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

doi:10.1021/acssynbio.5b00107

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^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

13 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 language	English
Pages (from-to)	1352-1360
Number of pages	9
Journal	ACS Synthetic Biology
Volume	4
Issue number	12
DOIs	https://doi.org/10.1021/acssynbio.5b00107
Publication status	Published - 2015 Dec 18
Externally published	Yes

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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10.1021/acssynbio.5b00107

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title = "Evolutionary Design of Choline-Inducible and -Repressible T7-Based Induction Systems",

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.",

keywords = "betI repressor, directed evolution, genetic switch, self-cloning, synthetic biology",

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

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year = "2015",

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doi = "10.1021/acssynbio.5b00107",

language = "English",

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T1 - Evolutionary Design of Choline-Inducible and -Repressible T7-Based Induction Systems

AU - Ike, Kohei

AU - Arasawa, Yusuke

AU - Koizumi, Satoshi

AU - Mihashi, Satoshi

AU - Kawai-Noma, Shigeko

AU - Saito, Kyoichi

AU - Umeno, Daisuke

PY - 2015/12/18

Y1 - 2015/12/18

N2 - 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.

AB - 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.

KW - betI repressor

KW - directed evolution

KW - genetic switch

KW - self-cloning

KW - synthetic biology

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

Abstract

Keywords

ASJC Scopus subject areas

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