Team:Cambridge/Bioluminescence

Project Firefly focussed on the construction of standalone light-generating BioBricks from genes involved in the light-producing pathways found in fireflies.

Fireflies in nature

Fireflies are one of the few 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. 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 Gomi and Kajiyama discovered another important enzyme involved the 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

In our experimental efforts we have concentrated on constructing Biobricks based on the following enzymes:

• The luciferase and LRE from the North American firefly, Photinus pyralis.
• The luciferase and LRE from the Japanese firefly, Luciola cruciata.

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 (BBa_I712019). We aimed to improve this part and to extend its applicability by pursuing the following three different routes:

• Codon optimisation for expression in E. coli to increase the rate of translation in the bacterium
• Mutagenesis to increase substrate affinity, thereby increasing the brightness of the emitted light
• Simultaneous expression of the Photinus pyralis LRE to increasing both intensity and duration of light emission

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

• Single amino acid mutagenesis to create BioBricks capable of emitting light of a range of different colours.

Luciola cruciata was specifically chosen for this purpose on the basis of a publication by Kajiyama and Nakano in 1991 that described a suite of single amino acid changes which significantly changed the wavelength of peak emission.

Photinus pyralis (source)

The luciferase from the North American firefly, P. pyralis, is a well-tested and characterised enzyme catalysing the creation of light using D-luciferin as a substrate. We were aware that a luciferase from this organism was already present in the registry (BBa_I712019). In order to improve this part and to further its applicability, we pursued three different routes:

• Codon optimisation for expression in E. coli to increase the rate of translation in E.coli.
• Creating 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, a phenomenon used by fishermen as a means of locating the shore. We selected Luciola cruciata because Kajiyama and Nakano (1991) had described a suite of single amino acid changes which significantly changed the wavelength of peak emission.