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Published on: June 25, 2015
Evolutionary Design of Choline-Inducible and -Repressible T7-Based Induction Systems
Kohei Ike1, Yusuke Arasawa1, Satoshi Koizumi2
1Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University , 1-33 Yayoi-Cyo, Inage-ku, Chiba 263-8522, Japan.
Abstract:
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.
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