Part:BBa_K3470013

Anti inflammation control mechanism

Circuit

Constitutive Promoter – RBS – SoxR - Double terminator - SoxS promoter – GFP - Double Terminator

Usage and Biology

SoxR is a transcription factor, activated in the conditions of oxidative stresses (presence of NO; pro-inflammatory signal) by reversible one-electron oxidation of its [2Fe-2S] cluster. In the active state, it activates the SoxS promoter by untwisting the promoter DNA by binding to the long spacer DNA (19- or 20-bp spacer between the -35 and -10 operator elements). (Miki et al., 2008). SoxS is a conditional promoter, regulated by the SoxR transcription factor (In Composite BioBrick 1). In the active state (when it is bound to NO; pro-inflammatory signal), SoxR activates the SoxS promoter and starts the transcription of the genes downstream to it. (Hidalgo et al., 1998)

Proposed Experimentation

Escherichia coli Nissle 1917 (bacteria) transformed with SoxR and SoxS genes must be tested to analyze the relationship between the amount of oxidative stress and transcription rates of the genes in response to methyl viologen dichloride hydrate which acts as an inducer by measuring GFP intensity. The intensity of GFP is expected to increase with time indicating that even if the concentration of the oxidative stress inducer is constant, the rate of activation of SoxR and thus the rate of transcription of SoxS promoter increases and then stabilizes to a constant rate. It is also expected that transcription rate of SoxS promoter increases with a higher inducer concentration.

It is also expected that the amount of oxidative stress required to activate SoxR and in turn activate SoxS promoter occurs even at low oxidative stress, preventing any unwanted inflammation due to our probiotic thus getting an anti-inflammatory response activation by composite bio-brick 2.

The GFP intensity is expected to increase with time for all the concentrations except the control and stabilize after a few hours. The culture fluorescence is hypothesized to be more for a higher concentration of paraquat and then decrease after a point due to bacterial death. Bacterial resistance to oxidative stress must be priorly checked.

Sequence and features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1266
  

References

Hidalgo, E., Leautaud, V., & Demple, B. (1998). The redox-regulated SoxR protein acts from a single DNA site as a repressor and an allosteric activator. EMBO Journal, 17(9), 2629–2636. https://doi.org/10.1093/emboj/17.9.2629

Miki, K., Watanabe, S., Kita, A., & Kobayashi, K. (2008). Crystal structure of the [2Fe-2S] transcriptional activator SoxR bound to DNA. Acta Crystallographica Section A Foundations of Crystallography, 64(a1), C89–C89. https://doi.org/10.1107/s0108767308097122