title: Rapid and liquid-based selection of genetic switches using nucleoside kinase fused with aminoglycoside phosphotransferase.
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.
authors: Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University, 1-33 Yayoi-Cyo, Inage-ku, Chiba, Japan.
authors: Masahiro
authors: M
authors: Tominaga
authors: Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University, 1-33 Yayoi-Cyo, Inage-ku, Chiba, Japan.
authors: Kohei
authors: K
authors: Ike
authors: Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University, 1-33 Yayoi-Cyo, Inage-ku, Chiba, Japan.
authors: Shigeko
authors: S
authors: Kawai-Noma
authors: Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University, 1-33 Yayoi-Cyo, Inage-ku, Chiba, Japan.
authors: Kyoichi
authors: K
authors: Saito
authors: Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University, 1-33 Yayoi-Cyo, Inage-ku, Chiba, Japan; Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama, Japan.
authors: Daisuke
authors: D
authors: Umeno
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D048908:Aliivibrio fischeri / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D005812:Genes, Switch; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D019868:Kanamycin Kinase / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics; D018139:Simplexvirus / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013937:Thymidine Kinase / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014764:Viral Proteins; C064778:LuxR autoinducer binding proteins; C088386:homoserine lactone; D013937:Thymidine Kinase; D019868:Kanamycin Kinase; D015107:4-Butyrolactone