Composite

Part:BBa_K569014

Designed by: David Golynskiy & Tyler Guinn   Group: iGEM11_UT_Dallas   (2011-09-27)

SCP+ToxR+FGFR

This part constitutively produces the ToxR+FGFR fusion protein. ToxR+FGFR dimerizes in response to FGF and can then activate the ctx promoter from the bacteria Vibrio cholera. ToxR by itself is a transcription factor, while FGFR is a immune system sensor.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1266
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 844
    Illegal NgoMIV site found at 1435
  • 1000
    COMPATIBLE WITH RFC[1000]


Preliminary Experiments

We transformed SCP+ToxR+FGFR (BBa_K569014) and ctx+GFP (BBa_J07011) into DH5a E.coli cells using two antibiotics: chloramphenicol and kanamycin. We selected a single colony and grew it in LB with chloramphenicol and kanamycin overnight at 37C and 220rpm. We made a 1:20 dilution of the sample and then grew it to a predetermined O.D. When the O.D. was reached induced with various amounts of heparin and fibroblast growth factor (FGF)and let it grow. We took measurements using fluorescence microscopy.

All images were taken with Olympus IX81 automated inverted microscope specially equipped for live cell imaging. The filter set we used is: 470/40x (excitation) and 525/50m (emission) for GFP. Data collection and processing was performed by the SlideBook software.


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