Plasmid

Part:BBa_K364332

Designed by: Endre Károly Kristóf   Group: iGEM10_Debrecen-Hungary   (2010-10-20)
Revision as of 17:21, 25 October 2010 by Mrtwister (Talk | contribs)

TRE-Gal4-PXR-PolyA in PSB1A3 (expression vector)

Expression vector from standard BioBrick parts, expressing Gal4 - human Pregnane X receptor LBD composite part.

The Gal4-PXR Chimeric nuclear receptor is an artificial eukaryotic TF made of Gal4 DBD (DNA Binding Domain) and H. sapiens nuclear hormone receptor LBD (Ligand Binding Domain) which can be expressed by this expression vector.

The minimal CMV promoter ensures a continuous expression of the chimeric construct, but furthermore it can be expressed in an inducible form using the Tetracyclin-controlled transcriptional activation system.

This composite artificial transcription factor will activate any reporter or any gene in general that has a UAS (Upper Activating Sequence) 3' of it's promoter. The usual binding sites of reporters, contain multiple UAS elements. In order to have a POPS output, the LBD has to recruit activators in the cell. This can be initiated by ligand binding or by recruiting a protein that has a fused strong activator like the VP activator.

With this system NHR (Nuclear Hormone Receptor) ligands or NHR interacting partners can be screened.

The NHR: cofactor-VP interaction should be also broken by a potential ligand binding, this is why this setup is also suitable for ligand identification. The benefit of the cofactor-VP interaction test is that the dynamic range of the assay is much higher than the dynamic range of the normal Gal4-NHR ligand activation assay.

More info about this project on the wiki pages of Team Debrecen-Hungary 2010. [http://2010.igem.org/Team:Debrecen-Hungary]

Picture of gel electrophoresis: TRE-Gal4-PXR-PolyA in pSB1A3 results a new expression vector from standard Biobrick parts. The TRE-Gal4-PXR LBD-PolyA insert is 1682 bp long, without polyA which was inserted in the last step is 191 bp shorter.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 912
    Illegal BglII site found at 1023
    Illegal BamHI site found at 1446
    Illegal XhoI site found at 547
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 466


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Categories
Parameters
n/aTRE-Gal4-PXR-PolyA in PSB1A3 (expression vector)