LAUSR

Giant unilamellar vesicles (GUVs) have been used as a material for bottom‐up synthetic biology. However, due to the semi‐permeability of the membrane, the need for methods to fuse GUVs has increased. To this aim, methods that are simple and show low leakage during fusion are important. In this study, we report a method of GUV fusion by a divalent cation (Ca2+) enhanced with a long chain polyethylene glycol (PEG20k). The methods showed significant GUV fusion without leakage of internal components of GUVs and maintained cell‐free transcription‐translation ability inside the GUVs without external supplementation of macromolecules. We demonstrate that the Ca‐PEG method can be applied for switching ON of transcription‐translation in GUVs in a fusion‐dependent manner. The method developed here can be applied to extend bottom‐up synthetic biology and molecular robotics that use GUVs as a chassis.