Team:UC Chile/Cyanolux/Results

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[[File: K743010.jpg| 400px| left]]
[[File: K743010.jpg| 400px| left]]
A couple of picked red colonies are shown in this plate.  
A couple of picked red colonies are shown in this plate.  
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[[File: C10 digestion.jpg| 400px| right]]
[[File: C10 digestion.jpg| 400px| right]]
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Digestion profile for [http://partsregistry.org/Part:BBa_K743018 K743018] & [http://partsregistry.org/Part:BBa_K743010 K743010 ]
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<h1>Synechocystis PCC 6803</h1>
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<h1>Transforming Synechocystis PCC 6803</h1>
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<h2>Transforming Synechocystis PCC 6803</h2>
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<h2>LuxABxl under transaldolase promoter transformation</h2>
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<h3>LuxABxl under transaldolase promoter transformation</h3>
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Transformation of Synechocystis PCC 6803 with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K743015 K743015] on the 14th day of transformation. The left plate is the control transformation plate (no DNA) on Kanamycin 25ug/mL and on the right, successfully transformed Synechocystis colonies growing throughout the Millipore membrane on the same antibiotic.  
Transformation of Synechocystis PCC 6803 with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K743015 K743015] on the 14th day of transformation. The left plate is the control transformation plate (no DNA) on Kanamycin 25ug/mL and on the right, successfully transformed Synechocystis colonies growing throughout the Millipore membrane on the same antibiotic.  
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<h2>Corroboration of integration</h2>
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To corroborate if the transformant colonies had integrated the actual insert, we did colony PCR for multiple parts contained in the insert.
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<h3>Integration of LuxABxl under transaldolase promoter</h3>
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We did a massive colony PCR to corroborate the presence of the parts which composed the original K743014 integration plasmid, with corresponding negative controls to avoid confusing a genomic amplicon with an insert amplicon.
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Regretably we had many negative controls which had amplicons and also lots of amplicons of the insert which we could not obtain... We are not certain about the repercussions of this result so we will try the experiment after the wiki freeze.
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<b>It is important to note that to our knowledge we are the first iGEM team to do a succesful direct transformation of Synechocystis PCC. 6803 with naked DNA.</b>
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<h3>Integration of LuxABvf under transaldolase promoter</h3>
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<div style="width: 400px; padding: 10px; margin-left: auto; margin-right: auto;">
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[[File:LuxABvf colony PCR.JPG| 400px| center]]
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This PCR was done using DNA from transformant LuxABvf colonies to amplify parts contained in the insert in lanes 2,4,6,8 and DNA from wild-type Synechocystis PCC 6803, used as a negative control in lanes 3,5,7,9. Ladder 1Kb is placed in lane 1 and 10.
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<p style="font-size:150%">We would like to make notice that to our knowledge we are the first iGEM team to do a succesful direct transformation of Synechocystis PCC. 6803 with naked DNA!</p>
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<h2>Testing constructions in Synechocystis</h2>
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To test our constructions, we managed to get access to a Luminometer which also has dispensing capabilities. To test the activity of the transformed Synechocystis with the luciferases we used decanal and dodecanal exogenously, using incremental concentrations of substrate.
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[[File:decanalxp.jpg| 400px | left]]
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[[File:dodecanalxp.jpg| 400px | right]]
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According to these results we are inclined to think that our the transaldolase promoter that we are using is not driving the expression of the luciferases. Furthermore, we have received recent advice to use much larger (up to 1 Kb) of promoter upstream of the +1 transcription site of the transaldolase gene.
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<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.
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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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<html><center><img src="https://static.igem.org/mediawiki/2012/1/1e/Results.post.colombia.camera.jpg" align="left" width="700"></center></html>
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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>
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<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. 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.
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<h2>Promoters</h2>
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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.
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<h3>sfGFP with LVA tag for describing circadian behaviour</h3>
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<h2>Luciferases</h2>
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To describe the circadian behaviour of the promoter we built a fast-degrading reporter consisting of sfGPF I746916 with a LVA degradation tag in the C-terminal end of the protein. This construct will serve as a real-time reporter of promoter activity, 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.  
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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.
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[[File: sfGFP.tag_digestion.jpg|300 px| right]]
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<h2> Susceptibility construct</h2>
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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