Team:Cambridge/Bioluminescence

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'''Project Firefly''' was the side of our project dedicated to taking amino-acid sequences from the light producing pathways found in firelies and placing them in the registry.  
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'''Project Firefly''' focussed on the construction of light-generating BioBricks from genes involved in the bioluminescence pathways found in fireflies.  
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Fireflies are some of the best known insects from the family of click beetles.  They have a light-emitting organ known as the lantern in their abdomen.
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==Fireflies in nature==
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== Natural history ==
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Fireflies are one of the few land animals on this planet equipped with the fascinating ability to emit light. They are insects from the family of click beetles and are thought to use the production of light for different purposes at distinct developmental stages. The glow of firefly larvae is thought to be an aposematic signal, advising predators of the glowworms bitter taste and toxicity. In contrast, adult fireflies use bioluminescence in courtship displays. Females respond to the flash patterns emitted by flying males with the generation of an attracting light flash from their own bodies.  
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The fireflies use their lantern in courtship displays, the flashing of a female's lantern attracts a male to her. However since larvae also glow the light is also thought to be an ''aposematic signal'', advising predators of the beetle's bitter taste.
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The chemical reactions that lead to the production of light occur in a specialised organ in the lower abdomen of fireflies, known as the lantern. Here the enzyme luciferase catalyses the oxidation of the pigment luciferin to oxyluciferin. Oxyluciferin is initially produced in an unstable chemically excited state, but quickly drops down into the more energetically favourable ground state. In this process a photon of light is emitted. In 2001 [http://www.ncbi.nlm.nih.gov/pubmed/11457857 Gomi and Kajiyama] discovered another important enzyme involved the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Background_Firefly light-producing pathway] in the lantern organ of fireflies. This luciferin-regenerating enzyme (LRE) is capable of converting oxyluciferin back into luciferin upon the addition of D-cysteine, thereby relieving inhibition of luciferase by oxyluciferin as well as fueling into the luciferin pool.
==Our work==
==Our work==
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The Cambridge team have exploited a number of proteins based on firefly bioluminescence:
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In our experimental efforts we have concentrated on constructing Biobricks based on the following enzymes:
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* The luciferase (light emitting enzyme) from the North American firefly, ''Photinus pyralis'' with three mutations to increase substrate affinity
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*The luciferase and LRE from the North American firefly, Photinus pyralis.
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* The luciferase from the Japansese firefly, ''Luciola cruciata'' for which we made a number of mutations to give 3 different colours.
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*The luciferase and LRE from the Japanese firefly, Luciola cruciata.
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* The '''luciferin-regenerating enzymes''' (LREs) from both these species.  Luciferin is the substrate used by luciferase to emit light.  Once a photon has been emitted this luciferin is released in an inactive from known as '''oxyluciferin'''. LRE is crucial for the regeneration of this oxyluciferin into new luciferin.
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The luciferase from the North American firefly, Photinus pyralis, is a well-tested and characterised enzyme that has found applications in several reporter assays in scientific research. We were aware that a luciferase from this organism was already present in the Registry ([http://partsregistry.org/Part:BBa_I712019 BBa_I712019]). We aimed to improve this part and to extend its applicability by pursuing the following three different routes:
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*[https://2010.igem.org/Team:Cambridge/Codons Codon optimisation] for expression in E. coli to increase the rate of translation in the bacterium
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*Mutagenesis to increase substrate affinity, thereby increasing the brightness of the emitted light
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*Simultaneous expression of the Photinus pyralis [https://2010.igem.org/Team:Cambridge/Bioluminescence/Luciferin_Regeneration LRE] to increasing both intensity and duration of light emission
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The luciferase of the North American firefly, ''P. pyralis'', is a tried and tested mechanism for creating bioluminescence. We were aware that a luciferase from this organism was already present in the registry ([http://partsregistry.org/Part:BBa_I712019 BBa_I712019]).  We wanted to improve on this by three techniques:
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Japanese fireflies of the species Luciola cruciata are notable for synchronising their flash patterns when in large groups, thereby making the trees on which they sit pulsate with green light. This phenomenon has been reported to be exploited by fisherman as a means of locating the shore. We wanted to add to the pre-existing luciferase in the Registry by
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* [https://2010.igem.org/Team:Cambridge/Codons Codon optimisation] for expression in E. coli to increase the rate of translation
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*Site-directed mutagenesis to create BioBricks capable of emitting light of a range of different colours.
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* Using a mutant with increased substrate affinity
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Luciola cruciata was specifically chosen for this purpose on the basis of a publication by [http://www.ncbi.nlm.nih.gov/pubmed/1946326 Kajiyama and Nakano] in 1991 that described a suite of single amino acid changes which significantly changed the wavelength of peak emission.  
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* Parallel use of the Photinus pyralis [https://2010.igem.org/Team:Cambridge/Bioluminescence/Luciferin_Regeneration luciferin regenerating enzyme] to both relieve inhibition by oxyluciferin and increase availability of luciferin.
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The Japanese firefly, ''Luciola cruciata'' is notable in that nearby fireflies flash in synchrony, making the trees pulsate with green light which fishermen use as a guide to bring them home.  The Cambridge team selected Luciola cruciata because [http://www.ncbi.nlm.nih.gov/pubmed/1946326 Kajiyama and Nakano (1991)] had described a suite of single amino acid changes which significantly changed the wavelength of peak emission.
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[[Image:Cam-luci-cycle.jpg|center|500px]]
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Latest revision as of 03:57, 28 October 2010