Nitrate as a Macro-nutrient

Nitrate is an essential nutrient that involves in 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). Second, it is a component of chlorophyll and 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 and yellowing of leaves, etc.

Nitrate sensor Design

yeaRp promoter (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 protein and relieve the repression on the yeaRp promoter. As a result, any genes that are downstream of the yeaRp promoter will be expressed. As a result, the reporter signal will increase with increasing nitrate concentration.

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Experiment performed

We have done 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.

We did quantitative characterization on the promoter by measuring the fluorescence signal intensity using an EnVision multilabel reader.

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.

The experiment were conducted three times and the final result was obtained by combining the 3 characterization trials.

Dynamic range characterization 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.

The experiment were conducted three times and the final result was obtained by combining the 3 characterization trials.

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.

The experiment was conducted three times and the final result was obtained by combining the 3 characterization trials.

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 further grew 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.

The experiment were conducted three times and the final result was obtained by combining the 3 characterization trials.

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Result obtained

Responsive range of promoter characterization in Luria Broth (LB)

After we have obtained 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 to obtain a result that, under low nitrate concentration, the Relative Fluorescence Unit (RFU) will be low and will increase according with increasing nitrate concentration.

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

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

Dynamic range characterization in Luria Broth (LB)

After we have obtained 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 to obtain a result that, under low nitrate concentration, the Relative Fluorescence Unit (RFU) will be low and will increase according with increasing nitrate concentration.

From the results obtained, the relative fluorescence level increases by 4.23 folds between 0mM and 10mM nitrate concentration. Moreover, it shows an upward slope from 0mM to 6mM nitrate concentration. At concentration point 8mM nitrate, it shows a downward slope and then rise again at 10mM nitrate. This result obtained is unexpected as according to previous work by BCCS-Bristol iGEM 2010, a continuous upward slope was obtained from 0mM to 9mM nitrate concentration. The discrepancy in the obtained result and the reference result could be due to use of different bacterial strain, since the strain used in BCCS-Bristol iGEM 2010 was different from ours, the behavior of the promoter may be different.

Responsive range of promoter characterization in M9

After we have obtained 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 to obtain a result that, under low nitrate concentration, the Relative Fluorescence Unit (RFU) will be low and will increase according with increasing nitrate concentration.

From the results obtained, the relative fluorescence level increases by 4.37 folds from 0mM and 2000μM nitrate concentration, and a plateau was shown from 500μM nitrate concentration point.

After obtaining the results of yeaRp promoter response behavior in the concentration of 0-2000μM nitrate, we find that the relative fluorescence level increases sharply between 0-500μM concentrations of nitrate. As a result, another characterization was done focusing on the dynamic range of the promoter, 0-500μM.

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Further Improvements

We were concerned with the endogenous nitrate will affect the sensitivity of the promoter, so, we have designed a method to reduce the endogenous noise.

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

As yeaRp promoter is regulated by Nar system and NsrR protein, by the overexpression of nsrR gene, the endogenous nitrate titrate against excess NsrR protein, so that nitrate could not 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, as a result, with the overexpression of nsrR gene, we expected to obtain a result that, the endogenous noise will be lowered, in which the relative fluorescence level at 0mM concentration of nitrate can be lowered to near 0.