A Temperature-Controlled Cell-Free Expression System by Dynamic Repressor

The cell-free protein synthesis (CFPS) system is a typical protein production platform in the field of synthetic biology. However, there are limitations in controlling protein synthesis in the CFPS system. Compared with the traditional method of adding chemicals, temperature is an ideal control switch to achieve precise spatiotemporal control with few side effects. Hence, the design of a temperature-controlled cell-free protein synthesis (tcCFPS) system based on E. coli was carried out with the repressor cI protein in this study. The corresponding tcCFPS achieved a 143-fold dynamic protein expression level at 37 °C than that at 30 °C. Besides, the artificial cell controlled by temperature was constructed to expand the applications of tcCFPS. This study provides a new effective method for active protein synthesis in a cell-free system and a potential means of drug synthesis and delivery.