We show the functional reconstitution of the post-translational biochemical oscillator, with a wild-type period of ~24 hours, from the circadian clock of Synechococcus elongatus into giant unilamellar vesicles (GUVs) assembled through the one-stepped modulated PAPYRUS (OSM-PAPYRUS) technique. This is the first instance of an autonomous molecular timekeeping system reconstituted into an in vitro synthetic cell. This core oscillator consists of a system of three clock proteins, KaiA, KaiB, and KaiC, with a subpopulation of fluorescently labeled KaiB. Quantitative analysis of time-lapse images of thousands of individual circadian oscillators shows sustained circadian rhythms with near ~24-hour oscillations for more than 4 days at the population level for vesicles prepared with wildtype and greater concentrations. Sub wild type concentrations resulted in lower oscillation amplitudes or the loss of population level oscillations. We found the variation in protein levels encapsulated with our technique mimics the natural variability in protein levels expressed by bacterial cells. However, despite varying stoichiometries induced by protein variation and varying loading concentrations, single vesicle analysis reveals the oscillator exhibits only small deviations, ±2-3 hours, from the median period of oscillation even in sub-wildtype concentrations. Instead, the clock reactions exhibit binary-like states where the clock can either oscillate at a near ~24-hour circadian period or fail to exhibit any detectable periodic behavior. The fraction of vesicles exhibiting the oscillatory state of the vesicles correlates positively with the concentration of encapsulated protein for a given loading concentration. When extended to above- and sub- wild type loading concentrations, the probability of oscillation across all vesicle sizes was strongly positively correlated with loading concentration. Our results suggest accurate timekeeping for the core oscillator necessitates high expression levels to buffer for intercell and intracell temporal variations of protein levels.

Anand Subramaniam

University of California, Merced

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