Crowding-induced spatial organization of gene expression in cell-sized vesicles

A major limitation of cell-free expression systems is the lack of a means to spatially organize gene expression components to mimic cellular environments. We used computer simulations to guide experimental efforts to control the spatial organization of DNA and ribosomes in cell-sized vesicles using macromolecular crowding. With a coarse-grained model of DNA plasmids and crowders, we showed that plasmids were uniformly distributed at low levels of crowding but, due to depletion interactions, became strongly adsorbed to confining surfaces at high levels of crowding. We validated these results using fluorescently-labelled DNA plasmids and ribosomes in cell-sized vesicles. At large concentrations of the crowding agent Ficoll 70, DNA plasmids preferentially localized near vesicle membranes while ribosomes remained uniformly distributed. We then used kinetic Monte Carlo simulations and a coupled mRNA/protein reporter technique to understand the dynamics of transcription and translation. Crowding-induced localization of DNA to vesicle surfaces resulted in lower protein abundance and decreasing translational efficiency with increasing system size. Our approach demonstrates a cell-free platform that provides a means to better understand spatial control of gene expression.