Part:BBa_K2637001

KaiA Protein of Circadian Rhythm

KaiA is one of the essential proteins in the circadian rhythm of cyanobacterias. It can regulate the process of KaiC’s phosphorylation and dephosphorylation together with KaiB and other proteins in our system, and then drive the cyanobacterias into subjective dawn or dusk. We have optimized it in yeast.

---

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 832
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Biology and Usage

Overview of Cyanobacterias' circadian rhythm

Figure 1. 机理图

Organisms are adapted to the relentless cycles of day and night, because they evolved timekeeping systems called circadian clocks, which regulate biological activities with ~24-hour rhythms. The clock of cyanobacteria is driven by a three-protein oscillator composed of only three protein KaiA, KaiB and KaiC, which together generate a circadian rhythm of KaiC phosphorylation at residues serine 431 and threonine 432 in the CII dimain. KaiA promotes KaiC (auto)phosphorylation during the subjective day, whereas KaiB provides negative feedback to inhibit KaiA and promotes KaiC (auto)dephosphorylation during the subjective night. The 24-h KaiC phosphorylation pattern can be reconstituted in vitro by merely combining the three Kai proteins and ATP, suggesting that the phosphorylation cycle is the fundamental timekeeping mechanism in cyanobacteria. Like KaiA, KaiB is also involved in regulating two antagonistic clock-output proteins--SasA and CikA, which reciprocally control the master regulator of transcription RpaA.

Usage of Life Tik Tok (Cyanobacterias' circadian rhythm in yeast)

About Life Tik Tok

Figure 2. 分子基础+下游应用

Life Tik Tok is a circadian rhythm that is established in yeast by iGEM Tianjin, 2018. It enables to regular the biological activities in yeast under the control of proteins in KaiABC oscillator. Learn more about LTT and click here.

Intereaction between the proteins

Figure 2. 蛋白相互作用

Stepwise binding of two KaiA dimers triggers KaiC autophosphorylation at Thr432 and Ser431 (I). These phosphorylation events enable cooperative binding of fold-switched KaiB monomers to the KaiC-CI domain, forming the KaiCB complex (II). KaiCB provides a scaffold for the successive sequestration of KaiA in ternary KaiCBA assemblies, concurring with a rearrangement of the KaiA PsR domains (III). KaiA sequestration promotes KaiC autodephosphorylation, resulting in the regeneration of free KaiC through release of KaiBA subcomplexes (IV).Temporal information from the oscillator is transmitted to downstream genes via the histidine protein kinase SasA (Synechococcus adaptive sensor A), whose autophosphorylation is stimulated by interaction with KaiC. Phosphorylated SasA in turn transfers a phosphoryl group to RpaA (regulator of phycobilisome association A) , a transcription factor that directly regulates the expression of approximately 100 genes. Moreover, RpaA indirectly regulates the expression of nearly all genes in the genome. Disruption of sasA also results in severely damped gene expression rhythms. Surprisingly, the phosphorylation state of RpaA, and subsequently its activity, have been shown to be dependent on CikA, which was primarily thought to be involved in entrainment.

Reconstruction of Life Tik Tok in yeast

When we reconstruct the KaiABC oscillator in yeast, our goals are： ·Characterize the combination between KaiABC changing over time via yeast two-hybrid system ·Explore the role of other proteins in stabilizing the oscillation without involving TTFL ·Discuss the effect of KaiC concentration on oscillation and select the proper promoters ·Research how heterologous circadian clock influences chromosome structure in yeast

We aim to integrate the KaiABC system into saccharomyces cerevisiae BY4741, and have it control circadian rhythm in yeast. To do this, we have designed three plasmids which can be transformed into yeast to produce a oscillation system. The first plasmid expresses KaiA, KaiB and KaiC which is fused to a Gal4 activation domain. The second plasmid is our reporter plasmid, which has fluorescent protein promoted by mutant Gal1p and luciferase promoted by Gal2p. The third plasmid contains CikA, SasA and RpaA. We respectively link a Gal4 binding domain with CikA or SasA to find the suitable binding protein that can characterize the oscillation. This system would conclusively show that the KaiABC system can work normally in yeast, which serves as a proof of concept for placing eukaryotic gene expression under the control of an exogenous circadian clock. In addition, to make sure that Gal1p and Gal2p can only be activited after the combination of proteins, we deleted two genes in wild-type BY4741. They are Gal4 and Gal80, which can activate or repress Gal1p and Gal2p, respectively. And then, we deleted gene

BarI......
Moreover, we detects the strength of several common promoters in yeast to find the proper one that provides suitable concentration. Thanks to our modeling, we can calculate the range of concentration of KaiC to support the oscillation, which contributes a lot to our selection of promoters.

===Origin of KaiA===

=Characterization of Life Tik Tok=

==Reporters' measurement in yeast==

==Characterization of Life Tik Tok==

=References=