Steps toward cell-like systems: spatio-temporal control of shared molecular resources for cell-free gene expression

The biosphere offers many promising economically and environmentally sustainable solutions to humanity’s increasing energy demand such as biomass conversion, chemical production, and pharmaceutical fermentation. These solutions could close the carbon loop and improve manufacturing efficiencies, but they all depend on accurate control of protein expression. To avoid limitations to protein control present in vivo such as membranes, homeostasis, and growth, artificial cell-like systems are being researched. These simplified systems are currently useful to study individual aspects of life such as regulating energy flux across membranes, responding to the environment, replication, and growth. These systems could be made more complex in the future to provide a simplified, engineered, cell-like platform for bioprocessing. Even at the single gene level, control of protein expression is hindered by resource sharing and bursting. To make proteins, genes require many reusable resources such as polymerase, ribosomes, and tRNAs which are shared among different genes. Resource sharing causes correlations in protein populations and limits steady state concentrations of competing genes. Bursty gene expression, periods of high expression, and thus high resource use, separated by periods of no expression, and thus no resource use, is a ubiquitous biological phenomenon that intimately links expression bursting and resource sharing. This dissertation investigates how gene expression bursting and variation is affected by expression resources being shared among genes and how the location of expression resources, either encapsulated or outside permeable lipid membranes, controls the level and the dynamics of cell-free protein expression. Cell-free protein synthesis systems, both crude and PURE, areused in combination with both physical PDMS barriers and defined lipid membranes to study the effects of shared and divided resource pools on gene expression bursting and protein production. Experimental results are supplemented with Gillespie simulations to add further insights. This work provides fundamental knowledge of protein expression and applied knowledge of the effects of resource sharing on cell-free gene expression bursting and variation in protein expression confined to cell-relevant volumes which are important steps toward artificial cell-like systems.