Composite

Part:BBa_K569013

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

PyeaR+ToxR+FGFR

PyeaR is a nitrate inducible promoter. ToxR+FGFR is a receptor fusion of two dimerizing proteins. FGFR responds to FGF by dimerizing; this causes ToxR to dimerize. When ToxR is dimerized it acts as a transcription factor for the ctx promoter.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1331
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 909
    Illegal NgoMIV site found at 1500
  • 1000
    COMPATIBLE WITH RFC[1000]



Preliminary Experiments

We transformed PyeaR+ToxR+FGFR (BBa_K569013) 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 we added potassium nitrate to a 10mM concentration and allowed the sample to grow. After 2 hours we 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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