Part:BBa_K1456015

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.

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.

Using the SimBiology toolbox for Matlab we created a framework of the NFKB system (Figure 1).

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

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.

(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]