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, 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. As a result, the reporter signal will increase with increasing nitrate concentration.

Experiment that we did

We have done two sets of characterization on pSB1C3-BBa_K381001 (BCCS-Britstol 2010), one using Luria broth (LB) medium and the other in M9 minimal medium. 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)

Responsive range of promoter characterization in Luria broth (LB)
The concentration of the characterization of yeaRp promoter was from 0 to 50mM nitrate, with an intervals of 10mM. Potassium nitrate (KNO₃) 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. Escherichia coli (E. coli) strain DH10B was used in the characterization of the promoter.

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

The experiment were conducted three times.This result was obtained by combining 3 characterization trials.

Dynamic range characterization in Luria broth (LB)
Escherichia coli (E. coli)strain DH10B was used in the characterization of the promoter. The concentration of the characterization of yeaRp 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.

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

The experiment were conducted three times.This result was obtained by combining 3 characterization trials.

Growth Medium: M9

Responsive range of promoter characterization in M9
The concentration of the characterization of yeaRp promoter was from 0 to 2000μM 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 Escherichia coli (E. coli) strain DH10B was used in the characterization of the promoter.

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

The experiment were conducted three times.This result was obtained by combining 3 characterization trials.

Dynamic range characterization in M9
The concentration of the characterization of yeaRp 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. Escherichia coli (E. coli)strain DH10B was used in the characterization of the promoter.

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

The experiment were conducted three times.This result was obtained by combining 3 characterization trials.

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, 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 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.

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, by the overexpression of NsrR, the endogenous nitrate titrate against excess NsrR, 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, 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.