Team:UC Chile/Cyanolux/Project

From 2012.igem.org

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<p>To try our approach, we selected various promoters which could serve the purpose.  Our rational for selecting candidate promoters involved amplitude of oscillation, peak activity, hour, absence of restriction sites, predicted strength of promoter according to the role of the gene and reproducibility between experiments (based on the literature available).  We looked for promoters which would have peak expression nearby dusk hours and that were slightly out of phase to optimize production of bioluminescence according to our mathematical models <b>(LINK OVER HERE!)</b>. We prioritized promoters from genes that would be involved in central energetic metabolism as we believe that their expression would be most robust and reliable.</p>
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<p>To try our approach, we selected various promoters which could serve the purpose.  Our rational for selecting candidate promoters involved amplitude of oscillation, peak activity, hour, absence of restriction sites, predicted strength of promoter according to the role of the gene and reproducibility between experiments (based on the literature available).  We looked for promoters which would have peak expression nearby dusk hours and that were slightly out of phase to optimize production of bioluminescence according to our [http://2012.igem.org/Team:UC_Chile/Cyanolux/Modelling mathematical models]. We prioritized promoters from genes that would be involved in central energetic metabolism as we believe that their expression would be most robust and reliable.</p>
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<p>We choose the transaldolase promoter <b>(specific name here and code in Synechocystis Genome)</b> to direct the expression of the LuxAB genes and we found a couple of other promoters which filled the other requirements from above. Pcaa3 (NAME HERE AND DESCRIPTION OF ENDOGENOUS ACTIVITY) and PsigE (NAME HERE AND DESCRIPTION OF ENDOGENOUS ACTIVITY), the former being already in Biobrick format (courtesy from the Utah team iGEM 2010).
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<p>We choose the transaldolase promoter <b>(specific name here and code in Synechocystis Genome)</b> to direct the expression of the LuxAB genes and we found a couple of other promoters which filled the other requirements from above. Pcaa3 and PsigE, the former being already in Biobrick format (courtesy from the Utah team iGEM 2010).
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<p>Due to issues mentioned in the [http://2012.igem.org/Team:UC_Chile/Cyanolux/Results results page] we designed a new plasmid backbone.
<p>Due to issues mentioned in the [http://2012.igem.org/Team:UC_Chile/Cyanolux/Results results page] we designed a new plasmid backbone.
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This is an integration plasmid which makes Synechocystis susceptible to copper concentrations higher than 0.75 uM [[#10|10]] by disrupting the CopS gene. We believe that this strategy serves as a biosafety measure to avoid the possibility of having a leakage of recombinant DNA to the environment. The plasmid uses Spectynomycin as a selectable marker. [PUT LINK TO CONSTRUCT HERE]
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This is an integration plasmid which makes Synechocystis susceptible to copper concentrations higher than 0.75 uM [[#10|10]] by disrupting the CopS gene. We believe that this strategy serves as a biosafety measure to avoid the possibility of having a leakage of recombinant DNA to the environment. The [http://partsregistry.org/Part:BBa_K743010 plasmid] uses Spectynomycin as a selectable marker.
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We plan on expressing LuxCDEG under the control of the promoters Pcaa3 and PsigE (mentioned above). These promoters have peak activities 1 hour before dusk. Based on our [http://2012.igem.org/Team:UC_Chile/Cyanolux/Modelling modelling] we believe that we might enhance bioluminescence yield initially by setting the substrate production/regeneration part of the operon prior to the expression of the luciferase.
We plan on expressing LuxCDEG under the control of the promoters Pcaa3 and PsigE (mentioned above). These promoters have peak activities 1 hour before dusk. Based on our [http://2012.igem.org/Team:UC_Chile/Cyanolux/Modelling modelling] we believe that we might enhance bioluminescence yield initially by setting the substrate production/regeneration part of the operon prior to the expression of the luciferase.

Revision as of 03:58, 27 September 2012

Project: Luxilla - Pontificia Universidad Católica de Chile, iGEM 2012