Team:HKUST-Rice/Nitrate Sensor PyeaR

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Nitrate Sensor - P_yeaR

Nitrate sensor (P_dcuS)

Parallel Sensors

E. coli that glows in adequacy of NO₃^- - at a glance

A. E. coli engineered with BBa_K381001 functions as a nitrate biosensor. High concentrations of NO3- activates the promoter PyeaR and increases the expression of GFP.		B. The nitrate sensing promoter BBa_K381001 can detect a gradient of NO3- concentrations and its activities were reported in Relative Fluorescence Units (RFU).

• Nitrate is an essential nutrient which plays multiple roles in plant growth and reproduction.
• This biosensor BBa_K381001 monitors NO₃^- concentration.
• Activity of NO₃^- sensing promoter (BBa_K216005) was re-characterized to further investigate on the behavior of it.

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An effort to make iGEM a better community

Nitrate is an essential nutrient which plays multiple roles in plant growth and reproduction. For example, it provides nitrogen that plants need for producing amino acids and nucleic acids (DNA and RNA). Also, it is a component of chlorophyll and is therefore essential for photosynthesis.

P_yeaR is first characterized and BioBricked by Edinburgh 2009 iGEM team and then further characterized by BCCS-Bristol 2010 iGEM team. To provide more characterization data on such a devices, we further characterize this promoter.

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Endogenous nitrate sensing system in E. coli

Figure 1. The NO₃^- uptake system in E. coli.

Escherichia coli (E. coli) detects environmental nitrate by the yeaR-yoaG operon. According to Figure 1, P_yeaR (Lin, et al., 2007) is regulated by the Nar two-component regulatory system (Nohno et al., 1989; Li et al., 1987) and NsrR regulatory protein (Partridge et al., 2009). When there is nitrate or nitrite, the repression from the Nar system on P_yeaR will be relieved due to the binding between the two. On the other hand, some nitrate will be converted into nitric oxide by nitrate reductase. Nitric oxide will bind to the NsrR protein and relieve the repression on P_yeaR. As a result, any genes that are downstream of P_yeaR will be expressed.

*The above text is our summarized understanding on NO₃^--sensing system. Please refer to our references section below for a full list of references cited.

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Results

After obtaining the quantitative results of GFP signal intensity using an EnVision® multilabel reader, the fluorescence signal were represented in fluorescence divided by biomass.

Dynamic range Characterization of P_yeaR in LB and M9

A. Characterization of PyeaR in LB.		B. Characterization of PyeaR in M9 minimal medium.

*GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 charaterization data obtained in 3 different days. Error bars were presented in SEM.

According to A, a plateau was shown starting from the 10 mM concentration point, suggesting that 10 mM nitrate concentration is the saturation point of P_yeaR and the dynamic range of P_yeaR is shown to be between 0-10 mM in our study. The relative fluorescence level increases 7.21 folds between 0 mM and 10 mM concentrations of nitrate.

According to B, a plateau was shown starting from the 500 μM concentration point, suggesting that 500 μM nitrate concentration is the saturation point of P_yeaR and the dynamic range of P_yeaR is shown to be between 0-500 μM in our study. The relative fluorescence level increases 3.12 folds from 0 μM and 500 μM nitrate concentrations.

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Materials and Methods

Please refer to our protocol page for the materials and methods used in characterization.

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References

Li, S. F., & DeMoss, J. A. (1987). Promoter region of the nar operon of Escherichia coli: nucleotide sequence and transcription initiation signals.Journal of bacteriology, 169(10), 4614-4620.

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.

Nohno, T., Noji, S., Taniguchi, S., & Saito, T. (1989). The narX and narL genes encoding the nitrate-sensing regulators of Escherichia coli are homologous to a family of prokaryotic two-component regulatory genes. Nucleic acids research,17(8), 2947-2957.

Partridge, J. D., Bodenmiller, D. M., Humphrys, M. S., & Spiro, S. (2009). NsrR targets in the Escherichia coli genome: new insights into DNA sequence requirements for binding and a role for NsrR in the regulation of motility.Molecular microbiology, 73(4), 680-694.

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