The Encapsulation of an E. coli TXTL in Cell-sized Compartments Towards Prototyping Synthetic Cells

Abstract

The bottom-up assembly of synthetic cell systems capable of recapitulating biological functions has become a means to understand living matter through construction. Cell-free protein synthesis platforms, which allow for the rapid prototyping of biological systems by reducing the design-build-test cycle relative to in vivo experiments, have emerged as a tool to achieve this goal. In this dissertation, I report on the most recent iteration of an all E. coli TXTL and its use towards the realization of a bottom-up synthetic cell. TXTL offers robust protein synthesis with access to the full complement of regulatory parts available in E. coli. I detail efforts to encapsulate TXTL into cell-sized liposomes, providing researchers a platform to carry out complex reactions in containers in which the local environment and membrane composition can be altered. While a functional E. coli divisome was not reconstructed, the FtsZ and MreB family of proteins were expressed in liposomes, with MreB showing significant deformation. Finally, I developed synthetic cell prototypes programmed to be mechanosensitive, coupling this function to create multiple synthetic cell prototypes that are biosensing or adaptive based on the local environment.