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Team:Cambridge/Bioluminescence
From 2010.igem.org
Project Firefly was the side of our project dedicated to taking amino-acid sequences from the light producing pathways found in fireflies and placing them in the registry.
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.
Natural history
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.
Our work
The Cambridge team have exploited a number of proteins based on firefly bioluminescence:
- The luciferase (light emitting enzyme) from the North American firefly, Photinus pyralis with three mutations to increase substrate affinity
- The luciferase from the Japansese firefly, Luciola cruciata for which we made a number of mutations to give 3 different colours.
- 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.
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:
- Codon optimisation for expression in E. coli to increase the rate of translation
- Using a mutant with increased substrate affinity
- Parallel use of the Photinus pyralis luciferin regenerating enzyme to both relieve inhibition by oxyluciferin and increase availability of luciferin.
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.
