An efficient platform for genetic selection and screening of gene switches in Escherichia coli.
Engineered gene switches and circuits that can sense various biochemical and physical signals, perform computation, and produce predictable outputs are expected to greatly advance our ability to program complex cellular behaviors. However, rational design of gene switches and circuits that function in living cells is challenging due to the complex intracellular milieu. Consequently, most successful designs of gene switches and circuits have relied, to some extent, on high-throughput screening and/or selection from combinatorial libraries of gene switch and circuit variants. In this study, we describe a generic and efficient platform for selection and screening of gene switches and circuits in Escherichia coli from large libraries. The single-gene dual selection marker tetA was translationally fused to green fluorescent protein (gfpuv) via a flexible peptide linker and used as a dual selection and screening marker for laboratory evolution of gene switches. Single-cycle (sequential positive and negative selections) enrichment efficiencies of >7000 were observed in mock selections of model libraries containing functional riboswitches in liquid culture. The technique was applied to optimize various parameters affecting the selection outcome, and to isolate novel thiamine pyrophosphate riboswitches from a complex library. Artificial riboswitches with excellent characteristics were isolated that exhibit up to 58-fold activation as measured by fluorescent reporter gene assay.
Muranaka
Norihito
N
Department of Biomedical Engineering, University of California, Davis, 451 Health Sciences Drive, Davis, CA 95616, USA.
Sharma
Vandana
V
Nomura
Yoko
Y
Yokobayashi
Yohei
Y
D017920:Antiporters / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005456:Fluorescent Dyes; D015870:Gene Expression; D015723:Gene Library; D005798:Genes, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry
D017920:Antiporters; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; C079051:tetA protein, Bacteria; D049452:Green Fluorescent Proteins