High-throughput screening of cell-free riboswitches by fluorescence-activated droplet sorting

• Published in: Nucleic acids research Vol. 50; no. 6; pp. 3535 - 3550
• Main Authors: Tabuchi, Takeshi, Yokobayashi, Yohei
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 08.04.2022
• Subjects: Bacteria - genetics; Biosensing Techniques; Cell-Free System; High-Throughput Screening Assays; Riboswitch; Synthetic Biology and Bioengineering
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkac152

Abstract Cell-free systems that display complex functions without using living cells are emerging as new platforms to test our understanding of biological systems as well as for practical applications such as biosensors and biomanufacturing. Those that use cell-free protein synthesis (CFPS) systems to enable genetically programmed protein synthesis have relied on genetic regulatory components found or engineered in living cells. However, biological constraints such as cell permeability, metabolic stability, and toxicity of signaling molecules prevent development of cell-free devices using living cells even if cell-free systems are not subject to such constraints. Efforts to engineer regulatory components directly in CFPS systems thus far have been based on low-throughput experimental approaches, limiting the availability of basic components to build cell-free systems with diverse functions. Here, we report a high-throughput screening method to engineer cell-free riboswitches that respond to small molecules. Droplet-sorting of riboswitch variants in a CFPS system rapidly identified cell-free riboswitches that respond to compounds that are not amenable to bacterial screening methods. Finally, we used a histamine riboswitch to demonstrate chemical communication between cell-sized droplets. Graphical Abstract Graphical Abstract Cell-free riboswitches that function in a cell-free protein synthesis system were engineered by fluorescence-activated droplet sorting. The riboswitches were used to enable chemical communication between cell-sized droplets.