Difference between revisions of "Part:BBa K364323: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)
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NHR-31
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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.
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Gal4 DBD
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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.
 +
 
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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.
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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===
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1. Hahn-Windgassen A, Van Gilst MR."The Caenorhabditis elegans HNF4alpha Homolog, NHR-31, mediates excretory tube growth and function through coordinate regulation of the vacuolar ATPase." PLoS Genet.2009 Jul;5(7):e1000553. Epub 2009 Jul 10.

Latest revision as of 22:43, 25 October 2010

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

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

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 (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. Hahn-Windgassen A, Van Gilst MR."The Caenorhabditis elegans HNF4alpha Homolog, NHR-31, mediates excretory tube growth and function through coordinate regulation of the vacuolar ATPase." PLoS Genet.2009 Jul;5(7):e1000553. Epub 2009 Jul 10.