Difference between revisions of "Team:HKUST-Rice/Nitrate Sensor PyeaR"

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Potassium Nitrate was being used as the source of nitrate in our experiments.
 
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.</p>
 
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.</p>
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                    <table border="1" style="width:100%; font-size: 150%">
 +
                              <tr>
 +
                                  <td><b>Nitrate concentration (μM)</b></td>
 +
                                  <td><b>LB (ml)</b></td>
 +
                                  <td><b>KNO3 (μl)</b></td>
 +
                                  <td><b>Antibiotics (μl)</b></td>
 +
                              </tr>
 +
                              <tr>
 +
                                  <td>0</td>
 +
                                  <td>10</td>
 +
                                  <td>0</td>
 +
                                  <td>10</td>
 +
                              </tr>
 +
                              <tr>
 +
                                  <td>20</td>
 +
                                  <td>10</td>
 +
                                  <td>0.2</td>
 +
                                  <td>10</td>
 +
                              </tr>
 +
                              <tr>
 +
                                  <td>200</td>
 +
                                  <td>10</td>
 +
                                  <td>2</td>
 +
                                  <td>10</td>
 +
                              </tr>
 +
                              <tr>
 +
                                  <td>2000</td>
 +
                                  <td>10</td>
 +
                                  <td>20</td>
 +
                                  <td>10</td>
 +
                              </tr>
 +
                            </table>
  
 
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Revision as of 03:41, 29 August 2015


Nitrate Sensor

Nitrate as a Macro-nutrient

Nitrate sensor Design

image caption

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.

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

Result obtained

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image caption

Lorem ipsum dolor sit amet, pro aeque temporibus eu, eum qualisque assueverit te. Ad est admodum epicuri suscipit, te alterum aliquando adversarium usu, pro ex omnesque luptatum comprehensam. In vix alia percipit gloriatur, no ferri lorem aliquando cum. Fugit concludaturque sed ne, ea sumo dico adolescens eos, quo eu pertinax expetendis. An his omnes instructior, vide possim eam id. Te cum enim sale offendit, vocent copiosae luptatum ut per. Eam in alienum accusamus, et probo reque vix. Vivendum necessitatibus qui ad, no vis enim veniam perpetua. Eu pri habemus senserit, dicit tation expetenda usu et. Sea eu dolor deserunt dissentias, sed an oportere moderatius assueverit. Usu te tation gloriatur, vidit tollit utinam mea id.