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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).
We used site-directed mutagenesis on the wild type to produce three different red light mutants:
- 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
Our site-directed mutagenesis and subsequent ligations and transformations produced cells which glowed green (wild type) and, wait for it... RED!! The two brightest red cultures were the 356R and S284T mutants which had been spun down and resuspended to give a higher concentration of cells. When measured in the luminometer, 356R and S284T gave readings of 3,689,393 RLU (relative luminescence units) and 4,060,513 RLU respectively. The wild type negative control gave a reading of 3,066,703 RLU, lower than both mutants.
The sequence of 356R shows that the insertion mutagenesis failed. Any difference in the colour of light seen was most likely misperception on behalf of the team members analysing the cultures in the dark room. One may even be inclined to believe it was wishful thinking. Alas, these qualitative assays are tricky things.
S284T also does not have an altered base in its sequence. This is quite confusing, since firstly, it was brighter than the wild type and secondly, several people confirmed a colour change.
356K, which we did not measure as it did not seem red enough or bright enough, is, as seen in the sequence data, an insertion mutant. We will have to retransform this and test it again with luciferin, then measure its luminosity, also remembering in future to take measurements of everything, even if we think they haven't worked.
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 that is not necessarily bright enough. RLUs are an arbitrary measurement and do not give a good enough indication of actual brightness for us to come to a conclusion on this yet. More characterisation is needed before this part can be used.
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 2: Results of spectrum analysis of our S284T mutated firefly luciferase.
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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.
