Composite

Part:BBa_K364328:Design

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

Gal4-DAF12


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


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)

DAF-12

The nuclear hormone receptor DAF-12 from Caenorhabditis elegans is activated by dafachronic acids, which derive from sterols upon oxidation by DAF-9, a cytochrome P450. DAF-12 activation is a critical checkpoint in C. elegans for acquisition of reproductive competence and for entry into adulthood rather than dauer diapause.

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

Antebi A, Yeh WH, Tait D, Hedgecock EM, Riddle DL. daf-12 encodes a nuclear receptor that regulates the dauer diapause and developmental age in C. elegans. Genes Dev. 2000 Jun 15;14(12):1512-27.

Snow MI, Larsen PL.Structure and expression of daf-12: a nuclear hormone receptor with three isoforms that are involved in development and aging in Caenorhabditis elegans. Biochim Biophys Acta. 2000 Nov 15;1494(1-2):104-16.

Matyash V, Entchev EV, Mende F, Wilsch-Bräuninger M, Thiele C, et al. (2004) Sterol-Derived Hormone(s) Controls Entry into Diapause in Caenorhabditis elegans by Consecutive Activation of DAF-12 and DAF-16. PLoS Biol 2(10): e280.

Motola D.L., Cummins C.L., Rottiers V., Sharma K.K., Li T., Li Y., Suino-Powell K., (...), Mangelsdorf D.J. Identification of Ligands for DAF-12 that Govern Dauer Formation and Reproduction in C. elegans. (2006) Cell, 124 (6), pp. 1209-1223.