loading . . . Eukaryotic-like Synthetic Cells with Chemically Controlled Protein Localization Compartmentalization by organelles and the dynamic control of protein localization within these compartmentalized spaces are key mechanisms for regulating biological processes in eukaryotic cells. Here, we present a bottom-up approach for constructing cell-sized liposomes (giant unilamellar vesicles, GUVs) encapsulating an artificial organelle with chemically controlled protein localization. In this system, proteins fused to Escherichia coli dihydrofolate reductase are rapidly recruited on demand from the inner solution to the interior of a DNA-droplet-based (ânucleusâ-like) organelle within GUVs upon addition of a synthetic, DNA-binding trimethoprim derivative to the external solution. By coupling this system with a sequence-specific protease, we constructed a synthetic cell platform that enables chemically induced, multistep cascade reactionsâincluding protein relocalization, organelle-specific enzymatic activity, and product release from the organelleâthat culminate in the control of synthetic-cell phenotypes, such as pore formation in the GUV membrane. This work provides a versatile platform for the bottom-up creation of eukaryotic-like synthetic cells with sophisticated and programmable functions. https://pubs.acs.org/doi/full/10.1021/acssynbio.5c00754
