Engineering small molecule-mediated communication between light-activatable synthetic cells and bacterial cells

Gene-expressing compartments are a minimal representation of living cells. By using DNA blueprints to inform an assembly line of biological machinery how to manufacture the proteins required for a particular function, these cell-like compartments, often referred to as synthetic cells, have been engineered to exhibit life-like behaviours, such as division and communication. Although, despite these advances, little emphasis has been placed on how their activities are regulated. For synthetic cells to become a mature and valuable technology, it is essential that we precisely control where and when their functions are executed. Should this be achieved, synthetic cells could be used for the controlled production of drugs or signalling molecules, and be interfaced with living cells/tissues to modulate their behaviours. This thesis delineates progress towards creating gene-expressing synthetic cells that are activated only in the presence of UV light and describes how light-activated synthetic cells could be interfaced with living cells. Synthetic cells were constructed by functionalising giant unilamellar vesicles with a cell-free protein synthesis system and DNA template. The DNA template dictated which proteins were synthesised inside the vesicle and, therefore, the overall function of the compartment. Photo-sensitive blocking groups were subsequently installed into the promoter regions of the DNA templates to regulate in situ gene expression; transcription, and in turn, translation, was prevented until the promoter was uncaged with UV light. This method of regulation enabled the resulting light-activated synthetic cells to be controlled remotely and in a spatiotemporally-controlled manner. To demonstrate the versatility of this platform, light-activated synthetic cells that participated in one-way communication with living cells were constructed. These compartments expressed an N-acyl homoserine lactone synthase in response to UV light, which synthesised an N-acyl homoserine lactone that was released and detected by engineered E. coli cells.