Part:BBa_K364322

Gal4-NHR23

Gal4 DBD - NHR 23 C. elagans LBD

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

NHR-23

Nuclear hormone receptor family member nhr-23. The nhr-23 gene encodes a nuclear hormone receptor homolog that is required in all larval molts; NHR-23 is highly similar to Drosophila DHR3, an ecdysone-inducible gene product involved in metamorphosis. The NHR-23 protein is nuclear, and is present in all blastomeres during early embryogenesis; during later stages of morphogenesis, NHR-23 is restricted to epidermal cells. nhr-23 expression cycles between stages of larval development; during each intermolt period, levels of nhr-23 transcripts are 2-5 times greater than levels at each molt. NHR-23 binds the DRS-type hormone response sequence in vitro.

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-NHR4 in pSB1C3 resulting an insert of 944 bp. Circular pSB1C3-RFP, pSB1C3-Gal4-NHR23 and restricted PMAT-NHR23 can be seen as control on the gel.

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 525
  Illegal BsaI.rc site found at 831
  Illegal BsaI.rc site found at 915