Team:UC Chile/Cyanolux/Project

From 2012.igem.org

(Difference between revisions)
 
(6 intermediate revisions not shown)
Line 83: Line 83:
<h1>Experimental Strategy</h1>
<h1>Experimental Strategy</h1>
<p>We have devised different strategies to achieve bioluminescence controlled under circadian rhythms. Here we describe the strategies used for building the constructs to reach our goals.</p>
<p>We have devised different strategies to achieve bioluminescence controlled under circadian rhythms. Here we describe the strategies used for building the constructs to reach our goals.</p>
-
 
-
[[File:strategyboard_uc_chile.jpg|600px|center]]
 
<h2>Splitting the Lux operon and choosing promoters</h2>
<h2>Splitting the Lux operon and choosing promoters</h2>
Line 93: Line 91:
<br />
<br />
-
 
<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 [https://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>
<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 [https://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>
<br />
<br />
-
<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).
+
<p>We choose the transaldolase promoter 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).
</p>
</p>
Line 111: Line 108:
<br>
<br>
<div id="boxright" style="width:500px;">
<div id="boxright" style="width:500px;">
-
[[File: pSB1C3_IntK.jpg | 500px | right]]
+
[[File:UC_Chile-IntKplasmidResult.jpg | 500px | right]]
</div>
</div>
<p>This construct is an integrative plasmid which targets neutral recombination sites (slr0370 and sll0337). We selected this locus because it has been extensively used in the literature ([[#11| 11]]) and it shown to have no deleterious effects on Synechocystis viability. We selected Kanamycin resistance as our selectable marker.
<p>This construct is an integrative plasmid which targets neutral recombination sites (slr0370 and sll0337). We selected this locus because it has been extensively used in the literature ([[#11| 11]]) and it shown to have no deleterious effects on Synechocystis viability. We selected Kanamycin resistance as our selectable marker.
Line 122: Line 119:
<h3>pSB1A3_IntC (Utah 2010 iGEM Team integration plasmid)</h3>
<h3>pSB1A3_IntC (Utah 2010 iGEM Team integration plasmid)</h3>
<br />
<br />
-
<p>We plan on using this plasmid to express the LuxCDEG contructs under the regulation of the Pcaa3 and PsigE promoters mentioned above. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K390200 pSB1A3_IntC].</p>
+
We plan on using this plasmid to express the LuxCDEG contructs under the regulation of the Pcaa3 and PsigE promoters mentioned above. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K390200 pSB1A3_IntC].
<h3>pSB1C3_IntS</h3>
<h3>pSB1C3_IntS</h3>
 +
<br />
<br />
<div id="boxright" style="width:500px;">
<div id="boxright" style="width:500px;">
-
[[File: pSB1C3_IntS.jpg | 500px | right]]
+
[[File:UC_Chile-IntSplasmidResult.jpg | 500px | right]]
</div>
</div>
Line 135: Line 133:
</p>
</p>
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 [https://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 [https://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.
 +
 +
 +
<h2>Implementation</h2>
 +
 +
Synthetic biology is inspired in nature to make abstractions of its principles and mechanisms. We thought this moto could be applied beyond mollecular genetics...
 +
 +
With the relevance of context in mind, a biomimetic biolamp structure was designed that resembles the organ in which Vibrio fischeri -the bacteria from which the lux genes were biobricked- lives.
 +
<html><center><img src="https://static.igem.org/mediawiki/2012/b/b3/Biomimetic.jpg" align="middle" width="860"></center></html>
 +
 +
 +
[https://2012.igem.org/Team:UC_Chile/Cyanolux/Biolamp Full description of the device here]
 +
 +
<html>
 +
<a href="https://2012.igem.org/Team:UC_Chile/Cyanolux/Results"><img src="https://static.igem.org/mediawiki/2012/a/ab/UC_Chile-Continue_button.jpg" align="right">
 +
</html>
 +
<h1>References</h1>
<h1>References</h1>
Line 208: Line 222:
<div id="16">
<div id="16">
(16)Tehrani, G. A., Mirzaahmadi, S., Bandehpour, M., & Laloei, F. (2011). Molecular cloning and expression of the luciferase coding genes of Vibrio fischeri. Journal of Biotechnology, 10(20), 4018-4023. doi:10.5897/AJB10.2363
(16)Tehrani, G. A., Mirzaahmadi, S., Bandehpour, M., & Laloei, F. (2011). Molecular cloning and expression of the luciferase coding genes of Vibrio fischeri. Journal of Biotechnology, 10(20), 4018-4023. doi:10.5897/AJB10.2363
-
 
-
<html>
 
-
<a href="https://2012.igem.org/Team:UC_Chile/Cyanolux/Results"><img src="https://static.igem.org/mediawiki/2012/a/ab/UC_Chile-Continue_button.jpg" align="right">
 
-
</html>
 
{{UC_Chilefooter}}
{{UC_Chilefooter}}

Latest revision as of 12:26, 26 October 2012

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