Part:BBa_K203119

NfKB Responsive promoter

A synthetic promoter upregulated by NF-κB activation. Manufactured by [http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters RA-PCR].

Usage and Biology

NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a transcription factor (TF) which regulates many different target genes resulting in the expression of various proteins. In most cell types (with the exception of B cells and Dentritic cells) NF-κB is bound to the Inhibitor of κB (IκB), which withhold NF-κB from entering the nucleus. When the cell becomes activated by an extra cellular stimluli, IκB is degraded and NF-κB can enter the nucleus. Within the nucleus NF-κB is able to enhance transcription of genes which are involved in immune response, cell proliferation or cell survival, depending on cell type and extra cellular stimuli. In many cells NF-κB regulates anti-apoptotic proteins (e.g. TRAF1/2) and therby preventing cell death. Therefore mutations of NF-κB resulting in a constitutively active form are often associated with unregulated cell proliferation and cancer. In macrophages the NF-κB signalling pathway could be activated by binding of bacterial lipopolysacchride (LPS). There NF-κB activation leads to secretion of cytokines which influence other lymphocytes. For references see [http://2009.igem.org/Team:Heidelberg/Eukaryopedia#Transcription_factors Eukaryopedia].

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]

Functional Parameters

NF-κB was induced with 2,5µM TNF-α in [http://2009.igem.org/Team:Heidelberg/Eucaryopedia#U2-OS U2-OS] cells. Expression level was determined in [http://2009.igem.org/Team:Heidelberg/Measurement REU] by flow cytometry (3 experiments on different days, 3 replicates / wells per experiment) 10 hours after induction. Promoter was shown to be specific to NF-κB

Figure 1: BBa_K203119 is a promoter induced approximately by 100% by TNF-α. [http://2009.igem.org/Team:Heidelberg/Measurement REU] was determined by flow cytometry, 3 experiments à three replicates

Figure 2: Timecourse of induction. Measured by image analysis. See [http://2009.igem.org/Team:Heidelberg/Project_Measurement#Flow_Cytometry/Fluorescence_Microscopy discussion] for why the results obtained by the two methods deviate.

Figure3: BBa_K203119 responds only to TNF-α and is not induced by other conditions. U2-OS Cells transfected with BBa_K203119 in BBa_K203100 were subjected to a variety of conditions (full medium DMEM+++, full medium lacking growth factors / serum DMEM++, starvation by minimal medium and [http://2009.igem.org/Team:Heidelberg/Eucaryopedia#HIF-1 pPARγ] induction by Thiazolidinedione) and did not show any significant induction or repression.

Figure 4: BBa_K203119 is only active if NF-κB is localized to the nucleus. Cell were cotransfected with BBa_K203119 in Part:BBa_K203100 and a p65-mcherry-fusion protein (kind gift of N. Brady, BioQuant). Only if the red fluorescence is localized to the nucleus, bright green fluorescence can be observed.