Evolutionary Design of Choline-Inducible and -Repressible T7-Based Induction Systems

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• 作者：Kohei Ike ; Yusuke Arasawa ; Satoshi Koizumi ; Satoshi Mihashi ; Shigeko Kawai-Noma ; Kyoichi Saito ; Daisuke Umeno
• 刊名：ACS Synthetic Biology
• 出版年：2015
• 出版时间：December 18, 2015
• 年：2015
• 卷：4
• 期：12
• 页码：1352-1360
• 全文大小：445K
• ISSN：2161-5063

文摘

By assembly and evolutionary engineering of T7-phage-based transcriptional switches made from endogenous components of the bet operon on the Escherichia coli chromosome, genetic switches inducible by choline, a safe and inexpensive compound, were constructed. The functional plasticity of the BetI repressor was revealed by rapid and high-frequency identification of functional variants with various properties, including those with high stringency, high maximum expression level, and reversed phenotypes, from a pool of BetI mutants. The plasmid expression of BetI mutants resulted in the choline-inducible (Bet-ON) or choline-repressible (Bet-OFF) switching of genes under the pT7/betO sequence at unprecedentedly high levels, while keeping the minimal leaky expression in uninduced conditions.