Part:BBa_K779607

Tre-Tight-mKate MammoBlock Device

This device consists of the TreT promoter and mKate, a red fluorescent protein. With rTTA3 and different concentrations of doxycycline, we can modulate the mKate expression.

<img src=""> 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.

To move from transfecting RNA into cells to transcribing RNA in vivo, we needed to control the expression of RNA, whether mRNA or short non-coding RNAs used in strand-displacement circuits. We achieved this by using inducible expression systems.

To characterize these systems, we constructed a system where an inducible promoter drives the expression of a fluorescent protein, which allows for quantification of the protein expression levels. Protein expression in turn informs us of mRNA levels.

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. <img src='https://static.igem.org/mediawiki/2012/b/bf/DOXCURVEpics.png' style="width: 575px"/>
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. </p>

"'"
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
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 193
  Illegal BsaI.rc site found at 859
  Illegal SapI.rc site found at 241