Team:UC Chile/Cyanolux/Results

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<h1>K743015 Debugging</h1>
 
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As we have no certainty that the transaldolase promoter we are currently using is driving the expression of our constructs we decided to have a direct approach to obtain clear answers.
 
<h2>sfGFP with degradation tag to characterize transaldolase promoter</h2>
<h2>sfGFP with degradation tag to characterize transaldolase promoter</h2>
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To describe the circadian behaviour of the transaldolase promoter without having the dificulties of equipment (luminometer and substrate), we built a fast-degrading reporter consisting of sfGPF(I746916) with a LVA degradation tag in the C-terminal end of the protein,  [http://partsregistry.org/Part:BBa_K743019 BBa_K743019]. This construct will serve as a real-time reporter of promoter activity. As it is a reporter plasmid with fast-degradation tags, verificating if sfGFP is being expressed circadianly by Synechocystis should be effortless.  
To describe the circadian behaviour of the transaldolase promoter without having the dificulties of equipment (luminometer and substrate), we built a fast-degrading reporter consisting of sfGPF(I746916) with a LVA degradation tag in the C-terminal end of the protein,  [http://partsregistry.org/Part:BBa_K743019 BBa_K743019]. This construct will serve as a real-time reporter of promoter activity. As it is a reporter plasmid with fast-degradation tags, verificating if sfGFP is being expressed circadianly by Synechocystis should be effortless.  
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You may find more information about the half-life of proteins with the LVA tag  [http://partsregistry.org/wiki/index.php?title=Part:BBa_M0050 | here]. The construct has been verified by digestion and corroborated by sequencing. Check construct digestion [[Team:UC_Chile/Cyanolux/Results#C10 here]]
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You may find more information about the half-life of proteins with the LVA tag  [http://partsregistry.org/wiki/index.php?title=Part:BBa_M0050 | here]. The construct has been verified by digestion and corroborated by sequencing. Check construct digestion [[Team:UC_Chile/Cyanolux/Results#C10 | here]]
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<h3>High-sensitive camera</h3>
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During the Latin America Jamboree, we had a chat with a couple of judges and a student from the Universidad de los Andes, David Olarte, on how to induce Synechocystis with n-decanal.
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David sent us some work he had done on luminescence assays in cyanobacteria. Following his methods we finally were able to see light emittion, confirming prescence of catalytically active luciferase.
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<h1>Experimental Highlights</h1>
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- Constructed two functional integration plasmids for <i>Synechocystis</i> PCC. 6803
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- Transformed <i>Synechocystis</i> PCC. 6803 with an optimized transformation protocol, available [https://2012.igem.org/Team:UC_Chile/Protocols#Transformation_of_Synechocystis_PCC._6803 here]
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- Verified integration of constructs
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- Confirmed expression of genes and activity of luciferase through a bioluminescence assay with exogenous substrate.
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<h1>Conclusions</h1>
<h1>Conclusions</h1>
<h2>Transformation in Synechocystis PCC. 6803</h2>
<h2>Transformation in Synechocystis PCC. 6803</h2>
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We have successfully transformed Synechocystis PCC. 6803. We have been able to standarize growth conditions and transformation protocols.
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We have successfully transformed Synechocystis PCC. 6803. We have been able to standarize growth conditions and transformation protocols. We believe that we are the first iGEM team to transform  Synechocystis PCC. 6803 directly with naked DNA.
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<h2>Verification of integrations</h2>
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We have been able to verify the integration of the construct into Synechocystis's genome.
<h2>Promoters</h2>
<h2>Promoters</h2>
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We have not been able to demonstrate te driving of expression by the transaldolase promoter but with our new sfGFP construct with degradation tag we will be able to determinate if the promoter we are using is not large enough. If that is the case then we will clone 1 Kb upstream from the +1 transcription site of the transaldolase.
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We have been able to demonstrate that the transaldolase is driving the expression of the constructs but we have no certainty as if it is behaving circadianly. With our sfGFP construct with degradation tag we will be able to determinate if that is so.
<h2>Luciferases</h2>
<h2>Luciferases</h2>
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Due to unexpected results of the colony PCR for K743014 and recent information in the [http://partsregistry.org/Part:BBa_K325905  registry page ] specifying it as a failed experience, we have decided to focus on K743015 with the LuxAB from Vibrio fisherii which we have already have success in bioluminescence experiments with the LuxBrick (K325909).
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With our new results with the High-sensitivity CCD camera we have been able to observe bioluminescence from our transformed Synechocystis. The luciferase is catalytically active, however we need to see if we can enhance light output by changing the length of the promoter.
<h2> Susceptibility construct</h2>
<h2> Susceptibility construct</h2>
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We are now ready to clone the LuxCDEG portion of the Lux operon with our newly acquired susceptibility construct. We have all parts readily amplified and Gibson assembly of the parts will be done after the Wiki freeze.
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We have been unable to obtain a correct assembly of the LuxCDEG (substrate production) into our suceptibility construct. Due to repetitive failures, we will try a new strategy of cloning, by ordering new primers with longer overlapping ends.
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Latest revision as of 10:05, 26 October 2012

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