Part:BBa_K779604

Hef1a-rTTA3 MammoBlock Device

This is a composite part with a Hef1A constitutive promoter with rtTA3, which is a tetracycline-analogue-inducible transactivator. We created this part through the gateway cloning protocol
Testing Hef1A:rtTA3:

The circuit that MIT iGEM 2012 tested:

In this circuit, a constitutive mammalian promoter, Hef1A, drives expression of a trans-activator, rtTA3. rtTA3 activates the Tre-Tight promoter in the prescence of doxycycline (DOX), a small molecule. Tre-Tight drives expression of mKate, a red fluorescent protein. In addition, a plasmid containing the Hef1A promoter driving expression of TagBFP, a blue fluorescent protein, is used as a transfection marker.


Experimental Data:
In this system, we have constitutive expression of rtTA3 driven by the Hef1A promoter. In the presence of doxycycline (DOX), rtTA3 can activate the Tre-Tight (TreT) promoter, which is driving expression of mKate, a red fluorescent protein.

100,000 HEK293 cells were transfected with equimolar rations of Hef1a:rTTA3, TreT:mKate, and Hef1a:TagBFP (as a transfection marker), standardized to 500 ng total plasmid DNA, with 1.65 uL Lipofectamine 2000. After 48 hours, cells were imaged on a Leica Confocal Microscope at 10x. We can see that we have transfection, since cells are fluorescing blue. Also, we can see that as we increase the concentration of DOX present, we see an increase in red fluorescence.


100,000 HEK293 cells were transfected with equimolar rations of Hef1a:rTTA3, TreT:mKate, and Hef1a:TagBFP (as a transfection marker), standardized to 500 ng total plasmid DNA, with 1.65 uL Lipofectamine 2000. After 24 hours, DOX was then added to 16 different concentrations ranging from 0.1 nM to 5000 nM. Lastly, cells were harvested for flow cytometry after 48 hrs and allowed to count 10,000 events. As we increase concentrations of DOX, the mean red fluorescence increases.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 315
  Illegal PstI site found at 820
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 315
  Illegal PstI site found at 820
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 569
  Illegal XhoI site found at 968
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 315
  Illegal PstI site found at 820
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 315
  Illegal PstI site found at 820
  Illegal NgoMIV site found at 703
  Illegal AgeI site found at 81
  
• 1000
  COMPATIBLE WITH RFC[1000]