Part:BBa_K4010010
Pconst + QseC & Pconst + NarX & PyeaR + QseB + NarL
NOTE: This composite part is similar to BBa K4010007, although this part uses an extra constitutive promoter rather than a linker for NarX expression to minimize each module's sequence length to below 2000bp.
Figure 1: Sequence overview of BBa K4010010.
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 (Vine et al., 2011). When nitric oxide is present, NsrR will no longer bind to and repress PyeaR, allowing the QseB gene to be expressed (Lin et al., 2007). Once QseB is made, the system will react to AI-3 as sensed by QseC, and genes downstream flhDC promoter can be transcribed.
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). To prolong the effect of nitric oxide in our circuit, 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 1514
Illegal NheI site found at 1537
Illegal NheI site found at 2886
Illegal NotI site found at 2179 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1003
Illegal BglII site found at 4498
Illegal BglII site found at 4666 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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 figures 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
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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