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Team:Edinburgh/Bacterial/Red light producer
From 2010.igem.org
Overview: The red light producer
Firefly luciferase (EC 1.13.12.7) from Photinus pyralis is one of the most efficient bioluminescent proteins known. Its emission peak is about 557nm at pH 7.8 (this is the ordinary internal pH of E. coli during growth). We attempted to produce a mutant luciferase which would produce red light, in order to activate the red light sensor part (which responds optimally to 660nm light).
Previous works such as Branchini et al. (2007) and Moradi et al. (2009) have already identified several luciferase mutants that produce red light. We used site-directed mutagenesis on the wild type to produce three different red light mutants, the emission spectra of which are shown in Figure 1 and Figure 2:
- Substitution mutant S284T
- Insertion mutant 356K
- Insertion mutant 356R
Figure 1: Emission spectra of the P. pyralis luciferase mutant S284T.
Image: Branchini et al. (2007)
Figure 2: Emission spectra of the P. pyralis luciferase mutants 356R (1) and 356K (2).
Image: Moradi et al. (2009)
Strategy
As stated above, we used site-directed mutagenesis on the wild type to produce three different red light mutants. We successfully produced two red mutants of the firefly luciferase: mutants 356K and S284T. Both of these glow a nice red colour, but S284T glows much brighter and should be used for work where red coloured bioluminescence is required. Measurements of bioluminescence / OD showed that the S284T luciferase glows at about 35% of the brightness of the wild type green one. The 356K luciferase glows significantly less and is very hard to see, even in a dark room.
Problems
This part is one of the only ones with no major setbacks. The main problem will be attempting to activate the red light sensor with something which might not be bright enough. We did not have time to attempt this over the summer. If this does not work, it would be interesting to see if one can combine the codon optimised green luciferase which has been mutated for increased brightness with the mutations for red light.
Another evident problem is that the luciferin necessary for luciferase activity cannot yet be produced within an E. coli chassis, and thus needs to be added externally. This is mitigated somewhat by the development of BioBricked luciferin-recycling enzymes by Cambridge 2010. The genes required for not known, but when they are discovered their addition to this system would improve it greatly.
BioBricks
Our BioBricks for this component of the project consist of the two successfully mutated luciferases S284T and 356K, and their composite constructs.
BBa_K322246: firefly luciferase from Photinus pyralis, S284T mutant.
BBa_K322211: firefly luciferase from Photinus pyralis, 356K mutant.
BBa_K322247: S284T mutant luciferase under lac promoter
BBa_K322212: 356K mutant luciferase under lac promoter
Characterisation
Figure 3: Results of spectrum analysis of our S284T mutated firefly luciferase, BioBricked as BBa_K322246.
Figure 3 shows the results of the spectral analysis of the S284T mutated firefly luciferase BBa_K322246. The emission spectrum is very close to that shown in the literature (Figure 1), which proves that our mutations have been successful.
Two major points that need to be emphasised when using this BioBrick are the temperature sensitivity of the luciferase, and its pH sensitivity. During the first part of the project, the cells were grown at 37C. When we tested growing them at 30C, the temperature sensitivity of the protein became evident, since the cells were a lot brighter. Rather than waiting 10 minutes in the dark room to get our eyes accustomed, they were visible before the door was closed.
pH sensitivity of the Photinus pyralis luciferase has been reported previously (Seliger and McElroy, 1964). The cells were usually suspended in citrate buffer, pH 4.8, as this allows the luciferin to enter the cells faster. This has an effect on the colour emitted, though not as marked as for the wildtype.
References
Branchini, B. R., Southworth, T. L., Khattak, N. F., Michelini, E. & Roda, A. (2005). Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications. Analytical Biochemistry 345, 140-148.
Branchini, B. R., Ablamsky, D. M., Murtiashaw, M. H., Uzasci, L., Fraga, H. & Southworth, T. L. (2007). Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications. Analytical Biochemistry 361, 253-262.
Moradi, A., Hosseinkhani, S., Naderi-Manesh, H., Sadeghizadeh, M. & Alipour, B. S. (2009). Effect of Charge Distribution in a Flexible Loop on the Bioluminescence Color of Firefly Luciferases Biochemistry 48, 575-582.
Seliger, H. H., & McElroy, W. D. (1964). The Colors of Firefly Bioluminescence: Enzyme Configuration and Species Specificity. PNAS 52 (1) 75-81
