Engineering semi-permeable giant liposomes.

Giant unilamellar vesicles (GUVs) with a semi-permeable nature are prerequisites for constructing synthetic cells. Here we engineer semi-permeable GUVs by the inclusion of DOTAP lipid in vesicles. Diffusion of molecules of different charge and size across GUVs are reported. Control over size-selective permeability is demonstrated by modulating the DOTAP lipid composition in different lipid systems without reconstituting membrane proteins. Such semi-permeable GUVs have immense applications for constructing synthetic cells.

Dynamic Control of Functional Coacervates in Synthetic Cells.

Membrane-less compartments formed via liquid-liquid phase separation (LLPS) are regulated dynamically via enzyme reactions in cells. Giant unilamellar vesicles (GUVs) provide a promising chassis to control, mimic, and understand the LLPS process; however, they are challenging to construct. Here, we engineer the dynamic assembly and disassembly of LLPS compartments using complex coacervates as models inside synthetic cells. Semipermeable GUVs constructed with defined lipid composition encapsulate the biomolecules, including enzymes required to regulate coacervates. Assembly and disassembly of coacervates are triggered in independent systems by the diffusion of substrates through the membrane into the vesicle lumen. The coupling of enzyme networks in a single synthetic cell system allows for reversible and out-of-equilibrium regulation of coacervates. The functional properties of the coacervates are revealed by sequestering biomolecules, including drugs and enzymes. GUVs, with functional LLPS compartment assembly, open avenues in constructing programmable autonomous synthetic cells with membrane-less organelles. The coacervate-in-vesicle platform has significant implications for understanding LLPS regulation mechanisms in cells.