Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells, as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of biological systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (protein synthesis using recombinant elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.

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ACS
The Netherlands Organisation for Scientific Research (NWO)
doi.org/10.1021/acssynbio.1c00068
ACS Synth. Biol.
Physics Cellular Interactions

Van de Cauter, L, Fanalista, F, van Buren, L, De Franceschi, N, Godino, E, Bouw, S, … Ganzinger, K.A. (2021). Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles. ACS Synth. Biol., 10(7), 1690–1702. doi:10.1021/acssynbio.1c00068