Rapid and liquid-based selection of genetic switches using nucleoside kinase fused with aminoglycoside phosphotransferase

Masahiro Tominaga, Kohei Ike, Shigeko Kawai-Noma, Kyoichi Saito, Daisuke Umeno

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)

Abstract

The evolutionary design of genetic switches and circuits requires iterative rounds of positive (ON-) and negative (OFF-) selection. We previously reported a rapid OFF selection system based on the kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the artificial mutator nucleoside dP. By fusing hsvTK with the kanamycin resistance marker aminoglycoside-(3′)-phosphotransferase (APH), we established a novel selector system for genetic switches. Due to the bactericidal nature of kanamycin and nucleoside-based lethal mutagenesis, both positive and negative selection could be completed within several hours. Using this new selector system, we isolated a series of homoserine lactone-inducible genetic switches with different expression efficiencies from libraries of the Vibrio fischeri lux promoter in two days, using only liquid handling.

Original languageEnglish
Article numbere0120243
JournalPloS one
Volume10
Issue number3
DOIs
Publication statusPublished - 2015 Mar 19
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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