Team:HKUST-Rice/Nitrate Sensor PyeaR

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Nitrate sensor - P_yeaR

Nitrate sensor (dcuSp)

Signal Coexpression

Nitrate as a Macro-nutrient

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. Lack of nitrogen will lead to stunted growth, yellowing of leaves, etc.

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Nitrate sensor Design

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 (T. 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. The 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.

By ligating the promoter together with the GFP generator ( pSB1C3-BBa_K381001), upward increase in reporter signal with increasing nitrate concentrations is expected.

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Experiment performed

Two sets of characterization on pSB1C3-BBa_K381001 (BCCS-Bristol iGEM 2010) in two different growth media, Luria Broth (LB) medium and M9 minimal medium were performed. M9 minimal medium was used as it does not contain nitrate and has a lower auto-fluorescence level, thus providing more accurate results. Potassium nitrate (KNO₃) was used as a source of nitrate in the experiments. E. coli strain DH10B was used in the characterization of the promoter. Quantitative characterization on the promoter was done by measuring the fluorescence signal intensity using an EnVision multilabel reader. All experiments were conducted three times at different days and the final results were obtained by combining the 3 characterization results together.

Please visit yeaRp Experiment Protocol for more details.

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Results

After obtaining the quantitative results of GFP signal intensity using an EnVision multilabel reader, we processed the data with relative fluorescence level (in OD₆₀₀) against nitrate concentration.

Dynamic range Characterization of P_yeaR in LB

Figure 2. Characterization of P_yeaR in LB. Bacteria hosting pSB1C3-BBa_K381001 weregrown overnight at 37^oC in LB. They were then washed with 0.85% NaCl solution. Washed candidates were resuspend in LB medium with different concentrations and grew for another 2.5 hours at 37^oC incubator with shaking. GFP emission measurements were made using EnVision multilabel reader. This result was obtained by combining together 3 charaterization data obtained in 3 different days.

According to Figure 2a, the relative fluorescence level increases by 7.21 folds between 0 mM and 10 mM concentrations of nitrate. Furthermore, a plateau was shown from the 10 mM nitrate concentration point. The result obtained was as expected, according to previous experiments by the Edinburgh iGEM 2009 team and the BCCS-Bristol iGEM 2010 team, where the dynamic range of P_yeaR was from 0-10 mM nitrate concentration.

After obtaining the results of P_yeaR response behavior within 0-50 mM nitrate concentration, it shows that the fluorescence signal increases sharply between 0-10 mM nitrate concentration. Therefore, another characterization was done focusing on this concentration range.

According to Figure 2b, the relative fluorescence level increases by 4.23 folds between 0 mM and 10 mM nitrate concentrations. It shows an upward slope from 0 mM to 6 mM nitrate concentration, however, at 8 mM nitrate concentration point, it shows a downward slope. It then rises again at 10 mM nitrate. The result obtained is unexpected as according to previous experiments by the BCCS-Bristol iGEM 2010 team, where a continuous upward slope was obtained from 0 mM to 9 mM nitrate concentration. The discrepancy between the obtained and reference results could be due to the use of different bacterial strains. The strain used by the BCCS-Bristol iGEM 2010 team was MG1655, which may have been the cause in the promoter behavior difference.

Dynamic range Characterization of P_yeaR in M9

Figure 3. Characterization of P_yeaR in M9. Bacteria hosting pSB1C3-BBa_K381001 weregrown overnight at 37^oC in LB. They were then washed with 0.85% NaCl solution. Washed candidates were resuspend in M9 medium with different concentrations and grew for another 4.5 hours at 37^oC incubator with shaking. GFP emission measurements were made using EnVision multilabel reader. This result was obtained by combining together 3 charaterization data obtained in 3 different days.

According to Figure 3a, the relative fluorescence level increases by 4.37 folds from 0 μM and 1000 μM nitrate concentrations, and a plateau was shown from 500 μM nitrate concentration point.

After obtaining the results of P_yeaR response behavior in the concentrations of 0-1000 μM nitrate, we find that the relative fluorescence level increases sharply between 0-500 μM concentrations of nitrate. As a result, another characterization was done focusing on this concentration range.

According to Figure 3b, the relative fluorescence level increases 3.12 folds between 0 and 500 μM. Furthermore, with higher nitrate concentration, the relative fluorescence level increases accordingly. The result obtained is expected as according to the previous characterization with concentration gradient of 0 to 1000 μM nitrate, which also shows an upward slope between 0 to 500 μM.

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Further Improvements

Since we were concerned that endogenous nitrate would affect the sensitivity of the promoter, a method in reducing the endogenous noise was designed.

With araBADp as an inducible promoter, experiments were carried out to find the concentration of L-arabinose which could reduce endogenous noise most effectively, so that the promoter could be more sensitive.

Rationale

As P_yeaR is regulated by the Nar system and NsrR protein, by overexpressing NsrR protein, the endogenous nitrate will titrate against the excess NsrR protein, so that less nitrate will drive the transcription of P_yeaR. On the other hand, when there is nitrate in the environment, the amount of nitrate is enough to relieve the repression from the Nar system and NsrR protein, and transcription could be resulted. With this method, less effects from the endogenous noise on the promoter is expected.

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Reference

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.

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.

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.

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.