Team:UC Chile/Cyanolux/Project

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<h2>Synechocystis PCC 6803</h2>
<h2>Synechocystis PCC 6803</h2>
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<p>Cyanobacteria are prokaryotic photoautotrophs and they are believed to have
<p>Cyanobacteria are prokaryotic photoautotrophs and they are believed to have
evolved oxygenic photosynthesis about 2.4 billion years ago [[#1|1]]. Although this biochemical breakthrough can not
evolved oxygenic photosynthesis about 2.4 billion years ago [[#1|1]]. Although this biochemical breakthrough can not
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<h2>Lux Operon </h2>
<h2>Lux Operon </h2>
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<p>The lux operon is a group of genes that are responsible for density-dependent bioluminescent behavior
<p>The lux operon is a group of genes that are responsible for density-dependent bioluminescent behavior
in various prokariotic organisms such as <i>Vibrio fischeri</i> and <i>Photorabdus luminescens</i> [[#12|12]]. In <i>V. fischeri</i>, the operon is composed of 8 genes: LuxA and LuxB encode for the monomers of a heterodimeric luciferase; LuxC, LuxD and LuxE code for fatty acid reductases enzymes and LuxR and LuxI are responsible for the regulation of the whole operon [[#13|13]].</p>
in various prokariotic organisms such as <i>Vibrio fischeri</i> and <i>Photorabdus luminescens</i> [[#12|12]]. In <i>V. fischeri</i>, the operon is composed of 8 genes: LuxA and LuxB encode for the monomers of a heterodimeric luciferase; LuxC, LuxD and LuxE code for fatty acid reductases enzymes and LuxR and LuxI are responsible for the regulation of the whole operon [[#13|13]].</p>
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<p>Lastly LuxG is believed to act as a FMNH2 dependent FADH reductase, although luminescence is barely affected
<p>Lastly LuxG is believed to act as a FMNH2 dependent FADH reductase, although luminescence is barely affected
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in its absence [[#14|14]]. The n-decanal ( n= 9 to 14) substrate oxidization to n-decanoic acid by the LuxAB heterodimer is coupled with the reduction of FMNH to FMNH2 and the releasing of oxygen and light.</p>
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in its absence [[#14|14]]. The n-decanal ( n= 9 to 14) substrate oxidization to n-decanoic acid by the LuxAB heterodimer is coupled with the reduction of FMNH to FMNH2 and the release of oxygen and light.</p>
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<p>The carboxylic group of the product is then reduced to aldehyde by CDE proteins allowing the reaction to
<p>The carboxylic group of the product is then reduced to aldehyde by CDE proteins allowing the reaction to
start over [[#15|15]].</p>
start over [[#15|15]].</p>
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<p>LuxAB genes have been widely used as reporters dependent on the addition of n-decanal to the culture
<p>LuxAB genes have been widely used as reporters dependent on the addition of n-decanal to the culture
media [[#16|16]] and in 2010, the [https://2010.igem.org/Team:Cambridge Cambridge iGEM team] engineered LuxABCDEG to an <i>E. coli</i>-optimized biobrick
media [[#16|16]] and in 2010, the [https://2010.igem.org/Team:Cambridge Cambridge iGEM team] engineered LuxABCDEG to an <i>E. coli</i>-optimized biobrick
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<p>As a team we decided to work with this operon for a number of reasons, first of all, the luminescence
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produced by this pathway is much more visually appealing than other systems from the registry (i.e XFPs),
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moreover, the light production doesn´t depend on a single peptide but on a whole pathway involving several genes, which makes it much more tunable, for instance, decoupling in time the substrate recovery from the luciferase reaction itself.</p>
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<p>As a team we decided to work with this operon for a number of reasons, first of all, the bioluminescence
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produced by this pathway is much stronger than other systems from the registry (i.e XFPs).
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Moreover, the light production does not depend on a single peptide but on a whole pathway involving several genes, which makes it much more tunable, for instance, decoupling in time the substrate recovery from the luciferase reaction itself.</p>
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<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>
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[[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>
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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 [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>
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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>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 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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<h3>pSB1C3_IntK</h3> [http://partsregistry.org/Part:BBa_K743006 BBa_K743006]
<h3>pSB1C3_IntK</h3> [http://partsregistry.org/Part:BBa_K743006 BBa_K743006]
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<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. [PUT LINK TO CONSTRUCT HERE].</p>
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<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.
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Using this backbone we have decided to put LuxAB under the transaldolase promoter.We have choose the transaldolase promoter to express the luciferase part of the operon, as in the literature the promoter is described as having a peak of expression at 2 hours past dusk, which we believe is just the right timing to "turn on the lamp".
Using this backbone we have decided to put LuxAB under the transaldolase promoter.We have choose the transaldolase promoter to express the luciferase part of the operon, as in the literature the promoter is described as having a peak of expression at 2 hours past dusk, which we believe is just the right timing to "turn on the lamp".
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<h3>pSB1A3_IntC (Utah 2010 iGEM Team integration plasmid)</h3>
<h3>pSB1A3_IntC (Utah 2010 iGEM Team integration plasmid)</h3>
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<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>
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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>
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<p>Due to issues mentioned in the results page (PUT LINK HERE) 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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<p>Due to issues mentioned in the [https://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 [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. 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.(LINK TO MODELLING?)
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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 [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.
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<h2>Implementation</h2>
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Synthetic biology is inspired in nature to make abstractions of its principles and mechanisms. We thought this moto could be applied beyond mollecular genetics...
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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.
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[https://2012.igem.org/Team:UC_Chile/Cyanolux/Biolamp Full description of the device here]
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<h1>References</h1>
<h1>References</h1>
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<div id="16">
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(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
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Latest revision as of 12:26, 26 October 2012

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