KaiB, on the other hand, binds to the CI domain of fully phosphorylated KaiC and sequesters KaiA away from the A-loop, preventing it from interacting with the loop and thus resulting in KaiC dephosphorylation [17; 18]. One crucial property of a mechanical clock is to run \"clockwise,\" although it is not an easy feat for a biological clock to keep this unidirectionality [19; 20]. There are two sites of phosphorylation in the CII domain of KaiC: S431 and T432 (Figure 2). When KaiA tethers to the exposed A-loop of KaiC and initiates kinase activity, T432 is phosphorylated first (pT432 S431). Then, S431 phosphorylation follows, and KaiC becomes fully phosphorylated (pT432 pS431). When KaiB binds KaiC and isolates KaiA from the A-loop, pT432 is dephosphorylated first (T432 pS431). Further dephosphorylation ensues, and KaiC returns to its unphosphorylated form (T432 S431) [21; 22]. This extremely slow rate of ordered phosphorylation and dephosphorylation spans 24 h.  
It is because of this free-running ability of the central oscillator that cyanobacteria can keep time, even in a constant environment, such as constant light, which makes sure that the internal clock is unperturbed by the environment [23]. Remarkably, the KaiC phosphorylation state stays the same among the divided cells while the cell undergoes this biologically \"noisy\" process of reproduction, maintaining the same phase of oscillation as a colony [24]. More surprising is KaiC's ability to allosterically induce other monomers in the same hexameric complex to reach the same phosphorylation state through intersubunit shuffling [25]. It is not difficult to glean the prominence of timekeeping in cyanobacteria's survival, considering their effort to synchronize the phase of the oscillator happens at both intercellular and intracellular levels. In an in vivo study, a cyanobacterial culture that harbor the circadian clock, where its period matches with that of the light and dark cycle, showed better reproductive fitness and outcompeted other populations that had different clock periods [26]. Moreover, in a constant light environment, a strain that had a defective circadian clock outcompeted one with a properly functioning clock, meaning that a circadian clock is only useful in a rhythmically oscillating environment and even renders a disadvantage when exposed to a constant environment [27]. A beneficial adaptation for cyanobacteria to survive in a rhythmically oscillating environment on this planet was to develop an internal clock that has a 24 h periodicity. However, although the in vivo mutational analyses provide an initial and basic insight on the phenotypic variation that results from a dysfunctional component, structural and functional analyses are needed in order to look at a more detailed picture of the biological timekeeping, phase resetting, and output signaling functions. By reconstituting the cyanobacterial circadian clock in vitro, we attempt to uncover detailed biochemical mechanisms behind the clockwork based on the most current structural data available in an attempt to answer how cyanobacteria manage to survive in various environmental niches. The circadian oscillation of KaiC phosphorylation is readily detected in vitro via SDS-PAGE, wherein phosphorylated KaiC can be viewed as an upper triple band and dephosphorylated KaiC can be viewed as a lower single band on the gel [28]. The ratio of phosphorylated KaiC to total KaiC is determined by the densitometry of each band, and the ratio is plotted as a function of the incubation time. Ideally, the graph produced should represent the self-running 24 h oscillations of circadian rhythm (Figure 3a) [29]. Figure 2: Ordered phosphorylation and dephosphorylation of KaiC. The initial unphosphorylated KaiC hexamer has the A-loops in their buried conformation by default. KaiA’s binding tethers the A-loops in their exposed conformation, initiating autokinase activity and phosphorylating T432 first. Then, KaiC reaches its fully phosphorylated state by phosphorylating its remaining S431.