Difference between revisions of "Part:BBa K1456005"

 
 
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<partinfo>BBa_K1456005 short</partinfo>
 
<partinfo>BBa_K1456005 short</partinfo>
  
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This part regulate protein degredation depend on oxygen successfully.
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<ul>
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  <li>The second mechanism includes a few understructure elements called TetR-VP16 complex and two different plasmids, pTet-off and pTRE vectors. In pTet-off plasmid, PCMV constitutive promoter with medium strength codes TetR-VP16 protein complex which can bind its respond element present in second plasmid, pTRE. Tetracyclin respond element (TetRE) is a protein binding domain which allows the binding of TetR component of the protein complex. Whereas, VP16 component works as a transcription activator for the weak constitutive promoter (PminiCMV) existing in pTRE vector. <b>TetR-VP16 protein complex can activate this weak promoter in pTRE vector.</b> </li>
 +
  <li>ODD is a peptide about 200 amino acid long which includes a hydroxylation site of prolyl hydroxylase. In normal oxygen levels, hydroxylation from this site causes peptide to be targeted by ubiquitine-proteosome degradation system to be eliminated swiftly. However, <b>in hypoxic conditions, hydroxyl subgroups cannot be added to ODD domain which results as the survival and accumulation of peptide and the components cohesive to it.</b> (Huang et al. 1998)</li>
 +
  <li>We aim to insert an additional peptide called oxygen dependent degradation domain (ODD domain) between TetR and VP16 components. With this mechanism, TetR-ODD-VP16 complex can only initiate the transcription of only weak promoter in hypoxic conditions because of the degradation of the complex by proteasome in oxygen presence. <b>ith this, protein complex is expected to behave like a hypoxia inducer.</b></li>
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 +
</ul>
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<center>https://static.igem.org/mediawiki/2014/thumb/9/9b/ATOMS-ODD_Complex.jpg/800px-ATOMS-ODD_Complex.jpg</center>
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 +
<ul>
 +
===Modeling===
 +
<li>The main goal of this part, being sensitive of TetR – VP16 system to hypoxia. In order to achieve this, ODD added between TetR and VP16 components.In this way, TetR-ODD-VP16 complex can only initiate the transcription of only weak promoter in hypoxic conditions because of the degradation of the complex by proteasome in oxygen presence. By using this system, we created a novel true/ false mechanism which triggers protein synthesis dependent on oxygen by modifying that has already present pTRE-Tet Off system.</li>
 +
<li>In order to realize how our system will work. We try to calculate how the protein synthesis will effect under different concentration of O2 at the TetR - ODD - VP16 system.
 +
</li>
 +
<li>Using the SimBiology toolbox for Matlab we created a framework of the pTRE-Tet Off System (Figure 2).
 +
</li></ul>
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<center>[[File:800px-ATOMS-Odd modeling 1.png|700px|]]</center><br/>
 +
<center>[[File:ATOMS-Odd modeling 2.png|700px|]]</center><br/>
 +
<ul>The symbol decleration is:</ul>
 +
<li>X1: pCMV
 +
</li>
 +
<li>Y: ODD (dependent on TetR – VP16)
 +
</li>
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<li>X2 : [TetRE -PminiCMV] Complex
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</li>
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<li>X2Y: ODD[TetRE -PminiCMV]
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</li>
 +
<li>X3 : Luciferase
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</li>
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<li>Z : PHD (prolyl hydroxylase)
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</li>
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<li>YZ: Hyroxylated ODD
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</li>
 +
<ul><li>After creating the basic framework for the model we needed to create mathematical equations for each reaction with appropriate rate constants. These equations and the corresponding values are shown below in Tables 3 and 4
 +
</li>
 +
<center>[[File:800px-ATOMS-Odd modeling 3.png|700px|]]</center><br/>
 +
<center>[[File:777px-ATOMS-Odd modeling 4.png|700px|]]</center><br/>
 +
===What did it show???===
 +
<li>After modified the TetR-VP16 system by adding ODD, we try to realize how our designed sytem will work under hypoxic and normoxic conditions. In normoxic condition, PDH is activated and hydroxylated the TetR-ODD-VP16 system, luciferase expression decreases.
 +
</li>
 +
<li>In our model approach,<b> we showed that luciferase expression increased %10 after exposed to 1nM O2 compared to 100 nM O2 concentration.</b>
 +
</li>
 +
===Modelling Results===
 +
<center>[[File:800px-ATOMS-Odd modeling 5.png|700px|]]</center><br/>
 +
<center>[[File:800px-ATOMS-Odd modeling 6.png|700px|]]</center><br/>
 +
<center>[[File:ATOMS-Odd modeling 7.png|700px|]]</center><br/>
 +
===Experimental Results:===
 +
<center>[[File:800px-ATOMS-Odd modeling 8.png|700px|]]</center><br/>
 +
<center>[[File:ATOMS-Odd modeling 9.png|700px|]]</center><br/>
 +
<li>We reached expected result in wet lab for HEK293 cell line. However, after we used HEPG2 cell line for this system, we had more reliable result about hydroxylation of ODD. The reason of this can be about life time of cells under hypoxia, because it will affect amount of PHD that enter nucleus during normoxia /hypoxia.
 +
</li>
 +
 
 +
 
 +
<center>[[File:Atoms odd 9.png|700px|]]</center><br/>
 +
 
 +
<li>When the Tet Off-ODD and pTRE-Luc vectors were cotranfected to Hep G2 cells in hypoxic medium, there was a 4 times increase in Luciferase concentration in respect to normoxic medium.
 +
<center>[[File:Atoms odd 10.png|700px|]]</center><br/>
 +
<li>In the light of these results, it is possible to say that the Tet-ODD-VP16 system was successfully synthesized and a novel hypoxia inducible system was introduced for future use in studies indulged into examining hypoxic conditions.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 15:29, 23 October 2014

Oxygen dependent degradation(ODD) domain from HIF-1a


  • The second mechanism includes a few understructure elements called TetR-VP16 complex and two different plasmids, pTet-off and pTRE vectors. In pTet-off plasmid, PCMV constitutive promoter with medium strength codes TetR-VP16 protein complex which can bind its respond element present in second plasmid, pTRE. Tetracyclin respond element (TetRE) is a protein binding domain which allows the binding of TetR component of the protein complex. Whereas, VP16 component works as a transcription activator for the weak constitutive promoter (PminiCMV) existing in pTRE vector. TetR-VP16 protein complex can activate this weak promoter in pTRE vector.
  • ODD is a peptide about 200 amino acid long which includes a hydroxylation site of prolyl hydroxylase. In normal oxygen levels, hydroxylation from this site causes peptide to be targeted by ubiquitine-proteosome degradation system to be eliminated swiftly. However, in hypoxic conditions, hydroxyl subgroups cannot be added to ODD domain which results as the survival and accumulation of peptide and the components cohesive to it. (Huang et al. 1998)
  • We aim to insert an additional peptide called oxygen dependent degradation domain (ODD domain) between TetR and VP16 components. With this mechanism, TetR-ODD-VP16 complex can only initiate the transcription of only weak promoter in hypoxic conditions because of the degradation of the complex by proteasome in oxygen presence. ith this, protein complex is expected to behave like a hypoxia inducer.
800px-ATOMS-ODD_Complex.jpg

    Modeling

  • The main goal of this part, being sensitive of TetR – VP16 system to hypoxia. In order to achieve this, ODD added between TetR and VP16 components.In this way, TetR-ODD-VP16 complex can only initiate the transcription of only weak promoter in hypoxic conditions because of the degradation of the complex by proteasome in oxygen presence. By using this system, we created a novel true/ false mechanism which triggers protein synthesis dependent on oxygen by modifying that has already present pTRE-Tet Off system.
  • In order to realize how our system will work. We try to calculate how the protein synthesis will effect under different concentration of O2 at the TetR - ODD - VP16 system.
  • Using the SimBiology toolbox for Matlab we created a framework of the pTRE-Tet Off System (Figure 2).
800px-ATOMS-Odd modeling 1.png

ATOMS-Odd modeling 2.png

    The symbol decleration is:
  • X1: pCMV
  • Y: ODD (dependent on TetR – VP16)
  • X2 : [TetRE -PminiCMV] Complex
  • X2Y: ODD[TetRE -PminiCMV]
  • X3 : Luciferase
  • Z : PHD (prolyl hydroxylase)
  • YZ: Hyroxylated ODD
    • After creating the basic framework for the model we needed to create mathematical equations for each reaction with appropriate rate constants. These equations and the corresponding values are shown below in Tables 3 and 4
      • 800px-ATOMS-Odd modeling 3.png

      777px-ATOMS-Odd modeling 4.png

      What did it show???

    • After modified the TetR-VP16 system by adding ODD, we try to realize how our designed sytem will work under hypoxic and normoxic conditions. In normoxic condition, PDH is activated and hydroxylated the TetR-ODD-VP16 system, luciferase expression decreases.
    • In our model approach, we showed that luciferase expression increased %10 after exposed to 1nM O2 compared to 100 nM O2 concentration.

      • Modelling Results

      800px-ATOMS-Odd modeling 5.png

      800px-ATOMS-Odd modeling 6.png

      ATOMS-Odd modeling 7.png

      Experimental Results:

      800px-ATOMS-Odd modeling 8.png

      ATOMS-Odd modeling 9.png

    • We reached expected result in wet lab for HEK293 cell line. However, after we used HEPG2 cell line for this system, we had more reliable result about hydroxylation of ODD. The reason of this can be about life time of cells under hypoxia, because it will affect amount of PHD that enter nucleus during normoxia /hypoxia.


      • Atoms odd 9.png

    • When the Tet Off-ODD and pTRE-Luc vectors were cotranfected to Hep G2 cells in hypoxic medium, there was a 4 times increase in Luciferase concentration in respect to normoxic medium.
      Atoms odd 10.png

    • In the light of these results, it is possible to say that the Tet-ODD-VP16 system was successfully synthesized and a novel hypoxia inducible system was introduced for future use in studies indulged into examining hypoxic conditions. 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
        COMPATIBLE WITH RFC[25]
      • 1000
        COMPATIBLE WITH RFC[1000]