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). Secondly, it is a component of chlorophyll and is therefore essential for photosynthesis. It is also the basic element of plant and animal proteins, and is important in periods of rapid plant growth. Lack of nitrogen will lead to stunted growth, yellowing of leaves, etc.

Nitrate sensor Design

yeaRp is regulated by the Nar two-component regulatory system and NsrR regulatory protein. When there is nitrate, the repression from the Nar system on yeaRp will be relieved due to the binding between the two. On the other hand, some nitrate will be converted into nitric oxide. The nitric oxide will bind to the NsrR protein and relieve the repression on yeaRp. As a result, any genes that are downstream of yeaRp will be expressed. Therefore, together with the GFP generator, the reporter signal will increase with increasing nitrate concentrations.

Experiment performed

We have performed two sets of characterization on pSB1C3-BBa_K381001 (BCCS-Britstol iGEM 2010), one using Luria Broth (LB) medium and the other in M9 minimal medium. Potassium nitrate (KNO₃) was used as a source of nitrate in the experiments. Escherichia coli (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 and the final result was obtained by combining together the 3 characterization trials.

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Results

After obtaining the quantitative results on 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 yeaRp in LB

Figure 2. Characterization of yeaRp in LB. Colonies transformed with pSB1C3-BBa_K381001 were picked and the candidates were grown in LB at 37oC overnight. They were then washed with 0.85% NaCl. Washed candidates were then added to LB medium in different concentrarions and grew for an addition 2.5 hours at 37oC incubator. GFP measurements were made using EnVision multilabel reader. This result was obtained by combining 3 characterization trials.

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

After obtaining the results of yeaRp response behavior within 0-50 mM nitrate concentration, we can see that between 0-10 mM nitrate concentration, the fluorescence signal increases sharply. Therefore, another characterization was done focusing on the dynamic range of the promoter, 0-10 mM.

According to Figure 3, the relative fluorescence level increases 4.23 folds between 0 mM and 10 mM nitrate concentrations. Moreover, it shows an upward slope from 0 mM to 6 mM nitrate concentration. At concentration point of 8 mM nitrate, it shows a downward slope which then rises again at 10 mM nitrate. The result obtained is unexpected as 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 in the obtained and reference results could be due to use of different bacterial strains, since the strain used by the BCCS-Bristol iGEM 2010 team was MG1655, which is different from ours, causing the behavior of the promoter to be different.

Characterization of promoter responsive range in M9

After obtaining the results of yeaRp response behavior in the concentrations of 0-2000 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 the dynamic range of the promoter, 0-500 μM.

Characterization of promoter dynamic range in M9 minimal medium

According to Figure 5, the relative fluorescence level increases 3.12 folds between 0 and 500μM. Furthermore, with higher nitrate concentration, the relative fluorescence level increases accordingly. The result obtained is expected as according to the characterization with concentration gradient of 0 to 2000μM nitrate that performed previously, it also shows an upward slope between 0 to 500μM.