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.