Difference between revisions of "Part:BBa K364322:Design"

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===Source===
 
===Source===
  
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Arteficial and C. elegans orphan nuclear receptor
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 +
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]
  
 
===References===
 
===References===
 +
1.Kostrouchova M., Kostrouch Z., Krause M., Rall J.E. "CHR3 genomic sequence." (APR-1996) to the EMBL/GenBank/DDBJ databases
 +
 +
2.Kostrouchova M, Krause M, Kostrouch Z, Rall JE. "Nuclear hormone receptor CHR3 is a critical regulator of all four larval molts of the nematode Caenorhabditis elegans" Natl Acad Sci U S A. 2001 Jun 19;98(13):7360-5

Latest revision as of 22:38, 25 October 2010

Gal4-NHR23


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


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-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]

References

1.Kostrouchova M., Kostrouch Z., Krause M., Rall J.E. "CHR3 genomic sequence." (APR-1996) to the EMBL/GenBank/DDBJ databases

2.Kostrouchova M, Krause M, Kostrouch Z, Rall JE. "Nuclear hormone receptor CHR3 is a critical regulator of all four larval molts of the nematode Caenorhabditis elegans" Natl Acad Sci U S A. 2001 Jun 19;98(13):7360-5