Coding

Part:BBa_K1680006

Designed by: Katharina Sporbeck   Group: iGEM15_Tuebingen   (2015-09-17)

Fluorescent protein dronpa

This part contains the protein coding sequence for a mutant (K145N) of the fluorescent protein Dronpa and is codon optimised for yeast. Dronpa is a photoswitchable protein. Fluorescence can be turned off by illumination with 488 nm light and switched back on by illumination with 405 nm. The K145N mutation leads to photoswitchable monomerisation/multimerisation behaviour (as described by Zhou et al. 2012). The part is supplied in RFC25. Fluorescence microscopy showed that the expression of this part works and photoswitching can be observed as expected.


Team Tuebingen microscpy-control.png Team Tuebingen microscpy-dronpa.png

Fluorescence, brightfield and overlay pictures of Saccharomyces cerevisiae wild type cells (top) and cells expressing dronpa from the pTUM104 plasmid (Part:BBa_K801004) grown overnight in SC medium containing 2% galactose.


Because Dronpa is a fluorescent protein that shows photo switching capabilities, it can be turned of by illumination at 488nm and turned back on by illumination at 405 nm. We were able to show this by illuminating dronpa expressing cells at the microscope.

Montage dronpa onoff scale3um.png

Fluorescence and brightfield overlay pictures of Dronpa expressing cells after consecutive illumination with 488nm and 405nm light. The scale bar represent 3 µm.

Usage and Biology

Dronpa is a fluorescent protein, that is an engineered version of a weakly fluorescent wild type protein occuring in Pectiniidae, a species of coral. (Ando et al. 2004)

An Ecoli optimized version of this part ia available here and a mutant version that shows better performance in TetR and β-galactosidase activity control is available here

Sequence and Features

This part is submitted in pSB1C3 with RFC25 standard. The part only sequence does therefore not contain a start and stop codon.

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 268
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//cds/reporter/chromoprotein
//cds/reporter/gfp
//chassis/eukaryote/yeast
//function/reporter/fluorescence
Parameters
colorGreen