Composite

Part:BBa_K4010012

Designed by: Yi Yang Fei   Group: iGEM21_McMaster   (2021-10-16)


Constitutively Expressed NarX


Figure 1: Sequence overview of BBa K4010012.

In NarX/L, Escherichia coli’s two-component regulatory system, NarX is a histidine kinase sensor which activates in response to nitrate (NO3-) via auto-phosphorylation (Noriega et al., 2008). When activated, it phosphorylates the NarL response regulator protein, which then binds to target operons to regulate gene expression (Lin et al., 2007; Noriega et al., 2008).

McMaster_2021

In iGEM McMaster 2021’s project, both nitric oxide and autoinducer-3 (AI-3) are used as biomarkers of adherent-invasive E. coli (AIEC) induced gut inflammation (Bretin et al., 2018). However, nitric oxide has a limited half-life and can be oxidized into nitrate in the presence of hemoglobins and oxygen (Hakim et al., 1996). Therefore, we proposed a positive feedback loop design using NarX/L’s ability to activate the yeaR-yoaG operon upon sensing nitrate. Specifically, the PyeaR promoter (BBa_K216005) in this operon contains a consensus sequence with the binding sites of the NsrR repressor (BBa_K1682011) and the NarL activator (BBa_K3411040) (Lin et al., 2007). In other words, the PyeaR promoter is simultaneously activated by NarL in the presence of nitrate (as sensed by NarX) and repressed by the NsrR protein in the absence of nitric oxide (Lin et al., 2007). As such, provided that NarX is constitutively expressed, NarL can be placed downstream of PyeaR along with a chosen sequence to ensure that oxidation of nitric oxide into nitrate would not pause downstream gene expression but instead amplify it.


Figure 2: Downstream reactions after nitric oxide activates the positive feedback loop.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 720
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal PstI site found at 720
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 321
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 720
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 720
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1226


References

Attribution Note: All images were created in Biorender.

Bretin, A., Lucas, C., Larabi, A., Dalmasso, G., Billard, E., Barnich, N., Bonnet, R., & Nguyen, H. T. T. (2018). AIEC infection triggers modification of gut microbiota composition in genetically predisposed mice, contributing to intestinal inflammation. Scientific Reports, 8(1), 12301. https://doi.org/10.1038/s41598-018-30055-y

Hakim, T. S., Sugimori, K., Camporesi, E. M., & Anderson, G. (1996). Half-life of nitric oxide in aqueous solutions with and without haemoglobin. Physiological Measurement, 17(4), 267–277. https://doi.org/10.1088/0967-3334/17/4/004

Lin, H.-Y., Bledsoe, P. J., & Stewart, V. (2007). Activation of yeaR-yoaG Operon Transcription by the Nitrate-Responsive Regulator NarL Is Independent of Oxygen- Responsive Regulator Fnr in Escherichia coli K-12. Journal of Bacteriology, 189(21), 7539–7548. https://doi.org/10.1128/JB.00953-07

Noriega, C. E., Schmidt, R., Gray, M. J., Chen, L.-L., & Stewart, V. (2008). Autophosphorylation and Dephosphorylation by Soluble Forms of the Nitrate-Responsive Sensors NarX and NarQ from Escherichia coli K-12. Journal of Bacteriology, 190(11), 3869–3876. https://doi.org/10.1128/JB.00092-08

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