Generator

Part:BBa_K325219

Designed by: Bill Collins and Emily Knott   Group: iGEM10_Cambridge   (2010-09-15)

Red Firefly Luciferase and LRE (under pBAD)
L. Cruciata
(E. coli optimised)

Input: L-Arabinose
Output: Light

pBad/araC
I0500
Luciferase/LRE
K325210
Cambridge-Eglowli.png

Part Main Page        Arabinose -> Light        Add Data       


Description
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.


Pictures

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

Derivative parts

The E.glowli team used site-directed mutagenesis to create a series of colour mutants from this BioBrick

Compatibility
Chassis: Device has been shown to work in Top 10 (Invitrogen)
Plasmids: Device has been shown to work on pSB1C3


References
[1]: S.M. Marques and J.C.G. Esteves da Silva, (2009) Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions,Life 61, 6-17.

[2]: T. Nakatsu et al. (2006) Structural Basis for the spectral difference in luciferase bioluminescence, Nature 440(16), 372-376.

[3]: K. Gomi and N. Kajiyama, (2001) Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin, The Journal of Biological Chemistry, 276(39), 36508-36513.
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Categories
Parameters
None