Next, we tested the critical fuel needed to initiate the vesicle formation. We used the MC540 assay four hours after the fuel addition as a proxy for vesicle formation (Figure ). For the reaction mixture with C6P and C8, we observed the first evidence of vesicles after adding a minimum of 4.5 mM of fuel. Using the kinetic model, we calculated that the reaction cycle had produced a maximum of 1.7 mM of the acyl phosphate C8C6P. We thus considered the critical vesicle concentration CVC of C8C6P to be 1.7 mM. We performed the same experiment with 20 mM C8P and 20 mM C8 and found that a minimum of 1.3 mM EDC was required for the first indication of vesicles corresponding to a CVC for C8C8P of 0.35 mM (Figure ). Noteworthy, the decrease of the CVC by roughly an order of magnitude follows the two-carbon rule for solubilities of aliphatic compounds. Finally, when C6 and C6P were fueled with a maximum of 20 mM EDC, we could not observe any evidence of vesicle formation (Supplementary Figure ). We further investigated the nature of vesicles by confocal fluorescence microscopy using MC540 as a fluorescent dye. We used the reaction mixture of 40 mM C8 with 40 mM C6P and 20 mM EDC. We obtained confocal micrographs of the reaction mixture at various time points (Figure , Supplementary Figure ). After 30 minutes, we found the first evidence of spherical assemblies that were too tiny to resolve-they were either oil droplets or vesicles. Given that MC540 is charged and preferentially stains bilayers, we assume they were small vesicles, which also aligns with our finding by Cryo-TEM. After 9 hours, the number of vesicles and their size had increased. Some larger vesicles had grown to multiple micrometers in diameter, and their membranes could easily be resolved. After 15 hours, as the vesicles were decaying, we observed that they underwent a morphological transition, i.e., only a few large vesicles were found, and most had merged with a large, spherical assembly that had incorporated a large amount of MC540, given their high fluorescence intensity. After 24 hours, no vesicles were detected in the reaction mixture; instead, there were just bright micrometer-sized spherical assemblies. We hypothesize that the spherical assembly is a sponge-like, continuous lipid phase given that it had incorporated MC540 than a typical bilayer. Moreover, cryo-TEM after 24 hours revealed the formation of a spongelike phase with interlamellar attachment points (See Supplementary Figure ). After 48 hours, the sample was mostly free of any assemblies corroborating the transient nature of the vesicles. To investigate the nature of the vesicles further, we isolated them from their surrounding solution by spinning them down in a centrifuge after 6, 15, and 24 hours. The pellet was resuspended in buffer and analyzed by HPLC and 31 P-qNMR for its composition.