Team:UC Chile/Results/LuxBrick

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[[#Temperature| Temperature]]
[[#Temperature| Temperature]]
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The results confirm our hypothesis: measured luminiscence output is growth-state dependent, low at the early lag phase, peaking at early log phase and decaying at late stationary phase.
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The results confirm our hypothesis: measured luminescence output is growth-state dependent, low at the early lag phase, peaking at early log phase and decaying at late stationary phase.
    
    
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Lux genes were originally cloned from <i>Vibrio fischerii</i>, a free living bacteria found in tropical sea water that also inhabits the poikilotherm mollusc <i>Euprymna scolopes</i> body in a symbiotic relationship, therefore we expect the Lux genes performance to be functional in the range of temperatures found in that environmental context.
Lux genes were originally cloned from <i>Vibrio fischerii</i>, a free living bacteria found in tropical sea water that also inhabits the poikilotherm mollusc <i>Euprymna scolopes</i> body in a symbiotic relationship, therefore we expect the Lux genes performance to be functional in the range of temperatures found in that environmental context.
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It has been shown that the optimal temperature conditions for bioluminescence in <i>V. harveyi</i> range from 20-26°C [[#5|5]] and the LuxBrick page states that luminescence is diminished in temperatures above 30°C. We aimed to characterize the luminiscence output of the LuxBrick in a set of temperature conditions used in <i>E.coli</i> cultivation.
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It has been shown that the optimal temperature conditions for bioluminescence in <i>V. harveyi</i> range from 20-26°C [[#5|5]] and the LuxBrick page states that luminescence is diminished in temperatures above 30°C. We aimed to characterize the luminescence output of the LuxBrick in a set of temperature conditions used in <i>E.coli</i> cultivation.
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<h1>N-decanal Effect Characterization - (BBa_K325909 and BBa_K325905)</h1>
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We decided to make a luciferase bioluminescence assay to put to test wether [http://partsregistry.org/Part:BBa_K743003 Pta promoter] is effectively driving LuxAB genes expression in [http://partsregistry.org/Part:BBa_K743014 BBa_K743014] and [http://partsregistry.org/Part:BBa_K743015 BBa_K743015] transformed into Synechocystis.
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At the same time, we made the same assay to test if the [http://partsregistry.org/Part:BBa_K325905 Bacterial Lux reporter CDEG pλ AB] from wich we cloned the LuxAB genes produces light emition in E. coli cells under different n-decanal concentrations.
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'''Results'''
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<html><img src="http://partsregistry.org/wiki/images/d/d6/Potopotopoto.jpg" width="500"></html>
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<html><img src="http://partsregistry.org/wiki/images/8/87/Cacacacax56t.jpg" width="500"></html>
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'''Analysis'''
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Results of our measurement showed production and expression of Luciferase only for LuxBrick. This BioBrick has a basal luminescence yield which is augmented in the presence of decanal and dodecanal.
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The results suggest that the limiting factor in the Luciferase reaction is the substrate production and not the catalytic activity of the enzyme, as is documented in literature.
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For the rest of the constructs measurements show total absence of luminescence for the whole range of aldehyde concentrations suggesting that luciferases are not being expressed. We believe this is caused by a failure in the [http://partsregistry.org/Part:BBa_K743003 Pta promoter] region which was taken 200 bp upstream from the original gene. We think that maybe the actual promoter could have been interrupted therefore it will be amplified again from chassis genome taking a much larger sequence upstream the atg start codon.
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Part [http://partsregistry.org/Part:BBa_K325905 K325905] is a Bacterial Lux reporter
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CDEG pλ AB that contains a luciferase gene(from Xenorhabdus luminescens) under plambda promoter. The presence of decanal or dodecanal should trigger luminescence production. However, luminescence levels are practically zero, which could be attributed to an error in the LuxAB gene or in the promoter.
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As [http://partsregistry.org/Part:BBa_K743014 BBa_K743014] was built from [http://partsregistry.org/Part:BBa_K325905 K325905] it is possible that our construct inherited the problems that make this biobrick fail
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<h1>References</h1>
<h1>References</h1>
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<a href="https://2012.igem.org/Team:UC_Chile/Protocols"><img src="https://static.igem.org/mediawiki/2012/a/ab/UC_Chile-Continue_button.jpg" align="right">
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Latest revision as of 02:53, 27 September 2012

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