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

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.

Synthetic RNA devices to expedite the evolution of metabolite-producing microbes

“…1a). Although this genetic device has been used for screening the genetic switches 16 or probing the genetic pathways 18 , we anticipated that this device might also be utilized as a platform for efficient strain improvement in the way that it can convert the cellular metabolic information into a conspicuous phenotype, such as survival under selection pressure.…”

“…1b). Although this lysineresponsive genetic device is a repressive type of regulator, the dual-selection modes of TetA enable cells that accumulate lysine in the cytoplasm to survive in the presence of toxic metal salts, such as NiCl 2 16 . As expected, cellular growth rates were severely affected by the intracellular concentration of lysine only if the functional Lysine Riboselector was implemented ( Supplementary Fig.…”

“…To demonstrate this, we chose L-tryptophan, whose naturally occurring riboswitch has not yet been discovered, as another inconspicuous target metabolite. As a starting point for the creation of a 'Tryptophan Riboselector' 16 , we selected one of the synthetic aptamers specific for L-tryptophan that are already known 33 . The readily obtained Tryptophan Riboselector (Fig.…”

“…Recently, various genetic devices combining the riboswitches with reporter genes have been described to modulate genetic switches, discover the genetic pathway and reprogram cellular behaviours 15,16,18,34,36 . However, none of them was directly used for evolving metabolite-producing microbes to increase the yield and the productivity of the target molecules.…”

“…RNA aptamers that bind to target molecules with high selectivity and sensitivity can be readily selected in vitro through systematic evolution of ligands by exponential enrichment 13 . Synthetic riboswitches that actually work in vivo can be generated from these in vitro-selected aptamers and can also be functionally manipulated using various high-throughput screening methods [14][15][16] .…”