Team:UC Chile2/Cyanolux/Results

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<h1>Construction of plasmids</h1>
<h1>Construction of plasmids</h1>
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Our first attempts to construct plasmid pSB1C3_IntK was through a Gibson Assembly reaction of 6 different parts (see construct detail [[http://partsregistry.org/Part:BBa_K743004| BBa_K743004]]) all at once, however we were unsuccessful due to many reasons, many which became clear afterwards (see NOTEPAD WEEK SOMETHING ABOUT GIBSON). We did many attempts betting on depletion of PpsbAB (140bp) due to T5 exonuclease where we changed concentration ratios of the parts, nevertheless we were not able to assemble the parts.
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<h2>pSB1C3_IntK</h2>
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<br>
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Our first attempts to build the construct ([[http://partsregistry.org/Part:BBa_K743004| BBa_K743004]]) through Gibson Assemly were unsuccessful due to problems involving high compexity of the reaction, however we were able to Biobrick the recombination sites ([[http://partsregistry.org/Part:BBa_K743000 | Part:BBa_K743000]] and [[http://partsregistry.org/Part:BBa_K743001 | Part:BBa_K743001]]). Afterwards we decided to build a simple backbone first before continuing with more complex assemblies.
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During late June we were able to Biobrick the recombination sites ([[http://partsregistry.org/Part:BBa_K743000 | Part:BBa_K743000]] and [[http://partsregistry.org/Part:BBa_K743001 | Part:BBa_K743001]]) and we decided to use standard assembly to construct a smaller version of pSB1C3_IntK [[http://partsregistry.org/Part:BBa_K743006| K743006]]  
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Through standard assembly we managed to build [[http://partsregistry.org/Part:BBa_K743006| K743006]] which led us to continue assembling our constructs through simpler Gibson Assemblies. In short time we were able to build our final constructs for the luciferase using 2 different versions of it available in the registry ([[http://partsregistry.org/Part:BBa_K743014 | From Photorhabdus luminiscent, Part:BBa_K743014]] and [[http://partsregistry.org/Part:BBa_K743015 | from Vibrio fisherii, Part:BBa_K743015]]) under an endogenous  Synechocystis's promoter (transaldolase Reference???)
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All constructs and parts have been verified by digestion and corroborated by sequencing.
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<h2>pSB1A3_IntC (From 2010 Utah's iGEM team)</h2>
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We decided to use this plasmid to place the LuxCDEG part of the Lux operon in this plasmid, however after various attempts to transform Synechocystis without success we reconsidered (see below SOMEWHERE!!! [[]]) due to problems regarding the design of this plasmid backbone (see below ANOTHERWHERE)
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<h2>pSB1C3_IntK</h2>
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When we reconsidered about using pSB1A3_IntC we designed a new plasmid backbone to place the LuxCDEG (substrate regeneration part of the Lux operon) under the Synechocystis
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<h2>Assembling DNA parts</h2>
 
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<h2>Confirming</h2>
 
<h2>Corroborating sequences</h2>
<h2>Corroborating sequences</h2>

Revision as of 16:06, 25 September 2012

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