Red Firefly Luciferase and LRE (under pBAD)
(E. coli optimised)
This part generates a red-mutant of the luciferase from the Japanese firefly (L.cruciata) as well as the luciferin regenerating enzyme (LRE). It is under the control of an Arabinose induced promoter.
D-Luciferin has to be added to obtain light output.
The light-emitting reaction involves the conversion of D-Luciferin into oxyluciferin. This compound competes with D-Luciferin for the lucifearase's binding site, causing strong inhibition of enzyme activity. LRE removes oxyluciferin from the system by converting it into 2-cyano- 6-hydroxybenzothiazole (CHBT). This compound is non-enzymatically converted into D-Luciferin in the presence of D-cysteine. It has been proposed that in the natural system, L-cysteine is used to produce L-Luciferin, which then isomerises into D-Luciferin, but this could not be reproduced by the 2010 Cambridge iGEM team.
Light output can also be achieved by addition of CHBT and D-cysteine instead of D-Luciferin, but D-cysteine might have detrimental effects on cell growth and physiology.
Figure 1 - E.Coli (Invitrogen TOP 10) cells transformed with BBa K325909 (blue light bulb) and BBa 325219 (red light bulb)
1Measured by the Cambridge iGEM team 2010
The E.glowli team used site-directed mutagenesis to create a series of colour mutants from this BioBrick
Chassis: Device has been shown to work in Top 10 (Invitrogen)
Plasmids: Device has been shown to work on pSB1C3
: S.M. Marques and J.C.G. Esteves da Silva, (2009) Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions,Life 61, 6-17.
: T. Nakatsu et al. (2006) Structural Basis for the spectral difference in luciferase bioluminescence, Nature 440(16), 372-376.
K. Gomi and N. Kajiyama, (2001) Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin, The Journal of Biological Chemistry