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

PloS One
Masahiro TominagaDaisuke Umeno

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.

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Citations

Mar 25, 2021·Nature Communications·Masahiro TominagaAkihiko Kondo
Jan 26, 2021·Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry·Daisuke UmenoShigeko Kawai-Noma

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Methods Mentioned

BETA
PCR
flow cytometry
phosphotransferase

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