Team:UChicago/Composite Part

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pMC001 (BBa_K1745001) is the main Kai ABC oscillator biobrick we submitted into the iGEM registry. It contains the three Kai proteins -KaiA, KaiB, and KaiC under the expression of two different promoters. KaiB and KaiC are under a constitutive promoter (Part J23100) and KaiA is under a L-rhamnose inducible promoter (Part K914003). The inducible promoter was chosen for KaiA in order to investigate the effect of altering the stoichiometric ratios between KaiA and KaiC. This ratio was found to be particularly significant in in vitro reactions, where the ratio of KaiA to KaiC was 1:3 (Nakajima, Science 2015).  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

pMC002 is one of the constructs for the read-out system. It contains the circadian promoter kaibc which is driven by the master transcription factor RpaA. RpaB is included as some studies have reported both RpaA and RpaB necessary for kaibc expression (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3406716/ Hanoaka, 2012). This was also to reconstitute the system used by the Silver lab. SasA, the RpaA activator is included as it directly binds to KaiC and phosphorylates RpaA, linking the KaiABC oscillator to an expression based read-out system. GFP is placed downstream the circadian promoter, acting as a reporter for kaibc expression. The GFP is LVA-Stop tagged in order to promote degradation, which is useful to quantify increases and decreases of GFP which we expect to see throughout the KaiC oscillations.  

 

 

 

 

 

 

 

 

 

 

 

 

 

pMC003 is another read-out plasmid that will be assayed independently of pMC002. This construct exhibits the same features as pMC002 with the addition of CikA, a negative regulator of RpaA which interacts directly with KaiB and dephosphorylates RpaA. We wanted to directly examine the effects of the read-out system with and without the CikA negative regulator to test the hypothesis that the inclusion of CikA leads to more robust regulation of RpaA.

Both pMC002 and pMC003 will be transformed in vitro with pMC004-7, representing the different phosphomimetics of KaiC.

 

 

 

 

 

 

 

 

 

 

 

pMC004-7 are supplemental plasmids to pMC002 and 3. They contain KaiB and KaiC phosphomimetics. The phosphomimetics represent different phosphorylation states of KaiC and emulate the oscillations of KaiC. KaiB is included as it interacts directly with CikA, the RpaA negative regulator. The phosphomimetics were generated by altering the amino acids at position 431 and 432: AA (unphosphorylated) (pMC004), AE (pMC005), EA (partially phosphorylated) (pMC006), EE (fully phosphorylated) (pMC007).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

***Note: Several parts that have no BBa plasmid were not submitted. We have several of these plasmids not in the registry on hand, but we forgot to put them onto a chloramphenicol backbone for submission.

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