Difference between revisions of "Part:BBa K1896001"

Latest revision as of 22:02, 21 October 2016

mGFPuv2

Variant of Green Fluorescent Protein (GFP) that contains the following mutations:

• F99S/M153T/V163A: GFPuv or cycle 3 GFP was optimised for excitation by UV light (360-400nm). [1]
• S208L: Increases the fluorescence intensity of GFPuv, resulting in GFPuv2. [2]
• A206K: Monomerizes most GFP variants by disrupting a hydrophobic patch. [3]

Usage and Biology

This combination of GFP mutations has not been reported in the literature. The UGent Belgium 2016 iGEM team used this part as a single protein and as a fusion partner with mSA2 and INP_RC

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  Colonies of E. coli containing BBa_K1896010 imaged under long-wave UV light.

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  Fluorescence spectrum recorded from culture expressing mGFPuv2.

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  Native PAGE analysis confirms that mGFPuv2 is indeed a monomeric protein. Left three bands: GFPuv, rightmost three bands: mGFPuv2.

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  MGFPuv2 can be used in difficult fusion proteins, from left to right: non-fluorescent control, INP_RC-mGFPuv2 (weak promoter), mGFPuv2-streptavidin, mGFPuv2-mSA2, mGFPuv2

Sequence and Features

References

1. von Stetten, D., Noirclerc-Savoye, M., Goedhart, J., Gadella, T. W., & Royant, A. (2012). Structure of a fluorescent protein from Aequorea victoria bearing the obligate-monomer mutation A206K. Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 68(8), 878-882.
2. Ito, Y., Suzuki, M., & Husimi, Y. (1999). A novel mutant of green fluorescent protein with enhanced sensitivity for microanalysis at 488 nm excitation. Biochemical and biophysical research communications, 264(2), 556-560.
3. Crameri, A., Whitehorn, E. A., Tate, E., Stemmer, W. P., Crameri, A., Kitts, P. A., & Kitts, P. A. (1996). Improved green fluorescent protein by molecular evolution using. Nat. Biotechnol, 14(3), 315-319.