Team:HKUST-Rice/Nitrate Sensor PyeaR

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Nitrate Sensor

Nitrate as a Macro-nutrient

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Nitrate sensor Design

PyeaR promoter (Lin, et al, 2007) is normally cross-regulated by the Nar two-component regulatory system (T.Nohno, et,al. , 1989) and nsrR, 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 PyeaR promoter. As a result, any genes that are downstream of the PyeaR promoter will be expressed. As a result, the reporter signal will increase with increasing nitrate concentration.

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Experiment that we did

We have done two sets of characterization on pSB1C3-BBa_K381001 (BCCS-Britstol 2010), one using LB medium and the other in M9 minimal medium. We did quantitative characterization on the promoter by measuring the fluorescence signal intensity using a plate reader.

pSB1C3-BBa_K381001 characterization

Medium: LB

Responsive range of promoter characterization in LB
The concentration of the characterization of PyeaR promoter was from 0 to 50mM Nitrate, with an intervals of 10mM. Potassium Nitrate was being used as the source of nitrate in our experiments. 1M KNO3 stock solution was first made and added in the following ratios to produce different concentrations of medium. E. coli strain DH10B was used in the characterization of the promoter.

Nitrate concentration (mM)	LB (ml)	KNO3 (μl)	Antibiotics (μl)
0	10	0	10
10	10	100	10
20	10	200	10
30	10	300	10
40	10	400	10
50	10	500	10

The test samples were first grown in LB overnight at 37oC. They were then washed for 3 times using NaCl. 100ul of samples were then added with 900ul of different concentrations of medium in the 96-well deep well plates and further grew for 2.5 hours at 37oC until the OD600 of the cells is between 0.4-0.6, the mid-log phase. The fluorescence output were then measured using plate reader.

This result was obtained by combing 3 characterization trials, with biological triplicates and technical triplicates for each trial.

Working range characterization in LB
E. coli strain DH10B was used in the characterization of the promoter. The concentration of the characterization of PyeaR promoter was from 0 to 10mM of nitrate, with an interval of 2mM. Potassium Nitrate was being used as the source of nitrate in our experiments. 1M KNO3 stock solution was first made and added in the following ratios to produce different concentrations of medium.

Nitrate concentration (mM)	LB (ml)	KNO3 (μl)	Antibiotics (μl)
0	10	0	10
2	10	20	10
4	10	40	10
6	10	60	10
8	10	80	10
10	10	100	10

The test samples were first grown in LB overnight at 37oC. They were then washed for 3 times using NaCl. 100ul of samples were then added with 900ul of different concentrations of medium in the 96-well deep well plates and further grew for 2.5 hours at 37oC until the OD600 of the cells is between 0.4-0.6, the mid-log phase. The fluorescence output were then measured using plate reader.

This result was obtained by combing 3 characterization trials, with biological triplicates and technical triplicates for each trial.

Medium: M9

Responsive range of promoter characterization in M9
The concentration of the characterization of PyeaR promoter was from 0 to 2mM Nitrate, with 10 folds increase for each concentration Potassium Nitrate was being used as the source of nitrate in our experiments. 1M KNO3 stock solution was first made and added in the following ratios to produce different concentrations of medium.E. coli strain DH10B was used in the characterization of the promoter.

Nitrate concentration (μM)	LB (ml)	KNO3 (μl)	Antibiotics (μl)
0	10	0	10
20	10	0.2	10
200	10	2	10
2000	10	20	10

The test samples were first grown in LB overnight at 37oC. They were then washed for 3 times using NaCl. 100ul of samples were then added with 900ul of different concentrations of medium in the 96-well deep well plates and further grew for 4.5 hours at 37oC until the OD600 of the cells is between 0.4-0.6, the mid-log phase. The fluorescence output were then measured using plate reader.

This result was obtained by combing 3 characterization trials, with biological triplicates and technical triplicates for each trial.

Working range characterization in M9
The concentration of the characterization of PyeaR promoter was from 0 to 500μM of nitrate, with an interval of 100μM . Potassium Nitrate was being used as the source of nitrate in our experiments. 1M KNO3 stock solution was first made and added in the following ratios to produce different concentrations of medium. E. coli strain DH10B was used in the characterization of the promoter.

Nitrate concentration (μM)	LB (ml)	KNO3 (μl)	Antibiotics (μl)
0	10	0	10
100	10	1	10
200	10	2	10
300	10	3	10
400	10	4	10
500	10	5	10

The test samples were first grown in LB overnight at 37oC. They were then washed for 3 times using NaCl. 100ul of samples were then added with 900ul of different concentrations of medium in the 96-well deep well plates and further grew for 4.5 hours at 37oC until the OD600 of the cells is between 0.4-0.6, the mid-log phase. The fluorescence output were then measured using plate reader.

This result was obtained by combing 3 characterization trials, with biological triplicates and technical triplicates for each trial.

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Result obtained

Responsive range of promoter characterization in LB

After we have obtained the quantitative results on GFP signal intensity using plate 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 RFU value will be low and will increase according with increasing nitrate concentration.

From the results obtained, the relative fluorescence level increases by 5 folds between 0mM and 10mM concentration of nitrate. Furthermore, a plateau was shown from 10mM nitrate concentration point. This result is expected as according to the previous works by Edinburgh 2009 and BCCS-Bristol 2010, the working range of the promoter was from 0-10mM nitrate concentration.

After obtaining the results of PyeaR 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 working range of the promoter, 0-10mM.

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