DNA

Part:BBa_K2100004

Designed by: Kathleen Brandes   Group: iGEM16_MIT   (2016-10-10)


pENTR pTRE

This construct is an entry vector containing the TRE promoter. The promoter is structured as six tetO sites upstream of a minimal CMV promoter. The induction of doxycycline activates a tetracycline transactivator protein which in turn binds to the tetO sites to initiate transcription of the gene.


Sequence and Features

Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal XbaI site found at 30
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal XbaI site found at 30
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal XbaI site found at 30
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal XbaI site found at 30

We characterized our TRE promoter induced with a fine sweep of Doxycycline from a concentration of 0 uM to 2000 uM. We transfected HEK293 cells with 250ng of TRE-mKate and 250 ng hEF1a-eYFP. We analyzed fluorescent output in the PE Texas Red channel to measure the activity of the TRE promoter under different DOX concentrations. We used 0 uM of DOX as a control.

T--MIT--TRE.png

The results indicate the optimal DOX concentration was 500 uM for maximum TRE activity. Beyond that point, adding a higher concentration of DOX does not significantly increase fluorescent output from the TRE-mKate construct.


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Categories
//awards/part_collection/2016
Parameters
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