An efficient platform for genetic selection and screening of gene switches in Escherichia coli

Norihito Muranaka, Vandana Sharma, Yoko Nomura, Yohei Yokobayashi

Research output: Contribution to journalArticle

69 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Article numbere39
JournalNucleic Acids Research
Volume37
Issue number5
DOIs
StatePublished - 2009

Fingerprint

Switch Genes
Gene Regulatory Networks
Genetic Selection
Genetic Testing
Riboswitch
Escherichia coli
Libraries
Thiamine Pyrophosphate
Green Fluorescent Proteins
Reporter Genes
Biomarkers
Peptides
Genes

ASJC Scopus subject areas

  • Genetics

Cite this

An efficient platform for genetic selection and screening of gene switches in Escherichia coli. / Muranaka, Norihito; Sharma, Vandana; Nomura, Yoko; Yokobayashi, Yohei.

In: Nucleic Acids Research, Vol. 37, No. 5, e39, 2009.

Research output: Contribution to journalArticle

Muranaka, Norihito ; Sharma, Vandana ; Nomura, Yoko ; Yokobayashi, Yohei. / An efficient platform for genetic selection and screening of gene switches in Escherichia coli. In: Nucleic Acids Research. 2009 ; Vol. 37, No. 5.
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