Part:BBa_K415505

VgEcR_2A_RxR L1L2 MammoBlock Entry Vector

Figure 1. Schematic of PonS Induction of the EGSH promoter, leading to transcriptional activation.

Mammalian L1L2 Entry Vector containing VgEcR_2A_RxR construct. retinoid X receptor (RxR) and ecdysone receptors (VgEcR) form a heterodimer to control expression of the EGSH promoter; they can be activated by the addition of the PonS compound. Here they are separated by a viral 2A site that allows for bicistronic expression.

Figure 1 describes this process:

1. Hef1a promoter leads to low level, constitutive expression of the two halves (VgEcR and RxR) of a receptor responsive to the chemical ponasterone A (PonS).
2. VgEcR, RxR, and PonS combine to form an active complex.
3. EGSH is induced by the VgECR-RxR-PonS complex.

                                                                                                                                                                                                                                                                                                  

Characterization

Figure 2. Brightfield image of a HEK293FT cell line stably infected with VgEcR_2A_RxR_2A_Hygro.

Figure 2 shows a bright field microscopy image taken of a HEK293FT cell line stably infected with VgEcR_2A_RxR_2A_Hygro, a variant construct (with the same DNA sequence up to the second 2A site as our construct) with a hygromycin selection marker. The marker was added for more efficient selection of the cell line.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        

Figure 3. Transcriptional Upregulation of EGSH in Response to PonS addition in the presence of EgVcR and RxR.

HEK293FT cell lines actively transcribing VgEcR, RxR, and Hygromycin under the low level constitutive promoter hEF1a were transfected with EGSH_EGFP constructs via calcium phosphate transient transfection. The micrographs shown in Figure 3 show characterization data for the cell line in the absence and presence of PonS.

Results

Introduction of PonS significantly upregulates the transcription activity of the EGSH_EGFP construct. This confirms the functionality of the EgVcR and RxR proteins.

                                                                                                                         

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 XbaI site found at 291
  Illegal PstI site found at 2272
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2091
  Illegal PstI site found at 2272
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1585
  Illegal BamHI site found at 486
  Illegal BamHI site found at 2962
  Illegal BamHI site found at 3115
  Illegal BamHI site found at 3229
  Illegal XhoI site found at 1578
  Illegal XhoI site found at 1659
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 291
  Illegal PstI site found at 2272
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 291
  Illegal PstI site found at 2272
  Illegal NgoMIV site found at 1707
  Illegal NgoMIV site found at 2054
  Illegal NgoMIV site found at 3527
  Illegal AgeI site found at 2815
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3000
  Illegal SapI.rc site found at 389
  Illegal SapI.rc site found at 521