Composite

Part:BBa_K364324

Designed by: Endre Károly Kristóf   Group: iGEM10_Debrecen-Hungary   (2010-10-20)

Gal4-NHR80

Gal4 DBD - NHR 80 C. elagans LBD


Artificial eukaryotic TF made of Gal4 DBD (DNA Binding Domain) and C. elegans orphan nuclear receptor LBD (Ligand Binding Domain)

NHR-80

Nuclear hormone receptor family member nhr-80.

The nhr-80 encodes a nuclear hormone receptor, expressed in the intestine, that regulates expression of the delta-9 desaturases FAT-5/-6/-7, and thus regulates fatty acid metabolism. The nhr-80 is specific to nematodes, being a divergent ortholog of HNF4 with many paralogs in C. elegans.

Gal4 DBD

This protein is a positive regulator for the gene expression of the galactose-induced genes such as GAL1, GAL2, GAL7, GAL10, and MEL1 which encode for the enzymes used to convert galactose to glucose. This protein contains a fungal Zn(2)-Cys(6) binuclear cluster domain.

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: Gal4-NHR80 in pSB1C3 resulting an insert of 995 bp.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 218
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 137
    Illegal BsaI.rc site found at 681


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