Genetically programmed synthetic cells for thermo-responsive protein synthesis and cargo release

Abstract

Synthetic cells containing genetic programs and protein expression machinery are increasingly recognized as powerful counterparts to engineered living cells in the context of biotechnology, therapeutics and cellular modelling. So far, genetic regulation of synthetic cell activity has been largely confined to chemical stimuli; to unlock their potential in applied settings, engineering stimuli-responsive synthetic cells under genetic regulation is imperative. Here we report the development of temperature-sensitive synthetic cells that control protein production by exploiting heat-responsive mRNA elements. This is achieved by combining RNA thermometer technology, cell-free protein expression and vesicle-based synthetic cell design to create cell-sized capsules able to initiate synthesis of both soluble proteins and membrane proteins at defined temperatures. We show that the latter allows for temperature-controlled cargo release phenomena with potential implications for biomedicine. Platforms like the one presented here can pave the way for customizable, genetically programmed synthetic cells under thermal control to be used in biotechnology. RNA-based genetic regulation has been used to control protein translation in synthetic cells in response to the external temperature. RNA thermometers enable in situ protein expression above permissive temperatures. Controlled expression of the membrane pore α-hemolysin allows small-molecule cargo to be released in a temperature-dependent manner.