Difference between revisions of "Part:BBa K5477026"

 
 
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<partinfo>BBa_K5477026 short</partinfo>
 
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The pRET2-NCOA composite part consists of the NCOA (Nuclear Receptor Coactivator) gene under the control of the pRET2 promoter, creating a module that enhances transcriptional activation by nuclear receptors like the Aryl hydrocarbon receptor (AhR). NCOA, also known as SRC (Steroid Receptor Coactivator), is a coactivator protein that interacts with activated nuclear receptors to promote transcription of target genes. NCOA achieves this by recruiting histone acetyltransferases (HATs) and other chromatin-remodeling complexes, which open the chromatin structure and allow transcription machinery to access DNA more effectively. In the pRET2-NCOA module, the pRET2 promoter drives the expression of NCOA, ensuring that it is available to function in pathways where enhanced nuclear receptor activity is required.
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The pRET2-NCOA composite part consists of the NCOA (Nuclear Receptor Coactivator) [https://parts.igem.org/Part:BBa_K5477012| BBa_K5477012] gene under the control of the pRET2 promoter [https://parts.igem.org/Part:BBa_K5477000| BBa_K5477000], creating a module that enhances transcriptional activation by nuclear receptors like the Aryl hydrocarbon receptor (AhR) [https://parts.igem.org/Part:BBa_K3793004 | BBa_K3793004]. NCOA, also known as SRC (Steroid Receptor Coactivator), is a coactivator protein that interacts with activated nuclear receptors to promote transcription of target genes. NCOA achieves this by recruiting histone acetyltransferases (HATs) and other chromatin-remodeling complexes, which open the chromatin structure and allow transcription machinery to access DNA more effectively (3) (4). In the pRET2-NCOA module, the pRET2 promoter drives the expression of NCOA, ensuring that it is available to function in pathways where enhanced nuclear receptor activity is required.
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This module supports the AhR-mediated response to toxic ligands like PAHs, dioxins and PCBs by binding to the XRE() in our reporter module facilitating the transcription of NanoLuc () (1) (2). The composite part was cloned using the method of USER-cloning into YCp-W. YCp-W is a centromeric plasmid used in yeast that carries a TRP1 marker, allowing for selection in tryptophan auxotrophic yeast strains. Like other CEN plasmids, YCp-W contains a CEN sequence, ensuring that it replicates and segregates similarly to yeast chromosomes. This low-copy plasmid (typically maintained at one to two copies per cell) provides stable maintenance within yeast cells. This composite part was used in our device:
  
  
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===References===
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1. Carambia, A., Schuran, F.A. The aryl hydrocarbon receptor in liver inflammation. Semin Immunopathol 43, 563–575 (2021). https://doi.org/10.1007/s00281-021-00867-8
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2. Goedtke L, Sprenger H, Hofmann U, Schmidt FF, Hammer HS, Zanger UM, Poetz O, Seidel A, Braeuning A, Hessel-Pras S. Polycyclic Aromatic Hydrocarbons Activate the Aryl Hydrocarbon Receptor and the Constitutive Androstane Receptor to Regulate Xenobiotic Metabolism in Human Liver Cells. Int J Mol Sci. 2020 Dec 31;22(1):372. doi: 10.3390/ijms22010372. PMID: 33396476; PMCID: PMC7796163.
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3. Onate SA, Boonyaratanakornkit V, Spencer TE, et al. The steroid receptor coactivator-1 contains multiple receptor interacting and activation domains that cooperatively enhance the activation function 1 (AF1) and AF2 domains of steroid receptors. J Biol Chem. 1998;273(20):12101-12108. doi:10.1074/jbc.273.20.12101
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4. Oñate SA, Tsai SY, Tsai MJ, O'Malley BW. Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science. 1995;270(5240):1354-1357. doi:10.1126/science.270.5240.1354

Latest revision as of 19:53, 26 September 2024


pRET2-NCOA - receptor module

The pRET2-NCOA composite part consists of the NCOA (Nuclear Receptor Coactivator) BBa_K5477012 gene under the control of the pRET2 promoter BBa_K5477000, creating a module that enhances transcriptional activation by nuclear receptors like the Aryl hydrocarbon receptor (AhR) | BBa_K3793004. NCOA, also known as SRC (Steroid Receptor Coactivator), is a coactivator protein that interacts with activated nuclear receptors to promote transcription of target genes. NCOA achieves this by recruiting histone acetyltransferases (HATs) and other chromatin-remodeling complexes, which open the chromatin structure and allow transcription machinery to access DNA more effectively (3) (4). In the pRET2-NCOA module, the pRET2 promoter drives the expression of NCOA, ensuring that it is available to function in pathways where enhanced nuclear receptor activity is required.

This module supports the AhR-mediated response to toxic ligands like PAHs, dioxins and PCBs by binding to the XRE() in our reporter module facilitating the transcription of NanoLuc () (1) (2). The composite part was cloned using the method of USER-cloning into YCp-W. YCp-W is a centromeric plasmid used in yeast that carries a TRP1 marker, allowing for selection in tryptophan auxotrophic yeast strains. Like other CEN plasmids, YCp-W contains a CEN sequence, ensuring that it replicates and segregates similarly to yeast chromosomes. This low-copy plasmid (typically maintained at one to two copies per cell) provides stable maintenance within yeast cells. This composite part was used in our device:


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1785
    Illegal EcoRI site found at 2407
    Illegal SpeI site found at 1234
    Illegal SpeI site found at 1535
    Illegal SpeI site found at 2602
    Illegal PstI site found at 1294
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1785
    Illegal EcoRI site found at 2407
    Illegal NheI site found at 182
    Illegal NheI site found at 3547
    Illegal SpeI site found at 1234
    Illegal SpeI site found at 1535
    Illegal SpeI site found at 2602
    Illegal PstI site found at 1294
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1785
    Illegal EcoRI site found at 2407
    Illegal BamHI site found at 3049
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1785
    Illegal EcoRI site found at 2407
    Illegal SpeI site found at 1234
    Illegal SpeI site found at 1535
    Illegal SpeI site found at 2602
    Illegal PstI site found at 1294
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1785
    Illegal EcoRI site found at 2407
    Illegal SpeI site found at 1234
    Illegal SpeI site found at 1535
    Illegal SpeI site found at 2602
    Illegal PstI site found at 1294
    Illegal AgeI site found at 37
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 3497
    Illegal SapI.rc site found at 4999


References

1. Carambia, A., Schuran, F.A. The aryl hydrocarbon receptor in liver inflammation. Semin Immunopathol 43, 563–575 (2021). https://doi.org/10.1007/s00281-021-00867-8

2. Goedtke L, Sprenger H, Hofmann U, Schmidt FF, Hammer HS, Zanger UM, Poetz O, Seidel A, Braeuning A, Hessel-Pras S. Polycyclic Aromatic Hydrocarbons Activate the Aryl Hydrocarbon Receptor and the Constitutive Androstane Receptor to Regulate Xenobiotic Metabolism in Human Liver Cells. Int J Mol Sci. 2020 Dec 31;22(1):372. doi: 10.3390/ijms22010372. PMID: 33396476; PMCID: PMC7796163.

3. Onate SA, Boonyaratanakornkit V, Spencer TE, et al. The steroid receptor coactivator-1 contains multiple receptor interacting and activation domains that cooperatively enhance the activation function 1 (AF1) and AF2 domains of steroid receptors. J Biol Chem. 1998;273(20):12101-12108. doi:10.1074/jbc.273.20.12101

4. Oñate SA, Tsai SY, Tsai MJ, O'Malley BW. Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science. 1995;270(5240):1354-1357. doi:10.1126/science.270.5240.1354