Abstract
Cell-free synthetic biology approaches enable engineering of biomolecular systems exhibiting complex, cell-like behaviors in the absence of living entities. Often essential to these systems are user-controllable mechanisms to regulate gene expression. Here we describe synthetic RNA thermometers that enable temperature-dependent translation in the PURExpress in vitro protein synthesis system. Previously described cellular thermometers lie wholly in the 5' untranslated region and do not retain their intended function in PURExpress. By contrast, we designed hairpins between the Shine-Dalgarno sequence and complementary sequences within the gene of interest. The resulting thermometers enable high-yield, cell-free protein expression in an inducible temperature range compatible with in vitro translation systems (30-37 °C). Moreover, expression efficiency and switching behavior are tunable via small variations to the coding sequence. Our approach and resulting thermometers provide new tools for exploiting temperature as a rapid, external trigger for in vitro gene regulation.