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 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-Bristol iGEM 2010) in two different growth media, Luria Broth (LB) medium and M9 minimal medium. 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. 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 results were obtained by combining the 3 characterization trials together.

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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 yeaRp in LB

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

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 yeaRp was from 0-10 mM nitrate concentration.

After obtaining the results of yeaRp 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 yeaRp in M9

Figure 3. Characterization of yeaRp in M9. Colonies transformed with pSB1C3-BBa_K381001 were picked and the candidates were grown overnight at 37^oC in LB. They were then washed with 0.85% NaCl. Washed candidates were then added to M9 medium in different concentrations and grew for an addition 4.5 hours at 37^oC incubator. GFP measurements were made using EnVision multilabel reader. This result was obtained by combining 3 characterization trials.

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 yeaRp 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.

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 would reduce endogenous noise most effectively, so that the promoter could be more sensitive.

As yeaRp 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 yeaRp. 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.

Methods

pSB1C3-BBa_K381001 characterization

Growth Medium: Luria Broth (LB)

The test samples were first grown in LB overnight at 37^oC. They were then washed 3 times using 0.85% NaCl. 100 μl of samples were then added to 900 μl of different concentrations of medium in a 96-well deep well plate and were further grown for 2.5 hours at 37^oC until the bacteria reached mid-log phase. The fluorescence output was then measured using an EnVision multilabel reader.

Characterization of yeaRp dynamic range in LB
The concentrations used for the characterization of yeaRp was from 0 to 50 mM nitrate, with intervals of 10 mM.

Characterization of the promoter dynamic range in Luria Broth (LB)
The concentration of the characterization of yeaRp was from 0 to 10 mM of nitrate, with intervals of 2 mM.

Growth Medium: M9

The test samples were first grown overnight at 37^oC in LB. They were then washed 3 times using 0.85% NaCl. 100 μl of samples were then added to 900 μl of different concentrations of medium in a 96-well deep well plate and were further grown for 4.5 hours at 37^oC until the bacteria reached mid-log phase. The fluorescence output was then measured using an EnVision multilabel reader.

Characterization of the promoter dynamic range in Luria Broth (LB)

The concentrations used for the characterization of yeaRp was from 0 to 2000 μM nitrate, with 10 folds increase for each interval.

Characterization of promoter dynamic range in M9
The concentrations of the characterization of yeaRp was from 0 to 500 μM of nitrate, with intervals of 100 μM.