Difference between revisions of "Part:BBa K364320: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-8 LBD
 
NHR-8 LBD
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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 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.  
  
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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.  
+
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.  
  
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With this system NHR-8 ligands or NHR-8 interacting partners can be screened.  
+
With this system NHR (Nuclear Hormone Receptor) ligands or NHR interacting partners can be screened.  
  
−
The NHR-8: 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.
+
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.
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 +
More info about this project on the wiki pages of Team Debrecen-Hungary 2010. [http://2010.igem.org/Team:Debrecen-Hungary]
  
 
===References===
 
===References===
 +
1. Lindblom TH, Pierce GJ, Sluder AE,"A C. elegans orphan nuclear receptor contributes to xenobiotic resistance" Curr Biol. 2001 Jun 5;11(11):864-8.
 +
 +
2. T. H. Lindblom, A. E. Sluder. "nhr-8 is Required for Gut Function and May Respond to the Food Signal". International C. elegans Meeting (1999).

Latest revision as of 22:56, 25 October 2010

Gal4-NHR8


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


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-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]

References

1. Lindblom TH, Pierce GJ, Sluder AE,"A C. elegans orphan nuclear receptor contributes to xenobiotic resistance" Curr Biol. 2001 Jun 5;11(11):864-8.

2. T. H. Lindblom, A. E. Sluder. "nhr-8 is Required for Gut Function and May Respond to the Food Signal". International C. elegans Meeting (1999).