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

PLoS One. 2015 Mar 19;10(3):e0120243. doi: 10.1371/journal.pone.0120243. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 4-Butyrolactone / analogs & derivatives
  • 4-Butyrolactone / chemistry
  • 4-Butyrolactone / metabolism
  • Aliivibrio fischeri / genetics
  • Bacterial Proteins / genetics*
  • Escherichia coli / metabolism
  • Gene Library
  • Genes, Switch*
  • Genetic Vectors / genetics
  • Genetic Vectors / metabolism
  • Kanamycin Kinase / genetics*
  • Promoter Regions, Genetic
  • Repressor Proteins / genetics
  • Simplexvirus / genetics
  • Spectrometry, Fluorescence
  • Thymidine Kinase / genetics*
  • Trans-Activators / genetics
  • Viral Proteins / genetics*

Substances

  • Bacterial Proteins
  • Repressor Proteins
  • Trans-Activators
  • Viral Proteins
  • LuxR autoinducer binding proteins
  • homoserine lactone
  • Thymidine Kinase
  • Kanamycin Kinase
  • 4-Butyrolactone

Grants and funding

This work is supported by the Precursory Research for Embryonic Science and Technology (PRESTO) program of the Japan Science and Technology Agency (JST), Grant-in-Aid for Scientific Research on Innovative Areas (23108507) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT)/ Japan Society for Promotion of Science (JSPS), and the commission for Development of Artificial Gene Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI), Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.