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

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

doi:10.1371/journal.pone.0120243

Masahiro Tominaga, Kohei Ike + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0120243

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Journal: PloS one	Publication Date: Mar 19, 2015
Citations: 8	License type: CC BY 4.0

Affiliation: Chiba University, Japan Science and Technology Agency

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.

Highlights

Genetic switches and their assemblies consist of multiple interacting component functions
TetA selection can be conducted in liquid media, but it requires overnight growth in both ON- and OFF-selection. We recently reported another single-gene dual selector system in which herpes simplex virus thymidine kinase, which has been used as a marker in gene therapy [10], was adapted for the OFF-selection of a genetic circuit [9]
We directly link the reading frame of herpes simplex virus thymidine kinase (hsvTK) except the three nucleotides coding stop codon with the reading frame of positive marker APH (Fig. 1A) or chloramphenicol acetyltransferase (CAT) (Fig. 2A), excluding their start codon

Summary

Introduction

Genetic switches and their assemblies (regulatory circuits) consist of multiple interacting component functions. The hsvTK::APH fusion selector was successfully applied to the rapid isolation of a set of variant Vibrio fischeri (V. fischeri) lux promoters with different expression efficiencies. The lux box libraries were constructed by oligonucleotide-based PCR mutagenesis of pACplux-hsvtk::aph-sfgfp.

Results

Conclusion