Team:Edinburgh/Bacterial

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Introduction: The original repressilator


In 2000, Elowitz and Leibler presented the first synthetic oscillatory system in E coli. Termed the repressilator, this combined three transcriptional repressor systems that were not naturally part of any oscillating system. This system oscillated but was not very precise (it had an oscillation period of 120 +/- 40min, and although mother cells transmitted their state to their daughters when dividing, the bacteria tended to get out of synchrony after time). Garcia-Ojalvo et al. (2004) presented a model of an improved repressilator which used quorum sensing to make the bacteria function as a single unit; Figure 1 shows a diagram of this system. Danino et al. (2009) produced an oscillating genetic circuit synchronised by quorum sensing, confirming that this method could be used to improve synthetic biological oscillators.




Figure 1: A quorum sensor synchronised repressilator as modelled by Garcia-Ojalvo et al (2004).



The repressilator presented by Danino et al. in 2009 was not only advantageous in that it was more stable than the original Elowitz repressilator, but also in that the oscillation period could be controlled. The bacteria were grown in a trapping chamber, and when the flow rate through the main tube was increased or decreased, the quorum sensing molecule diffused away faster or slower, altering the oscillation period (see figure 2).





Figure 2: Danino et al. oscillator in flow chamber. By increasing or lowering the flow rate of water through the tube, the autoinducer
(AI, which promotes transcription when bound to LuxR) diffuses more or less fast out of the chamber, allowing control of the oscillation rate.






Alignment of spectra



Figure 3: Normalised absorbance spectra of:
a. the lov2 domain of Avena sativa with bound FMN (adapted from Schüttrigkeit et al., 2003).
b. Green light absorbing form of cyanobacteriochrome CcaS from Synechocystis sp. PCC 6803. Adapted from Hirose et al, 2008.
c. Red light absorbing form of phytochrome Cph1-PCB adduct, from Synechocystis sp. PCC 6803. Adapted from Gambetta and Lagarias (2001).
Note that the relative absorbance of each spectrum is normalised to one.


The response of light receptors Cph8, lovTAP and our green light receptor to different wavelengths of light have not been measured. The absorbance spectra of the light sensitive proteins might not exactly mirror their response to light, but should give us a good idea until they are characterized.

In lovTAP, the light sensitive domain of the protein is the lov2 domain from the Avena sativa blue light receptor phototropin. This binds flavin mononucleotide (FMN) which is its co factor. Schüttrigkeit et al. (2003) measured the absorbance of the wild type lov2 bound to FMN (see Figure 3). Since this is the active part of lovTAP, we expect our blue light receptor to have a similar response in vivo. The red light absorbing form of Cph1 is what responds to red light in Cph8. The absorbance of Cph1 was measured by Gambetta and lagarias (2001). Similarly, the absorbance of the green light absorbing form of CcaS, which we are planning on using in our green light receptor, was measured by Hirose et al. (2008).





References


Elowitz, M. B. and S. Leibler (2000). "A synthetic oscillatory network of transcriptional regulators." Nature 403(6767): 335-338.
Danino, T., O. Mondragon-Palomino, et al (2009). "A synchronized quorum of genetic clocks." Nature 463(7279): 326-330.
Garcia-Ojalvo, J., M. B. Elowitz, et al. (2004). "Modeling a synthetic multicellular clock: Repressilators coupled by quorum sensing." Proceedings of the National Academy of Sciences of the United States of America 101(30): 10955-10960.
A Levskaya, OD Weiner, WA Lim, CA Voigt (2009), "Spatiotemporal control of cell signalling using a light-switchable protein interaction." Nature Vol 461| 15 October 2009| doi:10.1038/nature08446
YI Wu, D Frey, OI Lungu, A Jaehrig, I Schlichting, et al. (2009) "A genetically encoded photoactivatable Rac controls the motility of living cells" Nature, 2009 Vol 461| 3 September 2009| doi:10.1038/nature0824
F Zhang, AM Aravanis, A Adamantidis, et al. (2007) "Circuit-breakers: optical technologies for probing neural signals and systems" Nature Reviews VOLUME 8 | AUGUST 2007 | 577
Gambetta, G. A. & Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. Proceedings of the National Academy of Sciences of the United States of America 98, 10566-10571.
Hirose, Y., Shimada, T., Narikawa, R., Katayama, M. & Ikeuchi, M. (2008). Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. Proceedings of the National Academy of Sciences 105, 9528-9533.
Schüttrigkeit, T. A., Kompa, C. K., Salomon, M., Rüdiger, W. & Michel-Beyerle, M. E. (2003). Primary photophysics of the FMN binding LOV2 domain of the plant blue light receptor phototropin of Avena sativa. Chemical Physics 294, 501-508.