Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles

Lori Van De Cauter, Federico Fanalista, Lennard Van Buren, Nicola De Franceschi, Elisa Godino, Sharon Bouw, Christophe Danelon, Cees Dekker, Gijsje H. Koenderink, Kristina A. Ganzinger*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

6 Citations (Scopus)
73 Downloads (Pure)

Abstract

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.

Original languageEnglish
Pages (from-to)1690-1702
Number of pages13
JournalACS Synthetic Biology
Volume10
Issue number7
DOIs
Publication statusPublished - 2021

Keywords

  • actin cytoskeleton
  • bottom-up synthetic biology
  • emulsion transfer
  • GUVs
  • in vitro transcription-translation
  • synthetic cell

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