Description: The KaiABC system consists of three core proteins that comprise the 24-hour circadian clock endogenous to the cyanobacterium Synechocococcus elongatus. Oscillations are driven by the kinase activity of KaiC and its interactions with KaiA and KaiB which catalyze the cyclic auto-phosphorylation and auto-dephosphorylation of KaiC, respectively. The establishment of a modular circadian oscillator would be a useful tool for synthetic biology. The Kai oscillator can be reconstituted in vitro with purified protein, motivating a previous study to transplant the Kai system into E.coli (Chen et al, 2014). Our aim is to expand this line of research and transplant the Kai system into Saccharomyces cerevisiae, demonstrating this system in eukaryotes for the first time.We first aim to express the Kai system in yeast with an appropriate protein stoichiometry. Once the oscillator has been properly established, reporter constructs utilizing a LexA-SasA fusion protein will be introduced which respond to phosphorylated and dephosphorylated KaiC, respectively, with the production of two different fluorescent markers. This will allow oscillations to be viewed in real time as varying concentrations of these fluorophores. It will also serve as proof-of-concept for a system with the ability to synthesize two temporally separated protein products generated during alternating 12-hour time spans, a tool with potential downstream applications in basic research and medicine. It also serves as a first step towards introducing the KaiABC system into higher eukaryotic organisms such as Drosophila and human cell lines.
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Year: 2016Visit Wiki
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Updated at: 8/9/16