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Team:Edinburgh/Bacterial/Green light producer
From 2010.igem.org
Overview: The green light producer
For the production of green light, we relied on tried and trusted firefly luciferase from P. pyralis. This has a recorded luminescence emission peak of roughly 557nm, as recorded in Shapiro et al. (2009) and other references (Figure 1). This part was originally submitted by Ljubljana 2007. The sequence analysis revealed that there were 6 bases inserted upstream of the coding sequence. The distance between the ribosome binding site and the start codon was thus not optimal, and this resulted in a decreased luminescence intensity of the cells. We removed these 6 bases by PCR and resubmitted the part in pSB1C3.
Figure 1: Emission spectra of the P. pyralis luciferase (in solid black line).
Image: Shapiro et al. (2009)
In addition to the above, we created a codon-optimised mutant of the firefly luciferase, reported by Fujii et al. (2007) to be 12.5 times brighter than the wildtype. This may help to alleviate the problems foreseen with a lack of luminescence intensity failing to activate the light sensors of the corresponding wavelength.
An alternative approach involved the combination of LuxAB with the production of another protein: YFP from Vibrio fischeri. This will shift the wavelength of the blue light produced by LuxAB towards the yellow spectrum, making it the correct colour to activate a green light sensor. We expect our main collaborators, UNAM-Genomics Mexico to synthesise this DNA sequence and combine it with LuxAB. We also expect UNAM-Genomics to BioBrick it as part of their submission to the Registry.
Strategy
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Problems
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BioBricks
Firefly luciferase was originally deposited in the Registry by Ljubljana 2007. We modified this slightly and re-submitted in pSB1C3, along with a codon-optimised mutant and a simple reporter system.
BBa_K322237: firefly luciferase from Photinus pyralis, modified BBa_I712019.
BBa_K322238: firefly luciferase from Photinus pyralis under lac promoter.
BBa_K322451: firefly luciferase from Photinus pyralis, codon optimised and mutated for brighter bioluminescence.
Characterisation
Figure 2: Results of spectrum analysis of our wildtype firefly luciferase.
Figure 2 shows the results of the spectral analysis of our wildtype firefly luciferase BBa_K322237. Unlike the emission spectrum for our red-light producing S248T variant, the spectrum differs greatly from the expected; there is a small peak at roughly the correct location (557nm), but also a shoulder at approximately 580nm and a further peak at 600nm. Given that the difference between the aforementioned mutant and this wildtype is very noticeable to the naked eye, it is difficult to pinpoint the cause of this discrepancy; we hope to be able to re-run the analysis in an attempt to make sense of it.
References
Fujii, H., Noda, K., Asami, Y., Kuroda, A., Sakata, M. & Tokida, A. (2007). Increase in bioluminescence intensity of firefly luciferase using genetic modification. Analytical Biochemistry 366, 131-136.
Shapirol, E., Lu, C., Baneyx, F. (2009). Design and characterization of novel trypsin-resistant firefly luciferases by site-directed mutagenesis. Protein Eng Des Sel 22(11): 655-663.
Ljubljana 2007 team wiki, http://parts.mit.edu/igem07/index.php/Ljubljana.
