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 (Lin, et al., 2007) is normally regulated by the Nar two-component regulatory system (T. Nohno, et al., 1989) and NsrR protein, a regulatory protein. When there is nitrate, some will relieve the repression from the Nar system and others will be converted into nitric oxide. The nitric oxide will bind to NsrR and relieve the repression on the yeaRp promoter. As a result, any genes that are downstream of the yeaRp promoter will be expressed. Therefore, with the GFP generator ligated, the reporter signal will increase with increasing nitrate concentrations.

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Experiment performed

We 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 our 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 the 3 characterization trials.

pSB1C3-BBa_K381001 characterization

Growth Medium: Luria Broth (LB)

Characterization of the promoter responsive range in Luria Broth (LB)
The concentrations used for the characterization of yeaRp was from 0 to 50 mM nitrate, with intervals of 10 mM.

Final nitrate concentration (mM)	LB (ml)	1M KNO3 (μl)	Chloramphenicol (150ng/μl) (μl)
0	10	0	10
9.89	10	100	10
19.58	10	200	10
29.10	10	300	10
38.42	10	400	10
47.57	10	500	10

The test samples were first grown in Luria Broth (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 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.

Final nitrate concentration (mM)	LB (ml)	1M KNO3 (μl)	Chloramphenicol (150ng/μl) (μl)
0	10	0	10
1.99	10	20	10
3.98	10	40	10
5.96	10	60	10
7.93	10	80	10
9.89	10	100	10

The test samples were first grown in Luria Broth (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.

Growth Medium: M9

Characterization of the promoter responsive range in M9
The concentrations used for the characterization of yeaRp was from 0 to 2000 μM nitrate, with 10 folds increase for each interval.

Final nitrate concentration (μM)	LB (ml)	1M KNO3 added(μl)	Chloramphenicol (150ng/μl) (μl)
0	10	0	10
19.98	10	0.2	10
199.76	10	2	10
1994.02	10	20	10

The test samples were first grown in Luria Broth (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 4.5 hours at 37^oC until the bacteria reach mid-log phase. The fluorescence output was then measured using an EnVision multilabel reader.

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

Final nitrate concentration (μM)	LB (ml)	1M KNO3 (μl)	Chloramphenicol (150ng/μl) (μl)
0	10	0	10
99.89	10	1	10
199.76	10	2	10
299.61	10	3	10
399.44	10	4	10
499.25	10	5	10

The test samples were first grown in Luria Broth (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 werefurther grown for 4.5 hours at 37^oC until the bacteria reached mid-log phase. The fluorescence output were then measured using an EnVision multilabel reader.

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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.

We expected that under low nitrate concentrations, the Relative Fluorescence Unit (RFU) will be low and this will increase according with increasing nitrate concentrations.

Responsive range of promoter characterization in Luria Broth (LB)

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.

Characterization of promoter dynamic range in Luria Broth (LB)

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.

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Further Improvements

Since we were concerned that endogenous nitrate will affect the sensitivity of the promoter, we designed a method to reduce the endogenous noise.

With araBADp as an inducible promoter, we aimed to find the concentration of arabinose that will most effectively reduce the most amount of endogenous noise, so that the promoter can be more sensitive.

As yeaRp is regulated by Nar system and NsrR protein, by overexpressing of NsrR, the endogenous nitrate will titrate against the excess NsrR protein, so that less nitrate will drive the transcription of yeaRp promoter. When there is nitrate in the environment, the amount of nitrate is enough to relieve the repression from the Nar system and NsrR protein. By NsrR overexpression, we expected that the endogenous noise will be lowered, in which the relative fluorescence level at 0 mM nitrate concentration can be lowered to near 0.