## 5 Concluding Remarks and Future Perspectives  
Cyanobacteria rely on the circadian clock to generate a biological rhythm, staying in tune with the 24 h day/night transition in order to optimize survival and reproduction. The temperature-compensated post-translational modification that cycles both in vivo and in vitro maintains a stable oscillation, even without the transcriptional or translational feedback from the cell. At the onset of darkness, oxidized quinone becomes acutely abundant; depending on the timing of the dark pulse application, quinone can inactivate KaiA or CikA. Dissociation of the former results in a phase advance and that of the latter causes a delay in the KaiC phosphorylation rhythm, which is an effect that is equivalent to humans adjusting to a new time zone. The central oscillator transduces the temporal information to SasA and eventually to RpaA; the resultant cyclic RpaA phosphorylation periodically activates gene expression, controlling the physiological and behavioral patterns of cyanobacteria in such a way that they show a competitive advantage as primary producers in various ecological niches wherever sunlight reaches.  
Meanwhile, darkness also induces the accumulation of the stress response alarmone, ppGpp, and while this may not have a direct effect on the circadian clock, its different levels of abundance can affect gene expression patterns [75; 76]. Moreover, RpaB, another transcription factor, which gets phosphorylated in an antiphasic manner to RpaA phosphorylation, is also dark-induced; a clear link between the circadian clock and RpaB is still missing [5; 77]. In sum, more research is needed to move beyond RpaA- and RpaB-dependent transcription such that we can get a better understanding of how the rhythmic KaiC phosphorylation, as well as a light/dark pulse, controls and affects the organism as a whole. This could reveal other biologically crucial mechanisms that are potentially synergistic with the circadian clock's response to external stimuli.  
Cyanobacteria's billions of years of proliferation and presence on this planet are believed to have transformed Earth's atmosphere [78]. As time passed by, a common ancestor diverged into different species. However, the conserved _kai_Cs and _kai_Bs within a handful of cyanobacteria, as well as some eubacteria and archaea, suggest that perhaps cyanobacteria's long survival is attributed to a robust and resettable circadian clock that grants the host a competitive edge in any given environment that oscillates with a 24 h periodicity. As they have much left to show regarding how elaborately this biochemical clock runs, the biological and structural details are just beginning to unfold. We wish to get a glimpse of the functionality of each clock component by reanimating the entire circadian clock in vitro such that we can marvel even more at the hidden complexity of the biological gears of the evolutionary masterpiece.  
Conceptualization, P.K., M.K., H.-I.J. and Y.-I.K.; writing--original draft preparation, P.K. and M.K.; writing--review and editing, P.K., M.K., H.-I.J. and Y.-I.K.; supervision, H.-I.J. and Y.-I.K.; funding acquisition, Y.-I.K. All authors have read and agreed to the published version of the manuscript.  
This research was funded by the National Institute of General Medical Sciences of the National Institutes of Health Grant, 1R15 GM137311-01 (to Y.-I.K.).  
We thank [PERSON] for insightful discussions and [PERSON] and [PERSON] for proofreading. The authors declare no conflict of interest.