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

Kohei Ike, Yusuke Arasawa, Satoshi Koizumi, Satoshi Mihashi, Shigeko Kawai-Noma, Kyoichi Saito, Daisuke Umeno

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1352-1360
Number of pages9
JournalACS Synthetic Biology
Volume4
Issue number12
DOIs
Publication statusPublished - 2015 Dec 18
Externally publishedYes

Keywords

  • betI repressor
  • directed evolution
  • genetic switch
  • self-cloning
  • synthetic biology

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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