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).
To assay oscillations, phosphorylated and unphosphorylated KaiC can be tracked through Western Blots using a 7.5% SDS-Page gel.
Weights of KaiA, KaiB, and KaiC are 60kD, 32kD, and 12 kD respectively.
UChicago 2015 Results
Specific Experiments and Results
There were two essential goals of the assays we conducted for the oscillator plasmid (pMC001).
1. To characterize the rhamnose inducible promoter in order to manipulate the concentration of KaiA. Our hope is that altering the KaiA to KaiC ratio will help optimize the oscillations. In vitro, this KaiA : KaiC ratio was found to be ~ 1 : 3 (Nakijima, Science 2010).
2. To portray oscillations of KaiC phosphorylation. This was to be accomplished after goal 1, as an overabundance/lack of KaiA will prevent oscillations from occurring (See Figure below).
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(Lin et al. PNAS, 2014)
We first attempted to characterize the L-rhamnose inducible promoter, expanding on the investigation from the Paris-Bettencourt 2012 team. See characterization on design page.
We then conducted more tests on the L-Rhamnose inducible promoter and the stability of KaiA in the context of our oscillator biobrick. In consideration of the research we conducted about the L-rhamnose promoter, we determined 16 hours to be the maximum induction time to investigate. We developed an assay in which we induced cells with 0.5% L-rhamnose and took samples of E.coli cells expressing our oscillator biobrick every 2 hours for 16 hours. These cells had been induced with 0.5% L-rhamnose, as previous characterization had confirmed that this relatively high amount of Rhamnose would lead to KaiA expression. These samples were frozen in a -80C freezer to preserve the cell conditions. Over this time course experiment, we were seeking to observe the degradation/stability of the Kai proteins. Specifically, we wanted to determine the point at which KaiA expression reached steady state. This was of utmost importance before starting oscillations, as the KaiC oscillations are dependent on steady levels of Kai proteins.
Additionally, we had to deal with the issue of synchronizing our group of oscillating cells in order to properly assay for oscillations. To do so we referenced the Silver Lab paper (Chen et al. Science 2015), in which cells were first induced in M9 minimal media with carbon supplements (0.4% glycerol, 0.1% casamino acids), then shocked in M9 minimal media with no carbon supplements for 1 hour. The oscillations in this study were tracked using M9 minimal media with leucine and succinate supplements to promote slow growth. We decided to adopt a similar protocol throughout our time course experiment and induced in the M9 supplemented medium (w/glycerol and casamino acids). We also implemented a synchronization shock for 1 hour and 6 hours after the maximum induction time of 16 hours. We conducted a 1 hour shock to remain consistent with the paper, and a 6 hour shock upon receiving recommendation from our host lab (the Rust Lab) that had experience with synchronizing cyanobacterial cells. Our final sample was taken after 62 hours of starting the experiment to simulate the time length we would assay the oscillations for. The cell samples were analyzed using western blotting.
Results from Time Course experiment:
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Figure F: Densitometry analysis of Kai proteins during Time Course Western Blot. Densitometry conducted in Image J. Raw protein values were fit to a standard curve developed by purified protein samples (ng of standards listed). Values also corrected for OD600 of cells. Pre-shock refers to samples before synchronization in M9 minimal media only, Post-shock refers to samples after synchronization in M9 minimal media only.
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Supplementary Figure 1: Growth curves of pMC001 cells during Induction time course experiment. OD 600 readings taken at time points throughout assay.
Our results from the time course experiment show that the maximal steady state value of KaiA is reached after 16 hours. After the synchronization shock, the amount of KaiA decreases however only slightly. This decrease in KaiA was expected given the absence of rhamnose in the post-shock medium. This also indicates an inherent flaw in the set-up of our experiment -a decrease in KaiA will lead to dampened oscillations. However, the little range of decrease in KaiA indicates the potential of still being able to assay for robust oscillations. Accepting the fact that the oscillations may be dampened due to even a slight decrease in KaiA, we can still select for the most robust oscillations using different concentrations of KaiA.
Our results indicate that for KaiC, fluctuation in protein concentration is minimal compared to KaiA pre- and post-shock. This is a positive indication, as KaiC protein amounts need to remain constant for robust oscillations. The present rhamnose concentration of 0.5% however, is evidently too high as the levels (ng) of KaiC are very little compared to the levels (ng) of KaiA. This throws off the 1:3 KaiA:KaiC ratio seen in vitro. For our next experiment, we plan to obtain a better gradient characterization of the L-rhamnose promoter to obtain lower expression levels of KaiA.
Lastly, our results provide evidence that the Kai proteins are being expressed in E.coli!
Note: Please check out our notebook (http://2015.igem.org/Team:UChicago/Notebook), in order to see data and evidence for constructing out beloved oscillator biobrick.
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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