Part:BBa_K1745001
KaiABC Oscillator
pMC001 exhibits the three Kai proteins: KaiA, KaiB, and KaiC, which constitute a circadian oscillator. This biobrick can be used to drive circadian processes. Oscillations can be tracked through the phosphorylation states of KaiC. In vitro analysis of the Kai system found that the robustness of the oscillator was sensitive to KaiA:KaiC stoichiometry (Nakajima, 2010). In order to investigate this ratio, KaiA expression is driven under an L-rhamnose inducible promoter Part BBa_K914003 while KaiB and KaiA are driven under a constitutive promoter Part BBa_J23100.
The KaiABC System
The KaiABC proteins are endogenous to cyanobacteria and are responsible for globally regulating circadian processes. These proteins were first discovered as a cluster of genes by scientists Takao Kondo, Susan S. Golden, and Carl H. Johnson. The cyclical functioning of these proteins was particularly significant as they demonstrated a circadian rhythm -following an approximate 24 hour cycle in response to light and dark.Notably, the circadian oscillations of the KaiABC system have been reconstituted in vitro. The oscillations occur due to interactions on the protein level and arise due to the phosphorylation states of KaiC. KaiC exists in four possible states, dependent upon two possible sites of phosphorylation: T-432 and S-431. The protein KaiA acts as a direct kinase of KaiC while KaiB acts as a direct phosphatase of KaiC. KaiA and KaiB also exhibit different binding affinities based on the phosphorylation state of KaiC. KaiA has a higher binding affinity to unphosphorylated KaiC while KaiB has a higher binding affinity to S-431 phosphorylated KaiC. The antagonistic functions of KaiA and B and varied binding affinities result in oscillations in the four phosphorylation states of KaiC over a 24 hour period. Thus, the KaiABC system generates circadian rhythm used by cyanobacteria to drive clock based processes circadian processes in cyanobacteria is cell division.
UChicago 2015 iGEM Team
The purpose of the UChicago 2015 iGEM team was to reconstitute the KaiABC system into E.coli. They aimed to demonstrate the potential of applying this oscillatory system in the context of synthetic biology. While this system has been reconstituted in E.coli before (Chen et al., Science 2015) they hoped to optimize the KaiABC system by experimenting with the ratio of KaiA to KaiC concentrations-a ratio that has previously been shown to be highly sensitive in the robustness of oscillations in vitro (Nakajima, 2010).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3014
Illegal NheI site found at 3037 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 410
Illegal XhoI site found at 477 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2945
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 540
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