Team:HKUST-Rice/Nitrate Sensor PyeaR
Nitrate Sensor - PyeaR
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.
Nitrate sensor Mechanism
Escherichia coli (E. coli) detects environmental nitrate by the yeaR-yoaG operon. According to Figure 1, PyeaR (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 PyeaR 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 PyeaR. As a result, any genes that are downstream of PyeaR will be expressed.
Nitrate sensor construct
Figure 2. Construct for nitrate sensing. PyeaR with GFP generator.
With the positive relationship between the promoter activity and nitrate concentration, by ligating the promoter together with the GFP generator (pSB1C3-BBa_K381001), an upward trend for the reporter signal with increasing nitrate concentrations was expected.
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 on different days and the final results were obtained by combining the 3 characterization results together.
Please visit PyeaR Experiment Protocol for more details.
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 PyeaR in LB
Figure 3. Characterization of PyeaR in LB. 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. (a) showing characterization within 0-10 mM concentration of nitrate; (b) showing characterization within 0-50 mM concentration of nitrate.
According to Figure 3a, the relative fluorescence level increases 7.21 folds between 0 mM and 10 mM concentrations of nitrate. A continuous upward slope was obtained from 0 mM to 6 mM nitrate concentration. This result obtained was unexpected, according to previous experiments by the BCCS-Bristol iGEM 2010 team, 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, while we were using DH10B.
After obtaining the results of PyeaR response behavior within 0-10 mM nitrate concentration, another characterization on 0-50 mM nitrate concentration was performed to further examine the behavior of PyeaR. According to Figure 3b, a plateau was shown starting from the 10 mM concentration point, suggesting that 10 mM nitrate concentration is the saturation point of PyeaR and the dynamic range of PyeaR is shown to be between 0-10 mM in our study.
Dynamic range Characterization of PyeaR in M9
Figure 4. 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. (a) showing characterization within 0-500 μM concentration of nitrate; (b) showing characterization within 0-1000 μM concentration of nitrate.
According to Figure 4a, the relative fluorescence level increases 3.12 folds from 0 μM and 500 μM nitrate concentrations.
After obtaining the results of PyeaR response behavior in the concentrations of 0-500 μM nitrate, another characterization was was performed to further examine the behavior of PyeaR.
According to Figure 4b, a plateau was shown starting from the 500 μM concentration point, suggesting that 500 μM nitrate concentration is the saturation point of PyeaR and the dynamic range of PyeaR is shown to be between 0-500 μM in our study.
Further Improvements
Since endogenous nitrate would affect the sensitivity of the promoter, a method in reducing the endogenous noise was designed.
Figure 5. Construct for endogenous noise reduction of PyeaR.
Rationale
As PyeaR is regulated by the Nar system and NsrR protein, by overexpressing NsrR protein, endogenous nitrate alone is not likely to drive the transcription of PyeaR. 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 would result. With this method, less effects from the endogenous noise on the promoter is expected.
With the inducible promoter ParaBAD, 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 in detecting nitrate concentrations.
Result
Figure 6. Endogenous noise reduction of PyeaR. 0.01mM, 0.1mM, 1mM and 10mM of L-arabinose was added for inducing ParaBAD. With different concentrations of NsrR protein produced, the endogenous noise was reduced accordingly.
According to Figure 6, with L-arabinose added, the curve shifts downwards, suggesting the sensitivity of the promoter was being enhanced. However, as the result obtained is similar to that of Parallel Sensors, it is uncertain that the downward shifting was due to co-expression of promoters or the method for endogenous noise reduction.
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.