Team:Edinburgh/Bacterial/Red light producer

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Revision as of 11:12, 12 August 2010








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 different mutants:

  • Substitution mutant S248T
  • Insertion mutant 356K
  • Insertion mutant 356R





Emission spectra of the P. pyralis luciferase mutants 356R (1) and 356K (2).



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 S248T mutants which had been spun down and resuspended to give a higher concentration of cells. When measuered in the luminometer, 356R and S248T 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.





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 (if it ever works) with something which 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 either used or submitted.



Biobricks


This will be one of the parts that we submit to the registry this year. We need to change the promoter we're using, characterise the light output fully and insert it into pSB1C3 before it can be shipped.



References


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.