Coding

Part:BBa_K415504

Designed by: Laura Deming, Joy Jiao, Adrian Slusarczyk   Group: iGEM10_MIT   (2010-10-27)
Revision as of 04:05, 29 October 2010 by Joy (Talk | contribs)

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.
Comparison of TREt and EGSH Promoter 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].

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal EcoRI site found at 1372
    Illegal EcoRI site found at 2178
    Illegal XbaI site found at 30
    Illegal PstI site found at 1462
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal EcoRI site found at 1372
    Illegal EcoRI site found at 2178
    Illegal PstI site found at 1462
    Illegal NotI site found at 2170
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal EcoRI site found at 1372
    Illegal EcoRI site found at 2178
    Illegal BamHI site found at 339
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal EcoRI site found at 1372
    Illegal EcoRI site found at 2178
    Illegal XbaI site found at 30
    Illegal PstI site found at 1462
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal EcoRI site found at 1372
    Illegal EcoRI site found at 2178
    Illegal XbaI site found at 30
    Illegal PstI site found at 1462
    Illegal AgeI site found at 351
  • 1000
    COMPATIBLE WITH RFC[1000]


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