Composite

Part:BBa_K364323:Design

Designed by: Endre Károly Kristóf   Group: iGEM10_Debrecen-Hungary   (2010-10-20)
Revision as of 15:28, 22 October 2010 by Balintblaszlo (Talk | contribs) (→‎Source)

Gal4-NHR31


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 678
    Illegal SapI.rc site found at 548


Design Notes

Compatible with RFC-10 and RFC-25.


Source

Arteficial and C. elegans orphan nuclear receptor.

Arteficial TF made of a Gal4 DBD element and C. elegans orphan nuclear receptor LBD

NHR-31

Nuclear hormone receptor family member nhr-31 nhr-31 encodes one of over 200 C. elegans nuclear receptors; nhr-31 activity is required for proper growth, development, and function of the excretory cell; in regulating excretory cell development, NHR-31 appears to function by controlling the expression of genes encoding subunits of the vacuolar ATPase; an nhr-31::gfp promoter fusion is expressed at high levels in the excretory cell beginning at embryogenesis and continuing through adulthood, with lower levels of expression seen in the intestine and unidentified tail cells.

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 (Uper Activating Sequence) 3' of it's promoter. The usual binding sites of reporters, contain a multiple of the 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 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.

References