Part:BBa_K2520006

TRE-GFP-hGH

This part is an improvement of BBa_K1119008. This device allows inducible expression of GFP within mammalian cells. The TRE promoter is made up of Tet operator (TetO) sequence concatemers fused to a minimal hCMV. This promoter can be used for inducible expression of the downstream gene in two different schemes.

Tet-off

In the absence of Dox, tTA binds to the TRE promoter and activates transcription of the downstream gene. In the presence of Dox, tTA cannot bind to the TRE, and expression from the target gene remains minimal.

Figure 1: tet off system

Tet-on

In the presence of Dox, rtTA binds the TRE promoter and activates transcription of the downstream gene. In the absence of Dox, rtTA cannot bind the TRE promoter, and expression of the downstream gene is minimal.

Usage and Biology

In this device, the inducible protein is GFP, and can be used easily for calibration and characterization of inducible systems. The human growth hormone (hGH) terminator has been added to allow for efficient expression of the GFP.

Characterization

A demonstration of Tet-off system is shown below (figure 1). We co-transfected HEK-293 cells with two plasmids: BBa_K2520006 - TRE-GFP-hGH and BBa_K2520009 - CMV-tTA-hGH. The transfected cells expressed different levels of GFP depending on Doxycycline concentration, due to the binding of tTA to the TRE promoter. We set a negative controls: co-transfection of TRE-GFP-hGH and pUC19 - for measurement of the basal level of GFP expression without the binding of tTA to the TRE promoter.

Figure 2: Weighted median of cells expressing GFP depending on different concentrations of Doxycyline

Figure 3: Fluorescence intensity of cells expressing GFP in different concentrations of Doxycyline

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 site found at 1438
  Illegal BsaI.rc site found at 967