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Abstract The cyanobacterial circadian clock maintains remarkable precision and synchrony, even in cells with femtoliter volumes. Here, we reconstitute the KaiABC post-translational oscillator (PTO) in giant unilamellar vesicles (GUVs) to investigate underlying mechanisms of this fidelity. We show that our encapsulation methodology replicates native protein variability. With long-term, single-vesicle tracking of circadian rhythms using fluorescent KaiB and confocal microscopy, we find that oscillator fidelity decreases with lower protein levels and smaller vesicle sizes. KaiB membrane association, observed in cyanobacteria, was recapitulated in GUV membranes. A mathematical model incorporating protein stoichiometry limitations suggests that high expression of PTO components and associated regulators (CikA and SasA) buffers stochastic variations in protein levels. Additionally, while the transcription-translation feedback loop contributes minimally to overall fidelity, it is essential for maintaining phase synchrony. These findings demonstrate synthetic cells capable of autonomous circadian rhythms and highlight a generalizable strategy for dissecting emergent biological behavior using minimal systems. 

We report the discovery of a novel mechanism for the assembly of giant unilamellar vesicles, where fluid shear-induced fragmentation of a foam-like lamellar lipid mesophase occurs in lipid mixtures containing 3 mol% PEG2000-DSPE. 

Motivation Giant unilamellar vesicles (GUVs), cell-like synthetic micrometer size structures, assemble when thin lipid films are hydrated in aqueous solutions. Quantitative measurements of static yields and distribution of sizes of GUVs obtained from thin film hydration methods were recently reported. Dynamic data such as the time evolution of yields and distribution of sizes, however, is not known. Dynamic data can provide insights into the assembly pathway of GUVs and guidelines for choosing conditions to obtain populations with desired size distributions. Approach We develop the ‘stopped-time’ technique to characterize the time evolution of the distribution of sizes and molar yields of populations of free-floating GUVs. We additionally capture high resolution time-lapse images of surface-attached GUV buds on the lipid films. We systematically study the dynamics of assembly of GUVs from three widely used thin film hydration methods, PAPYRUS (Paper-Abetted amPhiphile hYdRation in aqUeous Solutions), gentle hydration, and electroformation. Findings We find that the molar yield versus time curves of GUVs demonstrate a characteristic sigmoidal shape, with an initial yield, a transient, and then a steady state plateau for all three methods. The population of GUVs showed a right-skewed distribution of diameters. The variance of the distributions increased with time. The systems reached steady state within 120 min. We rationalize the dynamics using the thermodynamically motivated budding and merging (BNM) model. These results further the understanding of lipid dynamics and provide for the first-time practical parameters to tailor the production of GUVs of specific sizes for applications.