Difference between revisions of "Part:BBa K1590007"

 
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<partinfo>BBa_K1590007 short</partinfo>
 
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Human Odorant Binding Protein 2A is ...
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Human Odorant Binding Protein 2A is a 155 amino acid (excluding the signal peptide) lipocalin of relatively low molecular weight (19318 Daltons). Structurally it forms an 8 sheet beta barrel flanked by a c-terminal alpha helix that  together forms an internal hydrophobic pore known as a calix. It is secreted by the olfactory epithelial cells of the nose where it lies in high abundance within nasal mucus. Its primary function in the human body is believed to be in the transport of hydrophobic odorant proteins across the otherwise impenetrable aqueous mucus layer to the olfactory receptors of the nose. Due to its high specificity and abundance within nasal mucus, OBP2A was selected as the protein for use in nasal mucus detection.
  
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===Usage and Biology===
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1590007 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1590007 SequenceAndFeatures</partinfo>
  
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===Usage and Biology===
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We aimed to separate the alpha helix c-terminal tail of OBP2A and use that as the substrate for fluorescent nanobead attachment. How would that detect the presence of nasal mucus? Through the use of PCR mutagenesis we aimed to improve the binding affinity that the separated c-terminal alpha helix would have for the rest of the OBP2A protein. This in turn we hoped would displace the natively conformed c-terminal tail of OBP2A in exchange for our tighter binding synthetic tail. P118 was decided to be the point of separation of the OBP2A - Barrel and the OBP2A - Helix.
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In order to probe the interaction between OBP2A - Barrel and OBP2A - Helix, a β-galactosidase assay was done. However, since the controls did not seem to work, no definite conclusions could be drawn from these experiments.
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https://static.igem.org/mediawiki/2015/6/61/Dundee15_obp_reuploadresizedobp.png
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Figure 1: illustrates the calculated miller’s activity of each control/sample and suggest that the OBP2A subunits are not interacting, as can be gauged from the sample on the far right of the graph. They suggest that OBP2A - Barrel and OBP2A - Helix have a lower interaction than the negative and regulatory controls. However since the controls do not seem to be functioning correctly no major conclusion can be drawn.
  
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K1590007 parameters</partinfo>
 
<partinfo>BBa_K1590007 parameters</partinfo>
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Latest revision as of 11:48, 19 September 2015

Human Odorant Binding Protein 2A

Human Odorant Binding Protein 2A is a 155 amino acid (excluding the signal peptide) lipocalin of relatively low molecular weight (19318 Daltons). Structurally it forms an 8 sheet beta barrel flanked by a c-terminal alpha helix that together forms an internal hydrophobic pore known as a calix. It is secreted by the olfactory epithelial cells of the nose where it lies in high abundance within nasal mucus. Its primary function in the human body is believed to be in the transport of hydrophobic odorant proteins across the otherwise impenetrable aqueous mucus layer to the olfactory receptors of the nose. Due to its high specificity and abundance within nasal mucus, OBP2A was selected as the protein for use in nasal mucus detection.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 445
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

We aimed to separate the alpha helix c-terminal tail of OBP2A and use that as the substrate for fluorescent nanobead attachment. How would that detect the presence of nasal mucus? Through the use of PCR mutagenesis we aimed to improve the binding affinity that the separated c-terminal alpha helix would have for the rest of the OBP2A protein. This in turn we hoped would displace the natively conformed c-terminal tail of OBP2A in exchange for our tighter binding synthetic tail. P118 was decided to be the point of separation of the OBP2A - Barrel and the OBP2A - Helix.

In order to probe the interaction between OBP2A - Barrel and OBP2A - Helix, a β-galactosidase assay was done. However, since the controls did not seem to work, no definite conclusions could be drawn from these experiments.

Dundee15_obp_reuploadresizedobp.png
Figure 1: illustrates the calculated miller’s activity of each control/sample and suggest that the OBP2A subunits are not interacting, as can be gauged from the sample on the far right of the graph. They suggest that OBP2A - Barrel and OBP2A - Helix have a lower interaction than the negative and regulatory controls. However since the controls do not seem to be functioning correctly no major conclusion can be drawn.

Functional Parameters