Difference between revisions of "Part:BBa K415504"

Latest revision as of 10:45, 8 November 2010

Rtta3_2a_Hygro L1L2 MammoBlock Entry Vector

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

L1L2 Mammoblock entry vector containing the Rtta3_2a_hygro construct. rtTA3 is a DOX-inducible transcription factor controlling expression of the TRE promoter; Hygro is a selection marker against the Hygromycin antibiotic used to select for stable mammalian cell lines. They are separated by the viral 2A sequence which allows for polycistronic expression of genes on the same mRNA molecule. The basic TREt system is depicted in Figure 1.

Characterization

Figure 2. Comparison of TREt and EGSH reporter constructs.

In Figure 2, 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

Negative control experienced very low level of basal transcription, or "leaky" expression. DOX addition led to 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