Part:BBa_K415507

pEGSH L4R1 MammoBlock Entry Vector

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

Schematic: Response of K415507 to the addition of PonS, in the presence of VgEcR and RxR.

L4R1 MammoBlock entry vector. EGSH is an inducible promoter. In the presence of the proteins retinoid X receptor (RxR) and ecdysone receptors (VgEcR), the addition of ponasterone S (PonS) leads to complex formation of RxR, VgEcR, and PonS. This activated complex is then capable of inducing the EGSH promoter and allows for dramatic upregulation in transcriptional activity.

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 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. EGSH_EGFP is shown to have very low basal expression rate and high fold change in transcription post PonS induction. This property making it ideal for use in sensitive toggles, expression of lethal or harmful proteins, as well as many other applications in genetic engineering and synthetic biology.

                                                                                                                                                                                                                        

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 6
  Illegal EcoRI site found at 671
  Illegal PstI site found at 485
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 671
  Illegal PstI site found at 485
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 671
  Illegal BglII site found at 115
  Illegal BamHI site found at 663
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 671
  Illegal PstI site found at 485
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 671
  Illegal PstI site found at 485
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 646