Difference between revisions of "Part:BBa K364322"

 
 
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<partinfo>BBa_K364322 short</partinfo>
 
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Gal4 DBD - NHR 23 C. elagans LBD
 
Gal4 DBD - NHR 23 C. elagans LBD
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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-23
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Nuclear hormone receptor family member 23.
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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. The expression of nhr-23 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. Furthermore NHR-23 binds the DRS-type hormone response sequence in vitro.
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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.
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With this system NHR (Nuclear Hormone Receptor) ligands or NHR interacting partners can be screened.
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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]
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[[Image:pSB1C3-Gal4-NHR23.gif|800px|thumb|center|Picture of gel electrophoresis: Gal4-NHR23 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.]]
  
 
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Latest revision as of 13:55, 25 October 2010

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 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. The expression of nhr-23 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. Furthermore 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-NHR23 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