Composite

Part:BBa_K364324:Design

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

Gal4-NHR80


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


Design Notes

Compatible with RFC-10 and RFC-25.


Source

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

NHR-80

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. nhr-80 is specific to nematodes, being a divergent ortholog of HNF4 with many paralogs in C. elegans Artificial eukaryotic TF made of Gal4 DBD (DNA Binding Domain) and C. elegans orphan nuclear receptor LBD (Ligand Binding Domain)

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]

References

1. Miyabayashi T, Palfreyman MT, Sluder AE, Slack F, Sengupta P. "Expression and function of members of a divergent nuclear receptor family in Caenorhabditis elegans." Dev Biol. 1999 Nov 15;215(2):314-31.