Evaluation of biological clock activity capsulated by lipid-mono-layer

In this paper, we try to establish newly technique that the components of the biological clock are reconstituted into the liposome. In other words, we try to produce a nano size clock, capsulated into the liposome, made by protein molecules. The circadian clock is a basic cellular system found in almost all organisms. This clock generates self-sustained oscillations under constant conditions with a ≈ 24-hour (circadian) period. In cyanobacteria, circadian clock could be reconstituted in vitro only by mixing the three clock proteins, KaiA, KaiB, KaiC, with adenosine triphosphate (ATP). So we reconstitute these proteins and adenosine triphosphate (ATP) into phospholipid-coated microdroplet and confirmed the clock function. The clocks in phospholipids-coated microdroplet indicate long period more than 24 hour. In this case, period length became 35 hour, self-sustaining oscillation was reaming with little dumping. To reveal why the time cycle became long period, we observed localization of Kai proteins in phospholipid-coated microdroplets by using fluorescents labeled Kai proteins under fluorescent (conforcal) microscopy. From the observation of localization of Kai proteins, we found KaiB protein was distributed equivalently, on the other hand, KaiC protein was located near membrane of phospholipid-coated microdroplet. These results indicate that deferent localization between Kai proteins cause long period oscillation.