The cell-free expression of proteins within GUVs was achieved using the Promega E. coli S30 Extract System for Circular DNA (L1020). 75 µl of plasmid, at different concentrations (pNCS-mClover3: 300 ng mL -1 ; ZP9A Actin: 98.4 ng ml -1 ; Chx10 3kb AP: 137 ng ml -1 ), 50 µL of S30 EXTRACT (cell lysate) were added to the reaction mixture. For ZP9A Actin, 10 µL of Phalloidin Atto565 (20 µM in MeOH) were added as well. After polymerization, to 70 µL of sample, 50 µl of S30 premix (containing the energy regenerating system) and 10 µL of complete amino acid mixture were added. The sample was incubated at 37 °C for 3 h, either split in 3 wells per condition (mClover synthesis) or in Eppendorf tubes. Afterwards, they were imaged via CLSM. Actin-containing vesicles were resuspended in 100 mM MgCl2, whereas ALP-containing ones were first incubated in CaGP according to the protocol described above.