TY - JOUR
T1 - Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles
AU - Van De Cauter, Lori
AU - Fanalista, Federico
AU - Van Buren, Lennard
AU - De Franceschi, Nicola
AU - Godino, Elisa
AU - Bouw, Sharon
AU - Danelon, Christophe
AU - Dekker, Cees
AU - Koenderink, Gijsje H.
AU - Ganzinger, Kristina A.
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
KW - actin cytoskeleton
KW - bottom-up synthetic biology
KW - emulsion transfer
KW - GUVs
KW - in vitro transcription-translation
KW - synthetic cell
UR - http://www.scopus.com/inward/record.url?scp=85110946153&partnerID=8YFLogxK
U2 - 10.1021/acssynbio.1c00068
DO - 10.1021/acssynbio.1c00068
M3 - Article
C2 - 34185516
AN - SCOPUS:85110946153
SN - 2161-5063
VL - 10
SP - 1690
EP - 1702
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
IS - 7
ER -