Part:BBa_K415503

Figure 1. Schematic depicting the schematic of the TREt system.

EYFP_2a_rtta3_ff5 L1L2 MammoBlock Entry Vector

EYFP_2a_rtTa3_FF5 is a construct containing two proteins - EYFP fluorescent protein, and rtTA3 (a DOX-inducible transcription factor that regulates the TRE promoter) seperated by a viral 2A sequence that allows for bicistronic expression of the two genes. FF5 is a micro-RNA controlled degradation tag. This construct is part of the 2010 iGEM mammalian team toggle switch. The basic TREt system, which is utilized in the toggle, is depicted in Figure 1.

Characterization

Figure 2. Expression of EGSH_EYFP_2a_rtTa3_FF5 Construct Post Ponasterone S Induction in HEK293FT Cells.

Expression of EGSH_EYFP_2a_rtTa3_FF5 Construct Post Ponasterone S Induction in HEK293FT Cells

The EGSH_EYFP_2a_rtTa3_FF construct was introduced into a HEK293FT cell line constitutively expressing VgEcR and RxR via calcium phosphate transient transfection. Fluorescent and brightfield micrographs were obtained 41 and 48 hours post transfection. PonS addition occured at 14 hours.

Figure 3. Comparison of TREt and EGSH reporter constructs.

Comparison of TREt and EGSH Promoter Constructs

In Figure 3, the inducibility of TREt and another inducible promoter, EGSH (Link:[1]), are compared. HEK293 FT cells were infected with reporter constructs for each system. The EGSH system is inducible with ponasterone, and expresses EGFP when induced. The TREt system controls expression of EYFP. Addition of DOX leads to activation of rtTA3, which then induces TREt_EYFP. Controls without inducing chemical factors are shown for both systems. For the EGSH system, (A) indicates the absence and (B) indicates the presence of ponesterone. For the TREt system, (C) indicate the absence and (D) indicates the presence of DOX. Scale bars (red) are 100 μm.

Results

DOX addition led to activation of rtTA3 and dramatic increase in transcriptional activity. This makes the TREt system ideal for inducible, high level expression of proteins, especially lethal or harmful proteins that must be tightly regulated. The TREt system is also widely used in synthetic systems for its rigorous positive feedback loop.

See also: TREt promoter [2].

Applications

The MIT iGEM 2010 team used this part to produce one of the few existing toggles in mammalian systems. A circuit diagram of this toggle is shown below, along with characterization data.

Figure 1. Circuit diagram of bistable toggle.

Our toggle involves a positive feedback loop between rtTA3+DOX and the promoter TREt. Addition of PonS into the system leads to the activation of EGSH, which then subsequently activates the positive feedback loop, propelling the system into a high output state (Figure 1).

Our system is bistable at a wide range of DOX levels. Figure 2 shows a rate plot (dX/dt vs. X) for rtTA3, where the time lapse dispalys the effect of increasing DOX levels on the system. The system is bistable when three intercepts occur on the ordinate, corresponding to a wide range of DOX levels. However, at high DOX levels the system becomes constitutively high/high for -PonS/+PonS.

Triple calcium phosphate transfections were performed on HEK293FT hEF1a_RxR_VgECR cell lines with constructs of our toggle: EGSH_rtTA3 and TREt_EYFP_rtTA3, as well as hEF1a_mKate serving as a fluorescent transfection efficiency control. Micrographs were obtained at 26 hours post transfection. The mKate fluorescence was converted to a binary mask. This mask was then applied to the EYFP fluorescence micrograph and pixel intensities were calculated. Figures 3 and 4 correspond to our sensitivity analysis performed in Figure 2. Qualitative data can be reviewed in figure 5.

Figure 3. Effect of -/+ PonS on the system under high DOX levels.

Figure 4. Effect of -/+ PonS on the system under low DOX levels.

Figure 5. Fluorescent micrographs showing -/+ PonS for: (left) overlay of mKate and EYFP fluorescence indicating both transfection efficiency and toggle output; (right) EYFP levels.

                                                                                                                                                                                                                                                                                                  

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1666
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  Illegal XbaI site found at 1709
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• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1666
  Illegal NheI site found at 804
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  Illegal PstI site found at 1671
  Illegal NotI site found at 1560
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1666
  Illegal BamHI site found at 1697
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1666
  Illegal XbaI site found at 816
  Illegal XbaI site found at 1709
  Illegal PstI site found at 225
  Illegal PstI site found at 1671
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1666
  Illegal XbaI site found at 816
  Illegal XbaI site found at 1709
  Illegal PstI site found at 225
  Illegal PstI site found at 1671
  Illegal NgoMIV site found at 774
  Illegal NgoMIV site found at 1433
  Illegal AgeI site found at 8
  
• 1000
  COMPATIBLE WITH RFC[1000]