Analysis of cytoplasmic and membrane molecular crowding in genetically programmed synthetic cells.

: Building genetically programmed synthetic cell systems by molecular integration is a powerful and effective approach to capture the synergies between biomolecules when they are put together. In this work, we characterized quantitatively the effects of molecular crowding on gene expression in the cytoplasm of minimal cells, when a crowding agent is added to the reaction, and on protein self-assembly at the membrane, when a crowding agent is attached to the lipid bilayer. We demonstrate that achieving membrane crowding only is sufficient to keep cytoplasmic expression at its highest and to promote the polymerization of the MreB cytoskeletal protein at the lipid bilayer into a network mechanically sturdy. Furthermore, we show that membrane crowding can be emulated by different types of macromolecules, supporting a purely entropic mode of action for supra-molecular assembly of cytoskeletal proteins at the bilayer. These unanticipated results provide quantitative and general insights relevant to synthetic cell builders.