Difference between revisions of "Part:BBa K415504"

Revision as of 03:55, 29 October 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 1. Comparison of TREt and EGSH reporter constructs.

In Figure 1, 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].

Sequence and Features