Composite

Part:BBa_K364320

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

Gal4-NHR8

Gal4 DBD - NHR 8 C. elagans LBD

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

NHR-8 LBD

Nuclear hormone receptor family member nhr-8.

The nhr-8 gene encodes a nuclear hormone receptor homolog; nhr-8 (ok186) mutants have abnormally low resistance to the toxins colchicine and chloroquine. NHR-8 functions in the nematode xenobiotic defense system.

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-NHR8 in pSB1C3 resulting an insert of 953 bp. Circular pSB1C3-RFP, pSB1C3-Gal4-NHR8 and restricted PMAT-NHR8 can be seen as control on the gel.
Activation of Gal-NHR8 and Gal-NHR64 to oil sand extractions.
Activation of NHR8 to Blesse bleu cheese extract.

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 SapI.rc site found at 545


[edit]
Categories
Parameters
n/aGal4-NHR8