Composite

Part:BBa_K4010008

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


Pconst + QseC + NarX

NOTE: This composite part is the histidine kinase sensor module of the BBa_K4010007 composite part.


Figure 1: BBa K4010008, the histidine kinase module of BBa K4010007.

QseBC and NarXL are both two-component regulatory systems found in Escherichia coli to regulate gene expression (Zhou et al., 2003). Both QseC and NarX are histidine kinase sensors, responsive to autoinducer-3 (AI-3) and nitrate (NO3-) respectively. Upon activation, they both auto-phosphorylate and subsequently phosphorylate QseB and NarL regulatory proteins respectively, if they are present. These proteins are responsible for controlling downstream gene expressions: QseB can bind to flhDC promoter (BBa_K554001), while NarL can bind to PyeaR promoter (BBa_K216005).

This composite part can be used to activate genes downstream of a flhDC promoter (BBa_K554001) in the presence of both AI-3, a quorum sensing molecule, and nitric oxide, which has a short half-life and is oxidized into nitrate in the presence of oxygen and hemoglobin (Hakim et al., 1996). In E. coli, NsrR repressors are naturally present to inhibit PyeaR expression unless nitric oxide is present (Vine et al., 2011), in which case it would attenuate NsrR’s ability to bind and repress PyeaR (Lin et al., 2007).

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: Positive feedback loop is not activated when no nitric oxide is present at first.




Figure 3: Downstream reactions after nitric oxide activates the positive feedback loop.

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
    Illegal NheI site found at 2806
    Illegal NotI site found at 2099
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1003
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This composite part assumes a chassis of Escherichia coli, where NsrR repressors are naturally present. If other bacteria are used, NsrR (BBa_K1682011) must be constitutively added to ensure PyeaR is being repressed when no nitric oxide is present.

While iGEM McMaster 2021 used QseBC to sense AI-3 and induce flhDC expression, we encourage future teams to switch out these components to replace AI-3 with other biomarkers alongside nitric oxide to provide targeted treatment or diagnoses to gut inflammation. They can do this by swapping out QseC for another constitutive histidine kinase sensor, QseB for another response regulator protein, and flhDC for another promoter that is activated by said response regulator.

References

Attribution Note: All images were created in Biorender.

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

Vine, C. E., Purewal, S. K., & Cole, J. A. (2011). NsrR-dependent method for detecting nitric oxide accumulation in the Escherichia coli cytoplasm and enzymes involved in NO production. FEMS Microbiology Letters, 325(2), 108–114. https://doi.org/10.1111/j.1574-6968.2011.02385.x

Zhou, L., Lei, X.-H., Bochner, B. R., & Wanner, B. L. (2003). Phenotype MicroArray Analysis of Escherichia coli K-12 Mutants with Deletions of All Two-Component Systems. Journal of Bacteriology, 185(16), 4956–4972. https://doi.org/10.1128/JB.185.16.4956-4972.2003

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