Team:Edinburgh/Bacterial/Core repressilator

From 2010.igem.org

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<p>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.</p><br>
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<p>In 2000, Elowitz and Leibler presented the first synthetic oscillatory system in <i>E. coli</i>. 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.</p><br>
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<center><p><img src="http://2010.igem.org/wiki/images/c/c4/Ed10-QuorumRepressilator.png" width="440" height="164" border="0" /></p><br>
<center><p><img src="http://2010.igem.org/wiki/images/c/c4/Ed10-QuorumRepressilator.png" width="440" height="164" border="0" /></p><br>
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<p>The overall oscillating design of our system is 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 signaling mechanism. The inner network works in the same manner as the original repressilator designed by Garcia-Ojalvo and M. B. Elowitz in 2004, containing three genes in an ordered circular fashion (λcI, lacI, tetR). The outer network, on the other hand, consists of the three light sensors and their associated light producers.</p>
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<p>The overall oscillating design of our system is 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 signaling mechanism. The inner network works in the same manner as the original 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, consists of the three light sensors and their associated light producers.</p>
<p>The integrated network can thus be considered in terms of its three separate components:</p>
<p>The integrated network can thus be considered in terms of its three separate components:</p>
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  <li> Component 1: red luciferase, lacI, red sensor. </li>
  <li> Component 1: red luciferase, lacI, red sensor. </li>
  <li> Component 2: blue luciferase, tetR, blue sensor. </li>
  <li> Component 2: blue luciferase, tetR, blue sensor. </li>
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  <li> Component 3: green luciferase, λcI, green sensor. </li>
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  <li> Component 3: green luciferase, lambda-cI, green sensor. </li>
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<p><b>Figure 3:</b> A sketch of the repressilator coupled to the light sensing and production pathways.</center><br><br>
<p><b>Figure 3:</b> A sketch of the repressilator coupled to the light sensing and production pathways.</center><br><br>
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<p>Within each component, the light (luciferase) and the repressor are both active or inactive at the same time. For example, in Component 1, high levels of λcI repress the activity of both the red luciferase and lacI; as soon as the levels of λcI begin to fall, the concentration of red luciferase and lacI in the system increases.</p>
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<p>Within each component, the light (luciferase) and the repressor are both active or inactive at the same time. For example, in Component 1, high levels of lambda-cI 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 increases.</p>
<p>The expression of the light luciferase, which produces light of the associated colour, inhibits the following promoter through the light sensor; the promoter, on the other hand, represses both the following light luciferase and the following promoter.</p>
<p>The expression of the light luciferase, which produces light of the associated colour, inhibits the following promoter through the light sensor; the promoter, on the other hand, represses both the following light luciferase and the following promoter.</p>
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<p>As for the light sensors, these work in two different ways. The red light sensor absorbs red photons and represses the phosphorylation of OmpR, thus inhibiting the fusion between the OmpR-dependent ompC promoter and tetR. The blue and green light sensors work in a similar manner, by modifying the α-helical domain linker of LovTAP as a conduit for an allosteric signal.</p>
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<p>As for the light sensors, these work in two different ways. The red light sensor absorbs red photons and represses the phosphorylation of OmpR, thus inhibiting the fusion between the OmpR-dependent <i>ompC</i> promoter and the <i>tetR</i> coding sequence. The blue light sensor works in a similar manner, by modifying the α-helical domain linker of LovTAP as a conduit for an allosteric signal, while the green light takes action upon the PhoR pathway.</p>
<p>The significance of our design is in the addition of the outer network (the light repressilator), thus introducing the light signal into the cell community and realising multicellular communication.</p>
<p>The significance of our design is in the addition of the outer network (the light repressilator), thus introducing the light signal into the cell community and realising multicellular communication.</p>
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<p>***</p>
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<p><a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a>: ribosome binding site based on the Elowitz repressilator.</p>
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<p><a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a>: the LacI-sensitive inhibitory promoter region.</p>
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<p><a href="http://partsregistry.org/Part:BBa_R0040">BBa_R0040</a>: the TetR-sensitive inhibitory promoter region.</p>
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<p><a href="http://partsregistry.org/Part:BBa_R0051">BBa_R0051</a>: the lambda-CI-sensitive inhibitory promoter region.</p>
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<p><a href="http://partsregistry.org/Part:BBa_C0012">BBa_C0012</a>: the coding region for the LacI protein.</p>
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<p><a href="http://partsregistry.org/Part:BBa_C0040">BBa_C0040</a>: the coding region for the TetR protein.</p>
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<p><a href="http://partsregistry.org/Part:BBa_C0051">BBa_C0051</a>: the coding region for the lambda-CI protein.</p>
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<p><a href="http://partsregistry.org/Part:BBa_B0011">BBa_B0011</a>: transcriptional terminator.</p>
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<p>Elowitz, M. B. and S. Leibler (2000). "A synthetic oscillatory network of transcriptional regulators." Nature 403(6767): 335-338.<br>
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<p><b>Elowitz, M. B. and S. Leibler (2000).</b> A synthetic oscillatory network of transcriptional regulators. <i>Nature</i> <b>403</b>(6767): 335-338.</p>
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Danino, T., O. Mondragon-Palomino, et al (2009). "A synchronized quorum of genetic clocks." Nature 463(7279): 326-330.<br>
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<p><b>Danino, T., O. Mondragon-Palomino, et al (2009).</b> A synchronized quorum of genetic clocks. <i>Nature</i> <b>463</b>(7279): 326-330.</p>
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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.<br></p>
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<p><b>Garcia-Ojalvo, J., M. B. Elowitz, et al. (2004).</b> Modeling a synthetic multicellular clock: Repressilators coupled by quorum sensing. <i>Proceedings of the National Academy of Sciences of the United States of America</i> <b>101</b>(30): 10955-10960.</p>
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Revision as of 11:02, 18 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 fast out of the chamber, allowing control of the oscillation rate.




Strategy


The overall oscillating design of our system is 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 signaling mechanism. The inner network works in the same manner as the original 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, consists of the three light sensors and their associated light producers.

The integrated network can 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 sketch of the repressilator coupled to the light sensing and production pathways.



Within each component, the light (luciferase) and the repressor are both active or inactive at the same time. For example, in Component 1, high levels of lambda-cI 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 increases.

The expression of the light luciferase, which produces light of the associated colour, inhibits the following promoter through the light sensor; the promoter, on the other hand, represses both the following light luciferase and the following promoter.

As for the light sensors, these work in two different ways. The red light sensor absorbs red photons and represses the phosphorylation of OmpR, thus inhibiting the fusion between the OmpR-dependent ompC promoter and the tetR coding sequence. The blue light sensor works in a similar manner, by modifying the α-helical domain linker of LovTAP as a conduit for an allosteric signal, while the green light takes action upon the PhoR pathway.

The significance of our design is in the addition of the outer network (the light repressilator), thus introducing the light signal into the cell community and realising multicellular communication.



Problems


Unfortunately, it seems as if this design was too ambitious for us to complete within the allocated time period, especially given the problems we have been experiencing with the other components. As an overall structure for the project we believe it is sound; it is unfortunate thus that we will not be able to pursue the design 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.