Phase Separation Dynamics in Cell-Free Expression System

The cytoplasm is a multi-component mixture and exhibits phase separation, suggesting a role in spatiotemporal control of biochemical reactions. However, the effect of physical constraints on intracellular phase separation, such as confinement, molecular crowding, and viscoelasticity, is not fully understood. We study cytoplasmic phase separation in a bottom-up approach using a cell-free expression system (TXTL). TXTL is composed of an E. coli lysate, salts, and buffers including molecular crowder PEG. We demonstrate that phase separation of TXTL is driven either by dehydration or an increase in PEG concentration. When concentrating the mixture in a cell-sized emulsion, the system exhibit phase separation and partitioning proteins produced by simultaneous gene expression. Combining cell-sized confinement and bulk experimental analysis, we show the interplay of membrane wetting and viscoelasticity on cytoplasmic droplet formation.