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Team:Edinburgh/Bacterial/Blue light producer
From 2010.igem.org
Overview: The blue light producer
To produce blue light, we used two systems using the bacterial luciferase from Xenorhabdus luminescens (LuxAB). The standalone LuxAB has an emission peak of 495nm. (Figure 1). According to O'Kane and Lee (1985) and others, the addition of lumazine protein shifts the wavelength towards blue (478 nm), to be in the correct region to activate the blue light sensor.
Figure 1: Emission spectra of LuxAB (in solid black line). The LuxAB depicted is that from Vibrio campbellii, but sources confirm that other bacterial luciferases produce similar spectral graphs.
Image: Suadee et al. (2008)
Strategy
Last year's Edinburgh team already made both of these parts individually, so all we had to do was fuse them together and add a promoter before. Individual parts were resubmitted in pSB1C3.
Problems
The LuxAB which last year's team made and stored is failing to transform correctly. Colonies that ought to be growing white are growing blue (LuxAB should have replaced the lacZ' in the vector, EdinbrickI). A similar thing is happening with LumP and RFP, where cells are still growing red, despite the fact that the RFP ought to have been removed when LumP was inserted last year.
We have come across several PCR products not yet inserted into vectors with which we are hoping to start this part of the project (i.e. the bit which should already have been done by last year according to their lab notes) again.
We eventually resolved these problems by using the alternative PCR products which were perfectly capable of being cloned into pSB1C3.
More recently, however, we have come up with the issue of our LuxAB-LumP fusions cooperating with neither camera nor spectrophotometer despite glowing very visibly blue, meaning that we could not take photographs of our blue light or characterise its emission spectrum. On the very last day before the wiki freeze, we finally managed to take a good photograph; with any luck, we will be able to characterise it more fully by the Jamboree.
BioBricks
Both lumP and luxAB have already been submitted as BioBricks in pSB1A3 by last year's Edinburgh team, but we are re-submitting corrected variants in pSB1C3.
We are also submitting a luxAB-lumP fusion construct in pSB1C3 with a constitutive promoter which can be used for blue light production. We have confirmation of working luxAB with the promoter in question.
BBa_K322139: bacterial luciferase luxAB, updated version of BBa_K216008.
BBa_K322312: luxCDE (required for luxAB expression), updated version of BBa_K216017.
BBa_K322007: lumP (shifts luxAB to blue), updated version of BBa_K216007.
BBa_K322140: luxAB under lac promoter.
BBa_K322141: luxAB and luxCDE under lac promoter.
BBa_K322149: luxAB and lumP composite part.
BBa_K322150: luxAB and lumP under lac promoter.
Characterisation
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References
O'Kane, D. J. & Lee, J. (1985). Chemical characterization of lumazine protein from Photobacterium leiognathi: comparison with lumazine protein from Photobacterium phosphoreum. Biochemistry 24, 1467-1475.
Suadee, C., Nijvipakul, S., et al. (2008). LuxG Is a Functioning Flavin Reductase for Bacterial Luminescence. J. Bacteriol. 190(5): 1531-1538
Edinburgh 2009 team wiki, https://2009.igem.org/Team:Edinburgh.
