Difference between revisions of "Part:BBa K1456015"
 
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<partinfo>BBa_K1456015 short</partinfo>
 
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<ul>
 
<ul>
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<center>https://static.igem.org/mediawiki/2014/thumb/9/93/KB-RE_2.jpg/800px-KB-RE_2.jpg</center>
 
<center>https://static.igem.org/mediawiki/2014/thumb/9/93/KB-RE_2.jpg/800px-KB-RE_2.jpg</center>
  
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===Modeling===
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<ul>Firstly, we built a model to help understand the gene expression dynamics of the whole circuit. In our promoter design, we added NFKB response element to upstream of the gene. During reperfusion, ROS increases. We assumed that this situation triggers the import of NFKB from cytoplasm to nucleus. Then, NF-kB integrates with compatible kB-RE sites in order to initiate transcription. We try to understand how this system effect of protein synthesis.
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</ul>
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<ul>Then we wrote down the four chemical reactions in represent of each process.
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</ul>
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<center>[[File:Atoms modeling 3 kbre.PNG|600px|]]</center><br/>
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<li>Using the SimBiology toolbox for Matlab we created a framework of the NFKB system (Figure 1).
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</li>
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<center>[[File:Atoms modeling kbre 4.jpg|600px|]]</center><br/>
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<ul>(Figure 1:)Diagram of reactions was created in the SimBiology toolbox for MATLAB.
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</ul>
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<center>[[File:Atoms modeling kbre.PNG|600px|]]</center><br/>
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===What did it show???===
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<ul>The effect of increasing ROS concentration on NFKB-KBRE association was changed and this situation triggers the import of NFKB from cytoplasm to nucleus. Therefore, protein expression of increases 2 times during reperfusion.
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</ul>
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<center>[[File:Atoms modeling 7 kbre.PNG|600px|]]</center><br/>
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<center>[[File:Atoms modeling 8 kbre.PNG|600px|]]</center><br/>
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<center>[[File:Atoms modeling 9 kbre.PNG|600px|]]</center><br/>
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<center>[[File:Atoms kb 5.png|600px|]]</center><br/>
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<ul>(p<0,05 and p<0,01 for H2O2 and TNFα induced kappaB-luc,respectively.)<br/>
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 15:40, 23 October 2014

NF-κB (Nuclear Factor kappa B) ROS response element


  • After removing the clot, we need an additional sensor system to compensate the hypoxia response elements transcription rates for antioxidant enzymes and proteins. Thus, we targeted to sense the ROS presence to conduct an efficient and swift solution.
  • Nuclear factor kappa-B (NF-kB) is a transcription factor complex of hypoxia inducible factor-1 alfa (HIF-1α) protein, which consists of two subunits, p50 and p65. This peptide is detained in cytoplasm by a complementary peptide called IkB. (Kabe et al. 2005) This detention prevents the activation of related promoter sequence existing in the cell nucleus which is named kappa-B response element (kB-RE).
  • In some circumstances, NF-kB/IkB complex can be separated by external effects such as radiation, cellular stress, pathogens, inflammation etc. In this case, NF-kB can enter into nucleus and integrate with compatible kB-RE sites in order to initiate transcription. (Gilmore 2006)
  • It is later described that one of these stimulants is the presence of ROS. (Bonello et al. 2007) We want to use this advantage to design a novel ROS inducible promoter in human cells in iGEM to be able to sense oxidative stress.
800px-KB-RE_2.jpg

Modeling

    Firstly, we built a model to help understand the gene expression dynamics of the whole circuit. In our promoter design, we added NFKB response element to upstream of the gene. During reperfusion, ROS increases. We assumed that this situation triggers the import of NFKB from cytoplasm to nucleus. Then, NF-kB integrates with compatible kB-RE sites in order to initiate transcription. We try to understand how this system effect of protein synthesis.
    Then we wrote down the four chemical reactions in represent of each process.
Atoms modeling 3 kbre.PNG

  • Using the SimBiology toolbox for Matlab we created a framework of the NFKB system (Figure 1).
    • Atoms modeling kbre 4.jpg

      (Figure 1:)Diagram of reactions was created in the SimBiology toolbox for MATLAB.
    Atoms modeling kbre.PNG

    What did it show???

      The effect of increasing ROS concentration on NFKB-KBRE association was changed and this situation triggers the import of NFKB from cytoplasm to nucleus. Therefore, protein expression of increases 2 times during reperfusion.
    Atoms modeling 7 kbre.PNG

    Atoms modeling 8 kbre.PNG


    Atoms modeling 9 kbre.PNG


    Atoms kb 5.png

      (p<0,05 and p<0,01 for H2O2 and TNFα induced kappaB-luc,respectively.)
      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 1
        Illegal XhoI site found at 47
      • 23
        COMPATIBLE WITH RFC[23]
      • 25
        COMPATIBLE WITH RFC[25]
      • 1000
        COMPATIBLE WITH RFC[1000]