Generator

Part:BBa_K325219

Designed by: Bill Collins and Emily Knott   Group: iGEM10_Cambridge   (2010-09-15)
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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 [http://2010.igem.org/Team:Cambridge 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
[http://www.ncbi.nlm.nih.gov/pubmed/18949818 [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.

[http://www.nature.com/nature/journal/v440/n7082/abs/nature04542.html [2]:] T. Nakatsu et al. (2006) Structural Basis for the spectral difference in luciferase bioluminescence, Nature 440(16), 372-376.

[http://www.ncbi.nlm.nih.gov/pubmed/11457857 [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