Team:Cambridge/Bioluminescence/Luciferin Regeneration

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{{:Team:Cambridge/Templates/RightImage|image=Cambridge-MixedPhenotype.jpg|caption=Colonies expressing luciferase and LRE}}
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===Introduction===
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Fireflies are easily visible in the evening, whereas colonies of bacteria expressing luciferase are not.  We hope to remove this divide by expressing further enzymes exploited by fireflies.  In <i>E. coli</i> and in eukaryotes, each molecule of luciferin that emits light is turned into oxyluciferin and cannot be regenerated.  A further problem is that this oxyluciferin inhibits the further reactions of luciferase.
Fireflies are easily visible in the evening, whereas colonies of bacteria expressing luciferase are not.  We hope to remove this divide by expressing further enzymes exploited by fireflies.  In <i>E. coli</i> and in eukaryotes, each molecule of luciferin that emits light is turned into oxyluciferin and cannot be regenerated.  A further problem is that this oxyluciferin inhibits the further reactions of luciferase.
[http://www.ncbi.nlm.nih.gov/pubmed/11457857 Gomi and Kajiyama (2001)] describe a <strong>luciferin regenerating enzyme (LRE)</strong>, which we believed may solve these problems and allow brighter longer lasting light output without further addition of substrate.
[http://www.ncbi.nlm.nih.gov/pubmed/11457857 Gomi and Kajiyama (2001)] describe a <strong>luciferin regenerating enzyme (LRE)</strong>, which we believed may solve these problems and allow brighter longer lasting light output without further addition of substrate.
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LRE transforms the inhibitory oxyluciferin to CHBT, which is converted back into the luciferin substrate required for light output. This was believed to occur in one of two ways: non-enzymatically requiring D-cysteine, or via L-luciferin requiring the L-cysteine. We aimed to verify these claims in our lab experiments and to quantify the difference in light output that LRE makes when expressed in ''E. coli''. To formally test the effect of the LRE we would have needed to add Oxyluciferin to the medium. Unfortunately, Oxyluciferin is not available commercially so we were not able to perform this experiment. We however tested other parts of the cycle, by adding CHBT and D-Cysteine to the medium.
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[[Image:Cam-luci-cycle.jpg|center|500px]]
[[Image:Cam-luci-cycle.jpg|center|500px]]
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LRE transforms the inhibitory oxyluciferin to CHBT, which is converted back into the luciferin substrate required for light output. This was believed to occur in one of two ways: non-enzymatically requiring D-cysteine, or via L-luciferin requiring the L-cysteine. We aimed to verify these claims in our lab experiments and to quantify the difference in light output that LRE makes when expressed in ''E. coli''. To formally test the effect of the LRE we would have needed to add Oxyluciferin to the medium. Unfortunately, Oxyluciferin is not available commercially so we were not able to perform this experiment. We however tested other parts of the cycle, by adding CHBT and D-Cysteine to the medium.
 
===Our Experiments===
===Our Experiments===

Latest revision as of 23:06, 27 October 2010