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

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 d...

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Bibliographic Details
Published in:PloS one 2015-03, Vol.10 (3), p.e0120243
Main Authors: Tominaga, Masahiro, Ike, Kohei, Kawai-Noma, Shigeko, Saito, Kyoichi, Umeno, Daisuke
Format: Article
Language:English
Subjects:
4-Butyrolactone - analogs & derivatives
4-Butyrolactone - chemistry
4-Butyrolactone - metabolism
Acids
Adenosine kinase
Aliivibrio fischeri - genetics
Aminoglycoside phosphotransferase
Antibacterial agents
Bacterial Proteins - genetics
Biological evolution
Biotechnology
Caprolactone
Chemistry
Circuit design
Circuits
Cloning
Design
E coli
Engineering
Escherichia coli
Escherichia coli - metabolism
Gene expression
Gene Library
Gene therapy
Genes, Switch
Genetic Vectors - genetics
Genetic Vectors - metabolism
Herpes simplex
Kanamycin
Kanamycin Kinase - genetics
Kinases
Mutagenesis
Negative selection
Nucleosides
Phosphotransferase
Plasmids
Promoter Regions, Genetic
Proteins
Repressor Proteins - genetics
Simplexvirus - genetics
Spectrometry, Fluorescence
Switches
Thymidine
Thymidine kinase
Thymidine Kinase - genetics
Trans-Activators - genetics
Vibrio fischeri
Viral Proteins - genetics
Viruses
Water-borne diseases
Waterborne diseases
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0120243