.

Key words: Targetron, group II intron, inducible system, Escherichia coli, lacZ

CHEN Xiang-hao, LIU Fang, WANG Cai-xia, CHEN Zheng-hong, HONG Wei, CAI Meng-di, ZHANG Zheng-rong, QI Ting-na, LIAO Yong-hui, GU Jun-ying, CUI Gu-zhen. Construction of Highly Efficient and Rigorous Targetron System in Escherichia coli[J]. Biotechnology Bulletin, 2019, 35(6): 213-220.

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