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.
University of Minnesota Ph.D. dissertation. May 2019. Major: Physics. Advisor: Vincent Noireaux. 1 computer file (PDF); viii, 116 pages.
The Encapsulation of an E. coli TXTL in Cell-sized Compartments Towards Prototyping Synthetic Cells.
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