Team:Tianjin/Description
<!DOCTYPE html>
Background
Many organisms contain a biological rhythm system to adapt to the Earth's circadian rhythm. The biological rhythm system in the cyanobacteria is designed to allow cyanobacterial cells to predict the time of daylighting in order to prepare for light reaction in advance.
In Synechococcus elongatus, the oscillator is composed of only three proteins KaiA, KaiB, and KaiC, which together generate the circadian rhythm of KaiC phosphorylation at residues serine 431 (S431) and threonine 432 (T432) in the CII domain. KaiA promotes KaiC (auto)phosphorylation during the subjective day, whereas KaiB provides negative regulation to inhibit KaiA and promotes KaiC (auto)dephosphorylation during the subjective night. [1]The 24-h KaiC phosphorylation pattern can be reconstituted in vitro by merely combining the three Kai proteins and ATP, suggesting that the oscillating system is a post-translational oscillation system. KaiA and KaiB are also involved in regulating two antagonistic clock-output proteins—SasA and CikA, which reciprocally control the final regulator of transcription, RpaA.
Molecular changes in the KaiABC circadian clock system: Stepwise binding of two KaiA dimers triggers KaiC autophosphorylation at Thr432 and Ser431. These phosphorylation events enable cooperative binding of fold-switched KaiB monomers to the KaiC-CI domain, forming the KaiBC complex. KaiBC provides a scaffold for the successive sequestration of KaiA in ternary KaiABC assemblies, concurring with a rearrangement of the KaiA PsR domains. KaiA sequestration promotes KaiC autodephosphorylation, resulting in the regeneration of free KaiC through release of KaiAB subcomplexes. [2]
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. [3]Disruption of sasA also results in severely damped gene expression rhythms. CikA acquires the ability to dephosphorylate RpaA by interacting with the KaiBC complex and acts on downstream genes via RpaA to complete the subjective night biochemical reaction.
Our team hopes to build a cyanobacteria biological rhythm system in yeast. Since yeast is a eukaryote, prokaryotic promoters cannot be used directly, so we abandoned the strategy of verifying oscillations by controlling downstream genes by RpaA. Finally, we used the yeast two-hybrid system to reporter gene expression.We selected KaiC-SasA, KaiC-CikA, and KaiC-KaiB three pairs of proteins for the yeast two-hybrid system. During the subjectively day, KaiC binds to SasA, causing spatial proximity of AD and BD leading to the initiation of downstream reporter genes. The KaiC and KaiB, CikA and KaiBC complexes are combined on subjective night, and downstream reporter genes are activated by the yeast two-hybrid system respectively.
Yeast Two-Hybrid System
Yeast two-hybrid system was put forward by Fields in 1980 according to his research about the transcription factor Gal4. Gal4 contains two separated but essential domain which is called AD (activation domain) and BD (binding domain). AD is located at 768-881 amino acid at C terminal of Gal4, which can recognize and bind to the upstream activation sequences (UAS) of effector genes of Gal4 and BD is located at 1-147 amino acid at N terminal of Gal4, which can act on some transcription factors such as SAGA ( Spt-Ada-Gcn5-Acetyltransferase complex)、TBP (TATA-box binding protein)、TFIIB (Transcription Factor IIB), thus promoting the transcription of downstream genes. Gal1 promoter and Gal2 promoter, which can be controlled by Gal4, are chosen as the promoters of downstream genes .[4-7]
Except for Gal4 AD and Gal4 BD, there are other types of yeast two-hybrid systems such as LexA BD and VP16 AD (LexA-VP16 system). By looking up related paper such as some applications about yeast two-hybrid system[8-10], we found that Gal4 system is the most common yeast two-hybrid system and it is more suitable for our project. For example, a study found that combinations between KaiC and SasA can’t be characterized by LexA-VP16 system.[8]
This system is often used to explore interactions between proteins and proteins, proteins and RNA, proteins and organic small molecule ligand. Usually, two proteins to be studied are fused with AD and BD respectively. The protein fused with BD is called “bait” and the protein fused with AD is called “prey”.When bait and prey combine with each other, it will make AD and BD spatially close enough. Thus the downstream genes will express. On the contrary, when bait and prey can’t combine, the downstream genes won’t express.[4]
All the galactose structural genes (GAL1, GAL10, GAL7, GAL2) are coordinately regulated at the level of transcription in response to galactose by Gal4, Gal80, and Gal3. In the presence of galactose, Gal3 sequesters the transcriptional repressor Gal80p in the cytoplasm, thereby relieving inhibition of Gal4 and resulting in GAL gene expression. In the absence of galactose, Gal80 remains bound as a dimer to Gal4, preventing Gal4 from recruiting other factors of the Pol II transcription machinery. When cells are grown on glucose, GAL1 and GAL2 is negatively regulated by catabolite repression at both the levels of transcription and protein degradation. Whatever the carbon source is, the Gal4 transcriptional activator is bound as a dimer to UAS(upstream activation sites) found in the promoters of the GAL genes. In general, Gal4 and Gal80 can act on Gal1 promoter and Gal2 promoter directly while Gal3 only can act them through Gal80. Besides, Mig1 and Sip1 can downregulate the expression of GAL2 and GAL1 respectively.[11-23]
We choose three periodically combined proteins to construct three sets of combinations. They are KaiC-SasA、KaiC-CikA、KaiC-KaiB respectively. During the subjective day, KaiC will combine with SasA. While during the subjective night, KaiC will combine with KaiB and CikA. Therefore different combinations will express their downstream genes at different times.[25]
| combination | bait | prey |
|---|---|---|
| KaiC-SasA | SasA | KaiC |
| KaiC-CikA | CikA | KaiC |
| KaiC-KaiB | KaiC | KaiB |
Thanks to the instruction books of Clontech, we know many experimental details about yeast two-hybrid system. First of all, we know that the false positive problem is the biggest problems of yeast two-hybrid system to some degree.To decrease the influence of that problem, we take many measures. For example, we set up negative control groups which only includes AD or BD and we choose the mutated Gal1 promoter which is synthesized by GeneScript according to the sequence of part:BBa_K801004. Since the false negative problem isn’t a big problem, we ignore it and don’t set up the positive control group including two proteins which can combine surely. Besides, we know that the strain to be used for yeast two-hybrid experiment should be knocked out GAL4 and GAL80 genes. Based on these information above, we knocked out GAL4 and GAL80 genes of our experimental strains through CRISPR-Cas9. The strains without GAL4 and GAL80 genes are named after d-two.