Team:Edinburgh/Bacterial/Core repressilator

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Revision as of 21:52, 24 October 2010







Overview: The core 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 quickly out of the chamber, allowing control of the oscillation rate.




Strategy


Our intentions were to build upon this pre-existing work on the familiar and famous repressilator system, only replacing the unwieldy quorum-sensing systems with a light-based solution. Communication via light would not only eliminate the need for chemical signalling, but also produce a highly visible and easily recognisable means of viewing and verifying the system in action. These dual benefits would hopefully serve as a proof of concept for a methodology that would have wide-ranging benefits throughout synthetic biology.

The overall oscillating design of our system would be composed of two parallel networks working in the same direction as an associated repressilator, with the addition of red, blue, and green light as a signalling mechanism. The inner network would function in the same manner as the repressilator designed by Garcia-Ojalvo and M. B. Elowitz in 2004, containing three genes in an ordered circular fashion (lambda-cI, lacI, tetR). The outer network, on the other hand, would consist of the three light sensors and their associated light producers.

The integrated network (Figure 3) could thus be considered in terms of its three separate components:

  • Component 1: red luciferase, LacI, red sensor.
  • Component 2: blue luciferase, TetR, blue sensor.
  • Component 3: green luciferase, lambda-cI, green sensor.





Figure 3: A diagrammatic representation of the repressilator coupled to the light sensing and production pathways.



Within each component, the light (the luciferase of the associated wavelength) and the repressor would be both either active or inactive at the same time. For example, in Component 1, high levels of lambda-cI would repress the activity of both the red luciferase and LacI; as soon as the levels of lambda-cI begin to fall, the concentration of red luciferase and LacI in the system would increase in tandem.

The expression of the light luciferase, which to the observer would produce light of a visible and distinguishable colour, would inhibit the following promoter through the action of the associated light sensor. The promoter thus inhibited would thus repress both the following light luciferase and the following promoter, similar to its functionality in the standard repressilator.

The light sensors would work in different ways according to the peculiarities of the associated pathways. The red light sensor would absorb red photons and repress the phosphorylation of OmpR, thus inhibiting the fusion between the OmpR-dependent ompC promoter and the tetR coding sequence. The green light sensor would work in a similar manner upon the PhoR pathway and the lacI coding sequence, whilst the blue light sensor would modify the α-helical domain linker of LovTAP as a conduit for an allosteric signal upon the lambda-CI coding sequence.

The significance of our design would be in the addition of the outer network (the light repressilator), thus theoretically introducing the light signal into the cell community and realising multicellular communication. Eventually, we hope to be able to demonstrate synchronous behaviour across populations of bacteria that communicate via light to maintain the same state in unison.



Problems


Unfortunately, the overall design was too ambitious for us to complete within the allocated time period, especially given the problems we experienced with the individual light-based components. As an overall structure for the project we believe it is both sound and interesting; it is unfortunate thus that we will not be able to pursue it any further.





BioBricks


BBa_B0034: ribosome binding site based on the Elowitz repressilator.

BBa_R0010: the LacI-sensitive inhibitory promoter region.

BBa_R0040: the TetR-sensitive inhibitory promoter region.

BBa_R0051: the lambda-CI-sensitive inhibitory promoter region.

BBa_C0012: the coding region for the LacI protein.

BBa_C0040: the coding region for the TetR protein.

BBa_C0051: the coding region for the lambda-CI protein.

BBa_B0011: transcriptional terminator.



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.




Throughout this wiki there are words in bold that indicate a relevance to human aspects. It will become obvious that human aspects are a part of almost everything in iGEM.