Composite
KaiABC

Part:BBa_K1745001:Design

Designed by: Mansi Chandra   Group: iGEM15_UChicago   (2015-09-17)

KaiABC Oscillator


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 3014
    Illegal NheI site found at 3037
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 410
    Illegal XhoI site found at 477
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2945
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 540


Design Notes

KaiB and KaiC are in reverse direction of KaiA to maximize the efficiency of the double terminator Part:BBa_B0014.

Characterization of pRha by UChicago iGEM 2015

One of the most significant design choices in this oscillator plasmid was the inducible L-rhamnose promoter (pRha). This choice was made by researching the characterization of a variety of different inducible promoters in the iGEM registry. The reason we chose this specific L-rhamnose inducible promoter, compared to a more standard IPTG inducible promoter was because the characterization for the L-rhamnose inducible promoter (Part:BBa_K914003), portrayed a more well characterized tight and graded response of pRha by the 2012 Paris-Bettencourt team. This leads us to believe that pRha is more suited to fine tune the concentrations of KaiA in order to investigate the KaiA : KaiC ratio in vivo.

We conducted our own characterization of this Biobrick by tracking KaiA expression for a gradient of L-rhamnose percentages during a 10 hour induction (10 hour induction time was reported by the 2012 Paris-Bettencourt team). In order to track KaiA expression we used western blotting techniques as well as protein densitometry analysis in Image J. We believe our characterization is an improvement on the characterization accomplished by the 2012 Paris-Bettencourt team which used a plate-reader.


http://i58.tinypic.com/dg5g0w.png

Induction for the characterization curve was accomplished in M9 minimal media with supplements (0.4%glycerol, 0.1% casamino acids).

The results from our western blots indicate the tightness of pRha. At 0% L-rhamnose, there is essentially no KaiA expressed compared to KaiB and KaiC. This supports our ability to tune KaiA concentrations to optimize oscillations.

http://i59.tinypic.com/2znzerd.png

Figure I: Gradient curve of ng of KaiA vs. %Rhamnose. ng KaiC vs. %Rhamnose and KaiA : KaiC ratio also included.

The gradient curve we developed portrays the ability to fine tune the concentrations of KaiA. Ideally, we would have run multiple such assays, with a higher gradient of L-rhamnose than listed above. However, the data presented gives us a good start to characterizing a usable gradient of KaiA expression. Additionally, the expression levels of KaiC are fairly stable relative to the gradient curve of KaiA. This means that the concentration of KaiA can be tuned without altering the concentration of KaiC.

Source

The KaiABC circadian oscillator is endogenous to the cynanobacterial species Synechococcus elongatus.

References

Chen, A. H., Lubkowicz, D., Yeong, V., Chang, R. L., & Silver, P. a. (2015a). Transplantability of a circadian clock to a noncircadian organism. Science Advance, 1(5), 1–6. http://doi.org/10.1126/sciadv.1500358

Nakajima, M., Imai, K., Ito, H., Nishiwaki, T., Murayama, Y., Iwasaki, H., … Kondo, T. (2005). Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science (New York, N.Y.), 308(5720), 414–415. http://doi.org/10.1126/science.1108451

Nakajima, M., Ito, H., & Kondo, T. (2010). In vitro regulation of circadian phosphorylation rhythm of cyanobacterial clock protein KaiC by KaiA and KaiB. FEBS Letters, 584(5), 898–902. http://doi.org/10.1016/j.febslet.2010.01.016

Pattanayak, G., & Rust, M. J. (2014). The cyanobacterial clock and metabolism. Current Opinion in Microbiology, 18(1), 90–95. http://doi.org/10.1016/j.mib.2014.02.010

Rust, M. J., Markson, J. S., Lane, W. S., Fisher, D. S., & O’Shea, E. K. (2007). Ordered phosphorylation governs oscillation of a three-protein circadian clock. Science (New York, N.Y.), 318(5851), 809–812. http://doi.org/10.1126/science.1148596

Taniguchi, Y., Takai, N., Katayama, M., Kondo, T., & Oyama, T. (2010). Three major output pathways from the KaiABC-based oscillator cooperate to generate robust circadian kaiBC expression in cyanobacteria. Proceedings of the National Academy of Sciences of the United States of America, 107(7), 3263–3268. http://doi.org/10.1073/pnas.0909924107