Difference between revisions of "Team:UChicago/Description"
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| − | Our goals for this project were threefold: | + | Our goals for this project were threefold:</span></p> |
| − | <br>1) show that the core oscillator proteins of the KaiABC system, KaiA, KaiB, and KaiC, can be expressed in S. cerevisiae <br>2) show that KaiA promotes autophosphorylation of KaiC as in the KaiABC system <br>3) show that reconstituted KaiABC can be synchronized across cells by an external stimulus<br>In combination, these goals constitute the first steps to a proof-of-concept that the cyanobacterial KaiABC system can be reconstituted in a eukaryotic organism. | + | <br><span class = "stitle" >1) show that the core oscillator proteins of the KaiABC system, KaiA, KaiB, and KaiC, can be expressed in S. cerevisiae</span> <br><span class = "stitle" >2) show that KaiA promotes autophosphorylation of KaiC as in the KaiABC system </span><br><span class = "stitle" >3) show that reconstituted KaiABC can be synchronized across cells by an external stimulus</span><br><p><span class="p">In combination, these goals constitute the first steps to a proof-of-concept that the cyanobacterial KaiABC system can be reconstituted in a eukaryotic organism. |
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Revision as of 00:57, 20 October 2016
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Project Overview
Our goals for this project were threefold:
1) show that the core oscillator proteins of the KaiABC system, KaiA, KaiB, and KaiC, can be expressed in S. cerevisiae
2) show that KaiA promotes autophosphorylation of KaiC as in the KaiABC system
3) show that reconstituted KaiABC can be synchronized across cells by an external stimulus
In combination, these goals constitute the first steps to a proof-of-concept that the cyanobacterial KaiABC system can be reconstituted in a eukaryotic organism.
Our Project Design
The goal of our project is to integrate the KaiABC system into yeast, and have it control gene expression in yeast. To do this, we have designed 3 plasmids which can be transformed into yeast to produce a system based off the Y2K model in yeast, to produce the desired effect. The first plasmid contains KaiC fused to a Gal4 activation domain, with a self-cleaving peptide linker, P2A, fusing it to KaiB. The second plasmid has KaiA linked by P2A to another fusion protein, SasA fused with the LexA binding domain. The third plasmid is our reporter plasmid, and contains GFP fused with a PEST degradation tag, and has a lexA operator region before the promoter. Together, this system would conclusively show that the KaiABC system can regulate gene expression in yeast, which serves as a proof of concept for placing eukaryotic gene expression under the control of an exogenous circadian clock. In the end, we were able to synthesize the KaiCKaiB construct and submit it to the registry.
