Evolutionary Design of Choline-Inducible and -Repressible T7-Based Induction Systems.
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.
Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University , 1-33 Yayoi-Cyo, Inage-ku, Chiba 263-8522, Japan.
Kohei
K
Ike
Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University , 1-33 Yayoi-Cyo, Inage-ku, Chiba 263-8522, Japan.
Yusuke
Y
Arasawa
Technology Development & Research Department, Kyowa Hakko Bio Co., Ltd. , 1-6-1, Ohtemachi, Chiyoda-ku, Tokyo 100-8185, Japan.
Satoshi
S
Koizumi
Technology Development & Research Department, Kyowa Hakko Bio Co., Ltd. , 1-6-1, Ohtemachi, Chiyoda-ku, Tokyo 100-8185, Japan.
Satoshi
S
Mihashi
Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University , 1-33 Yayoi-Cyo, Inage-ku, Chiba 263-8522, Japan.
Shigeko
S
Kawai-Noma
Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University , 1-33 Yayoi-Cyo, Inage-ku, Chiba 263-8522, Japan.
Kyoichi
K
Saito
Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University , 1-33 Yayoi-Cyo, Inage-ku, Chiba 263-8522, Japan.|Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST) , 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
Daisuke
D
Umeno
D017123:Bacteriophage T7 / Q000235:genetics / Q000378:metabolism; D002794:Choline / Q000494:pharmacology*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism
C098629:BetI protein, E coli; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D002794:Choline
betI repressor; directed evolution; genetic switch; self-cloning; synthetic biology