Coding

Part:BBa_K1896001

Designed by: Bob Van Hove, Maarten Van Brempt   Group: iGEM16_UGent_Belgium   (2016-10-12)


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 INPRC

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

  • Fluorescence spectrum recorded from culture expressing mGFPuv2.

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

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


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

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.


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