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 potassium 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. 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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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.

Nitrate Sensor Mechanism

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.

Nitrate Sensor construct

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 P_yeaR 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 P_yeaR in LB

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 P_yeaR response behavior within 0-10 mM nitrate concentration, another characterization on 0-50 mM nitrate concentration was performed to further examine the behavior of P_yeaR. 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 P_yeaR and the dynamic range of P_yeaR is shown to be between 0-10 mM in our study.

Dynamic range Characterization of P_yeaR in M9

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 P_yeaR response behavior in the concentrations of 0-500 μM nitrate, another characterization was was performed to further examine the behavior of P_yeaR.

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 P_yeaR and the dynamic range of P_yeaR is shown to be between 0-500 μM in our study.