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

 
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<div id= "page_title"><h1>Nitrate Sensor</h1>
+
<div id= "page_title"><h1>Nitrate Sensor - <i>P<sub>yeaR</sub></i> </h1>
 
</div>
 
</div>
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<div id="MYicon1">
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                      <a href="https://2015.igem.org/Team:HKUST-Rice/Nitrate_Sensor_PdcuS"><img src="https://static.igem.org/mediawiki/2015/e/ea/HKUST-Rice15_leftarrow.png">
 +
<p style="color:#5570b0; font-size: 130%"> Nitrate sensor (<i>P<sub>dcuS</sub></i>) </p></a>
 +
</div>
 +
                <div id="MYicon2">
 +
                      <a href="https://2015.igem.org/Team:HKUST-Rice/Expression"><img src="https://static.igem.org/mediawiki/2015/7/7a/HKUST-Rice15_rightarrow.png">
 +
<p style="color:#5570b0; font-size: 130%"> Parallel Sensors</p></a>
 +
</div>
 
 
 
<div class="project_content">
 
<div class="project_content">
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<!--<div class="project_row">
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<div class="project_row">  
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<h1>Nitrate as a Macro-nutrient</h1>
+
<h1><i>E. coli</i> that glows in adequacy of NO<sub>3</sub><sup>-</sup> - at a glance</h1>
−
<p>#</p>
+
 
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<div class="project_image">
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                                        <table>
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<td style="width:48.5%">
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<figure>
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<img  src="https://static.igem.org/mediawiki/2015/5/56/Team_HKUST-Rice_2015_pyeaRmech.PNG"style="width:100%;">
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</figure>
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</td>
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<td style="width:3%">
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</td>
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<td style="width:48.5%">
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<figure>
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<img src="https://static.igem.org/mediawiki/2015/6/60/Team_HKUST-Rice_2015_dumumpyear.PNG" style="width:100%">
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</figure>
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</td>
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<tr>
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<td style="width:48.5%">
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<p style="font-size:110%; padding-left:2%; padding-right: 2% ; height'90px';"><strong>A.</strong> <i>E. coli</i> engineered with <a href="http://parts.igem.org/Part:BBa_K381001"target="_blank">BBa_K381001</a> functions as a nitrate biosensor. High concentrations of NO<sub>3</sub><sup>-</sup> activates the promoter <i>P<sub>yeaR</sub></i> and increases the expression of GFP.</p>
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</td>
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<td style="width:3%">
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</td>
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<td style="width:48.5%">
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<p style="font-size:110%; padding-left:2;height:'90px'; padding-right: 2%"  ><strong>B.</strong> The nitrate sensing promoter <a href="http://parts.igem.org/Part:BBa_K381001"target="_blank">BBa_K381001</a> can detect a gradient of NO<sub>3</sub><sup>-</sup> concentrations</strong> and its activities were reported in Relative Fluorescence Units (RFU).</p>
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</td>
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</table>
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 +
                                        <p><ul style="text-align:left; font-size:1 em; line-height= 1 em; font-family: 'Helvetica Neue', Helvetica, sans-serif;"><li>Nitrate is an essential nutrient which plays multiple roles in plant growth and reproduction. </li>
 +
<li>This biosensor <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K381001">BBa_K381001</a> monitors NO<sub>3</sub><sup>-</sup> concentration.</li>
 +
<li>Activity of NO<sub>3</sub><sup>-</sup> sensing promoter (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K216005">BBa_K216005</a>) was re-characterized to further investigate on the behavior of it.</ul></p>
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</div>
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<div class="project_row">
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                                        <hr class="para">
 +
<h1> An effort to make iGEM a better community</h1>
 +
<p>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). Also, it is a component of chlorophyll and is therefore essential for photosynthesis.</p>
 +
<p><i>P<sub>yeaR</sub></i> is first characterized and BioBricked by <a href="https://2009.igem.org/Team:Edinburgh">Edinburgh 2009</a> iGEM team and then further characterized by <a href="https://2010.igem.org/Team:BCCS-Bristol/Wetlab/Part_Design/BioBricks/PyeaR"target="_blank">BCCS-Bristol 2010</a> iGEM team. To provide more characterization data on such a devices, we further characterize this promoter. </p>
 +
                               
 +
                                <hr class="para">                   
 +
                                <p class="subTitle">Endogenous nitrate sensing system in <i>E. coli</i></p>
 +
<img style="width:70%;" src="https://static.igem.org/mediawiki/2015/5/56/Team_HKUST-Rice_2015_pyeaRmech.PNG" alt="image caption">
 +
<p style="font-size:110%; padding-left:6%;"><strong>Figure 1. The NO<sub>3</sub><sup>-</sup> uptake system in  <i>E. coli</i>.</strong></p>
 
 
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<div class="project_row">
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<p><i>Escherichia coli</i> (<i>E. coli</i>) detects environmental nitrate by the <i>yeaR-yoaG</i> operon. According to Figure 1, <i>P<sub>yeaR</sub></i>  (Lin, <i>et al.</i>, 2007) is regulated by the Nar two-component regulatory system (Nohno et al., 1989; Li et al., 1987) and NsrR regulatory protein (Partridge et al., 2009). When there is nitrate or nitrite, the repression from the Nar system on <i>P<sub>yeaR</sub></i> will be relieved due to the binding between the two. On the other hand, some nitrate will be converted into nitric oxide by nitrate reductase. Nitric oxide will bind to the NsrR protein and relieve the repression on <i>P<sub>yeaR</sub></i>. As a result, any genes that are downstream of <i>P<sub>yeaR</sub></i> will be expressed.</p>
−
<h1>Nitrate sensor Design</h1>
+
<p style = "font-size:110%">*The above text is our summarized understanding on NO<sub>3</sub><sup>-</sup>-sensing system. Please refer to our references section below for a full list of references cited.</p></div>
 +
<!--<div class="project_row">
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<br><br><br>
 +
                                        <hr class="para">
 +
<h1>Nitrate Sensor construct</h1>
 +
                         
 
<div class="project_image">
 
<div class="project_image">
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<img src="https://static.igem.org/mediawiki/2015/7/7f/Team_HKUST_Rice_2015_PyeaR_mechanism.JPG" alt="image caption">
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<img style="width:50%;"src="https://static.igem.org/mediawiki/2015/f/f3/Team_HKUST-Rice_2015_pyearconstruct.PNG" alt="image caption">
 
</div>
 
</div>
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<p><i>yeaRp</i> promoter (Lin, et al, 2007) is normally regulated by the Nar two-component regulatory system (T.Nohno, et,al. , 1989) and <i>NsrR</i>, 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 <i>NsrR</i> and relieve the repression on the <i>yeaRp</i> promoter. As a result, any genes that are downstream of the <i>yeaRp</i> promoter will be expressed. As a result, the reporter signal will increase with increasing nitrate concentration.</p>
+
<div class="des">
−
+
<p style="font-size:110%"><strong>Figure 2. Construct for nitrate sensing. </strong> <i>P<sub>yeaR</sub></i> with GFP generator.</p></div>
−
+
<p>With the positive relationship between the promoter activity and nitrate concentration, by ligating the promoter together with the GFP generator (<a href="http://parts.igem.org/Part:BBa_K381001"target="_blank">pSB1C3-BBa_K381001</a>), an upward trend for the reporter signal with increasing nitrate concentrations was expected.</p>
−
</div>
+
</div>-->
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<div class="project_row">
+
<!--<div class="project_row">
 
<hr class="para">
 
<hr class="para">
−
<h1>Experiment that we did</h1>
+
−
<p>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.</P>
+
<h1>Experiment Performed</h1>
−
<h1 style="font-size: 180%"><b><u><font-size= "150%">pSB1C3-BBa_K381001 characterization</u></b><br><b></h1><p>Growth Medium: Luria broth (LB)</font></b><br><br><b>Responsive range of promoter characterization in Luria broth (LB)</b><br>
+
<p>Two sets of characterization on pSB1C3-BBa_K381001 (<a href="https://2010.igem.org/Team:BCCS-Bristol/Wetlab/Part_Design/BioBricks/PyeaR"target="_blank">BCCS-Bristol iGEM 2010</a>) in two different growth media, Luria Broth (LB) medium and M9 minimal medium were performed. M9 minimal medium was used as it does not contain nitrate and has a lower auto-fluorescence level, thus providing more accurate results.  
−
The concentration of the characterization of <i>yeaRp</i> promoter was from 0 to 50mM nitrate, with an intervals of 10mM.
+
Potassium nitrate (KNO<sub>3</sub>) was used as a source of nitrate in the experiments. <i>E. coli</i> 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 on different days and the final results were obtained by combining the 3 characterization results together.</p>
−
Potassium nitrate (KNO<sub>3</sub>) was being used as the source of nitrate in our experiments.
+
<p>Please visit <a href="https://static.igem.org/mediawiki/2015/b/b5/Team_HKUST-Rice_2015_2_pyearprotol.pdf"target="_blank"><i>P<sub>yeaR</sub></i> Experiment Protocol</a> for more details. </p>
−
1M KNO<sub>3</sub> stock solution was first made and added in the following ratios to produce different concentrations of medium.
+
−
Escherichia coli (<i>E. coli</i>) strain DH10B was used in the characterization of the promoter.</p>
+
−
                            <table border="1" style="width:100%; font-size: 150%">
+
−
                              <tr>
+
−
                                  <td><b>Final nitrate concentration (mM)</b></td>
+
−
                                  <td><b>LB (ml)</b></td>
+
−
                                  <td><b>1M KNO<sub>3</sub>added(μl)</b></td>
+
−
                                  <td><b>150ng/μl Chloramphenicol <br>Antibiotics added(μl)</b></td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>0</td>
+
−
                                  <td>10</td>
+
−
                                  <td>0</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>9.89</td>
+
−
                                  <td>10</td>
+
−
                                  <td>100</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>19.58</td>
+
−
                                  <td>10</td>
+
−
                                  <td>200</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>29.10</td>
+
−
                                  <td>10</td>
+
−
                                  <td>300</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>38.42</td>
+
−
                                  <td>10</td>
+
−
                                  <td>400</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>47.57</td>
+
−
                                  <td>10</td>
+
−
                                  <td>500</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                        </table>
+
−
                        <p>The test samples were first grown in Luria broth (LB) overnight at 37<sup>o</sup>C. 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<sup>o</sup>C until the bacteria reach mid-log phase. The fluorescence output were then measured using an EnVision multilabel reader.  
+
−
<br><br>The experiment were conducted three times.This result was obtained by combining 3 characterization trials.<br><br></p>
+
−
<p><b>Dynamic range characterization in Luria broth (LB)</b>
+
−
<br>Escherichia coli (<i>E. coli</i>)strain DH10B was used in the characterization of the promoter.
+
−
The concentration of the characterization of <i>yeaRp</i> 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 KNO<sub>3</sub> stock solution was first made and added in the following ratios to produce different concentrations of medium.</p>
+
−
                          <table border="1" style="width:100%; font-size: 150%">
+
−
                              <tr>
+
−
                                  <td><b>Final nitrate concentration (mM)</b></td>
+
−
                                  <td><b>LB (ml)</b></td>
+
−
                                  <td><b>1M KNO<sub>3</sub>added(μl)</b></td>
+
−
                                  <td><b>150ng/μl Chloramphenicol <br>Antibiotics added(μl)</b></td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>0</td>
+
−
                                  <td>10</td>
+
−
                                  <td>0</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>1.99</td>
+
−
                                  <td>10</td>
+
−
                                  <td>20</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>3.98</td>
+
−
                                  <td>10</td>
+
−
                                  <td>40</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>5.96</td>
+
−
                                  <td>10</td>
+
−
                                  <td>60</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>7.93</td>
+
−
                                  <td>10</td>
+
−
                                  <td>80</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>9.89</td>
+
−
                                  <td>10</td>
+
−
                                  <td>100</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                        </table>
+
−
 
+
−
<p>The test samples were first grown in Luria broth (LB) overnight at 37<sup>o</sup>C. 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<sup>o</sup>C until the bacteria reach mid-log phase. The fluorescence output were then measured using an EnVision multilabel reader.
+
−
<br><br>The experiment were conducted three times.This result was obtained by combining 3 characterization trials.</p>
+
−
 
+
−
<p><b>Growth Medium: M9</b>
+
−
<br><br><b>Responsive range of promoter characterization in M9</b>
+
−
<br>The concentration of the characterization of <i>yeaRp</i> 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 KNO<sub>3</sub> stock solution was first made and added in the following ratios to produce different concentrations of medium Escherichia coli (<i>E. coli</i>) strain DH10B was used in the characterization of the promoter.</p>
+
−
                    <table border="1" style="width:100%; font-size: 150%">
+
−
                              <tr>
+
−
                                  <td><b>Final nitrate concentration (μM)</b></td>
+
−
                                  <td><b>LB (ml)</b></td>
+
−
                                  <td><b>1M KNO<sub>3</sub>added(μl)</b></td>
+
−
                                  <td><b>150ng/μl Chloramphenicol <br>Antibiotics added(μl)</b></td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>0</td>
+
−
                                  <td>10</td>
+
−
                                  <td>0</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>19.98</td>
+
−
                                  <td>10</td>
+
−
                                  <td>0.2</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>199.76</td>
+
−
                                  <td>10</td>
+
−
                                  <td>2</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>1994.02</td>
+
−
                                  <td>10</td>
+
−
                                  <td>20</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                            </table>
+
−
<p>The test samples were first grown in Luria broth (LB) overnight at 37<sup>o</sup>C. 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<sup>o</sup>C until the bacteria reach mid-log phase. The fluorescence output were then measured using an EnVision multilabel reader.
+
−
<br><br>The experiment were conducted three times.This result was obtained by combining 3 characterization trials.</p>
+
−
 
+
−
<p><b>Dynamic range characterization in M9</b>
+
−
<br>The concentration of the characterization of <i>yeaRp</i> 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 KNO<sub>3</sub> stock solution was first made and added in the following ratios to produce different concentrations of medium.
+
−
Escherichia coli (<i>E. coli</i>)strain DH10B was used in the characterization of the promoter.
+
−
 
+
−
                            <table border="1" style="width:100%; font-size: 150%">
+
−
                              <tr>
+
−
                                  <td><b>Final nitrate concentration (μM)</b></td>
+
−
                                  <td><b>LB (ml)</b></td>
+
−
                                  <td><b>1M KNO<sub>3</sub>added(μl)</b></td>
+
−
                                  <td><b>150ng/μl Chloramphenicol <br>Antibiotics added(μl)</b></td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>0</td>
+
−
                                  <td>10</td>
+
−
                                  <td>0</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>99.89</td>
+
−
                                  <td>10</td>
+
−
                                  <td>1</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>199.76</td>
+
−
                                  <td>10</td>
+
−
                                  <td>2</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>299.61</td>
+
−
                                  <td>10</td>
+
−
                                  <td>3</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>399.44</td>
+
−
                                  <td>10</td>
+
−
                                  <td>4</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                              <tr>
+
−
                                  <td>499.25</td>
+
−
                                  <td>10</td>
+
−
                                  <td>5</td>
+
−
                                  <td>10</td>
+
−
                              </tr>
+
−
                            </table>
+
−
<p>The test samples were first grown in Luria broth (LB) overnight at 37<sup>o</sup>C. 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<sup>o</sup>C until of the bacteria reach mid-log phase. The fluorescence output were then measured using an EnVision multilabel reader.
+
−
<br><br>The experiment were conducted three times.This result was obtained by combining 3 characterization trials.</p>
+
  
  
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</div>-->
 
</div>-->
 
 
−
</div>
+
</div>-->
 
<div class="project_row">
 
<div class="project_row">
 
<hr class="para">
 
<hr class="para">
−
<h1>Result obtained</h1>
+
<h1>Results</h1>
−
<p><b>Responsive range of promoter characterization in Luria broth (LB)</b></P>
+
<p>After obtaining the quantitative results of GFP signal intensity using an EnVision® multilabel reader, the fluorescence signal were represented in fluorescence divided by biomass.
−
<div class="project_image">
+
 
−
<img src="https://static.igem.org/mediawiki/2015/6/6e/Team_HKUST-Rice_2015_LB_0-50_yeaRp.PNG" alt="image caption">
+
<p><b>Dynamic range Characterization of <i>P<sub>yeaR</sub></i>  in LB and M9</b></P>
−
</div>
+
 
−
<p></p>
+
<table>
 +
<tr>
 +
<td style="width:48.5%">
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2015/c/c6/Team_HKUST-Rice_2015_LBlallal.PNG"style="width:100%;">
 +
</figure>
 +
</td>
 +
<td style="width:3%">
 +
</td>
 +
<td style="width:48.5%">
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2015/a/a4/Team_HKUST-Rice_M9llallal.PNG" style="width:100%">
 +
</figure>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="width:48.5%">
 +
<p style="font-size:110%; padding-left:2%; padding-right: 2% ; height'90px';"><strong>A.</strong> Characterization of <i>P<sub>yeaR</sub></i> in LB. </p>
 +
</td>
 +
<td style="width:3%">
 +
</td>
 +
<td style="width:48.5%">
 +
<p style="font-size:110%; padding-left:10%;height:'90px'; padding-right: 2%"  ><strong>B.</strong> Characterization of <i>P<sub>yeaR</sub></i> in M9 minimal medium. </p>
 +
</td>
 +
</tr>
 +
</table>
 +
<p style="font-size:110%">*GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 charaterization data obtained in 3 different days. Error bars were presented in SEM.</p>
 +
 
 +
<p>
 +
According to <strong>A</strong>,<!-- the result obtained was unexpected, 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 between the obtained and reference results could be due to the use of different bacterial strains. The strain used by the BCCS-Bristol iGEM 2010 team was MG1655, while we were using DH10B.--> 
 +
a plateau was shown starting from the 10 mM concentration point, suggesting that 10 mM nitrate concentration is the saturation point of <i>P<sub>yeaR</sub></i> and the dynamic range of <i>P<sub>yeaR</sub></i> is shown to be between 0-10 mM in our study. The relative fluorescence level increases 7.21 folds between 0 mM and 10 mM concentrations of nitrate.</p>
 +
 
 +
<p>According to <strong>B</strong>, 
 +
<!--After obtaining the results of <i>P<sub>yeaR</sub></i> response behavior in the concentrations of 0-500 μM nitrate, another characterization was was performed to further examine the behavior of <i>P<sub>yeaR</sub></i>. <br><br>According to Figure 3b,-->a plateau was shown starting from the 500 μM concentration point, suggesting that 500 μM nitrate concentration is the saturation point of <i>P<sub>yeaR</sub></i> and the dynamic range of <i>P<sub>yeaR</sub></i> is shown to be between 0-500 μM in our study. The relative fluorescence level increases 3.12 folds from 0 μM and 500 μM nitrate concentrations.</p>  
 
 
−
<p>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 OD<sub>600</sub>) against nitrate concentration.
 
−
<br><br>We expected to obtain a result that, under low nitrate concentration, the Relative Fluorescence Unit will be low and will increase according with increasing nitrate concentration.
 
−
<br><br>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 <i>yeaRp</i> was from 0-10mM nitrate concentration.
 
−
<br><br>After obtaining the results of <i>yeaRp</i> 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.
 
−
</p>
 
−
<p><b>Dynamic range characterization in Luria broth (LB)</b></P>
 
−
                                        <div class="project_image">
 
−
<img src="https://static.igem.org/mediawiki/2015/7/71/Team_HKUST-Rice_2015-LB_0-10_yeaRp.PNG" alt="image caption">
 
−
</div>
 
−
<p>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 OD<sub>600</sub>) against nitrate concentration.
 
−
<br><br>We expected to obtain a result that, under low nitrate concentration, the Relative Fluorescence Unit will be low and will increase according with increasing nitrate concentration.
 
−
<br><br>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.
 
−
 
−
<p><b>Responsive range of promoter characterization in M9</b></P>
 
−
                                        <div class="project_image">
 
−
<img src="https://static.igem.org/mediawiki/2015/2/25/Team_HKUST1-Rice_2015_M9_0-2000_yeaRp.PNG" alt="image caption">
 
−
</div>
 
−
<p>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 OD<sub>600</sub>) against nitrate concentration.
 
−
<br><br>We expected to obtain a result that, under low nitrate concentration, the Relative Fluorescence Unit will be low and will increase according with increasing nitrate concentration.
 
−
<br><br>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.
 
−
<br><br>After obtaining the results of <i>yeaRp</i> 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.</p>
 
  
 
</div>
 
</div>
−
<div class="project_row">
+
</div>
 +
<!--<div class="project_row">
 
<hr class="para">
 
<hr class="para">
 
<h1>Further Improvements</h1>
 
<h1>Further Improvements</h1>
−
<p>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.  
+
<p>Since endogenous nitrate would affect the sensitivity of the promoter, a method in reducing the endogenous noise was designed.  
−
+
−
<br><br>With <i>AraBADp</i> 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.</p>
+
 
                                         <div class="project_image">
 
                                         <div class="project_image">
−
<img src="https://static.igem.org/mediawiki/2015/e/ee/Team_HKUST-Rice_2015_Pyear_debug_mechanism.JPG" alt="image caption">
+
<img style="width:80%;"src="https://static.igem.org/mediawiki/2015/d/d8/Team_HKUST-Rice_2015_nitrate_debug.PNG" alt="image caption">
 +
</div>
 +
<div class="des">
 +
<p style="font-size:110%"><strong>Figure 5. Construct for endogenous noise reduction of <i>P<sub>yeaR</sub></i>.</strong></p></div>
 +
<p style="font-size:180%"><b>Rationale</b></p>
 +
<p id="Methods">As <i>P<sub>yeaR</sub></i> is regulated by the Nar system and NsrR protein, by overexpressing NsrR protein, endogenous nitrate alone is not likely to drive the transcription of <i>P<sub>yeaR</sub></i>. On the other hand, when there is nitrate in the environment, the amount of nitrate is enough to relieve the repression from the Nar system and NsrR protein, and transcription would result. With this method, less effects from the endogenous noise on the promoter is expected.
 +
 +
<br><br>With the inducible promoter <i>P<sub>araBAD</sub></i>, experiments were carried out to find the concentration of L-Arabinose which could reduce endogenous noise most effectively, so that the promoter could be more sensitive in detecting nitrate concentrations.</p>
 +
 
 +
<p style="font-size:180%"><b>Result</b></p>
 +
<div class="project_image">
 +
<img style="margin-left:-5%;width:90%;height:300px" src="https://static.igem.org/mediawiki/2015/c/cf/Team_HKUST-Rice_2015_debug1vv.PNG" alt="image caption">
 +
</div>
 +
<div class="project_image">
 +
<img style="margin-left:-5%;width:90%;height:300px" src="https://static.igem.org/mediawiki/2015/0/00/Team_HKUST-Rice_2015_debug2vv.PNG" alt="image caption">
 
</div>
 
</div>
−
<p>As <i>yeaRp</i> promoter is regulated by Nar system and <i>NsrR</i>, by the overexpression of <i>NsrR</i>, the endogenous nitrate titrate against excess NsrR, so that nitrate could not drive the transcription of <i>yeaRp</i> promoter.When there is nitrate in the environment, the amount of nitrate is enough to relieve the repression from the Nar system and <i>NsrR</i>, as a result, with the overexpression of <i>NsrR</i>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. </p>
 
−
</div>
 
  
−
+
<div class="des">
−
+
<p style="font-size:110%"><strong>Figure 6. Endogenous noise reduction of <i>P<sub>yeaR</sub></i>.</strong> 0.01mM, 0.1mM, 1mM and 10mM of L-Arabinose was added for inducing <i>P<sub>araBAD</sub></i>. With different concentrations of NsrR protein produced, the endogenous noise was reduced accordingly.</p></div>
−
</div>
+
<p>According to Figure 6, with L-Arabinose added, the curve shifts downwards, suggesting the sensitivity of the promoter was being enhanced. However, as the result obtained is similar to that of <a href="https://2015.igem.org/Team:HKUST-Rice/Expression#co-expression">Parallel Sensors</a>, it is uncertain that the downward shifting was due to co-expression of promoters or the method for endogenous noise reduction. </p>
−
<hr class= "title">
+
</div>-->
−
</div>
+
                                <div class="project_row">
 +
                          <hr class="para">
 +
<h2>Materials and Methods</h2>
 +
<p>Please refer to <a href ="https://2015.igem.org/Team:HKUST-Rice/Protocol">our protocol page for the materials and methods used in characterization.</a></p>
 +
</div>
 +
                                <div class="project_row">
 +
<hr class="para">
 +
<h2>References</h2>
 +
                                <p style="font-size:125%">Li, S. F., & DeMoss, J. A. (1987). Promoter region of the nar operon of Escherichia coli: nucleotide sequence and transcription initiation signals.Journal of bacteriology, 169(10), 4614-4620.
 +
<br><br>Lin, H. Y., Bledsoe, P. J., & Stewart, V. (2007). Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen-responsive regulator Fnr in Escherichia coli K-12. Journal of bacteriology, 189(21), 7539-7548.
 +
<br><br>Nohno, T., Noji, S., Taniguchi, S., & Saito, T. (1989). The narX and narL genes encoding the nitrate-sensing regulators of Escherichia coli are homologous to a family of prokaryotic two-component regulatory genes. Nucleic acids research,17(8), 2947-2957.
 +
<br><br>Partridge, J. D., Bodenmiller, D. M., Humphrys, M. S., & Spiro, S. (2009). NsrR targets in the Escherichia coli genome: new insights into DNA sequence requirements for binding and a role for NsrR in the regulation of motility.Molecular microbiology, 73(4), 680-694.</p></div>
 +
 
 +
                               
 +
 
 +
 
 +
 +
 +
 
</div>
 
</div>
 
 
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{{HKUST-Rice Directory}}

Latest revision as of 02:23, 19 September 2015


Nitrate Sensor - PyeaR

E. coli that glows in adequacy of NO3- - at a glance

A. E. coli engineered with BBa_K381001 functions as a nitrate biosensor. High concentrations of NO3- activates the promoter PyeaR and increases the expression of GFP.

B. The nitrate sensing promoter BBa_K381001 can detect a gradient of NO3- concentrations and its activities were reported in Relative Fluorescence Units (RFU).

  • Nitrate is an essential nutrient which plays multiple roles in plant growth and reproduction.
  • This biosensor BBa_K381001 monitors NO3- concentration.
  • Activity of NO3- sensing promoter (BBa_K216005) was re-characterized to further investigate on the behavior of it.

An effort to make iGEM a better community

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). Also, it is a component of chlorophyll and is therefore essential for photosynthesis.

PyeaR is first characterized and BioBricked by Edinburgh 2009 iGEM team and then further characterized by BCCS-Bristol 2010 iGEM team. To provide more characterization data on such a devices, we further characterize this promoter.

Endogenous nitrate sensing system in E. coli

image caption

Figure 1. The NO3- uptake system in E. coli.

Escherichia coli (E. coli) detects environmental nitrate by the yeaR-yoaG operon. According to Figure 1, PyeaR (Lin, et al., 2007) is regulated by the Nar two-component regulatory system (Nohno et al., 1989; Li et al., 1987) and NsrR regulatory protein (Partridge et al., 2009). When there is nitrate or nitrite, the repression from the Nar system on PyeaR will be relieved due to the binding between the two. On the other hand, some nitrate will be converted into nitric oxide by nitrate reductase. Nitric oxide will bind to the NsrR protein and relieve the repression on PyeaR. As a result, any genes that are downstream of PyeaR will be expressed.

*The above text is our summarized understanding on NO3--sensing system. Please refer to our references section below for a full list of references cited.

-->

Results

After obtaining the quantitative results of GFP signal intensity using an EnVision® multilabel reader, the fluorescence signal were represented in fluorescence divided by biomass.

Dynamic range Characterization of PyeaR in LB and M9

A. Characterization of PyeaR in LB.

B. Characterization of PyeaR in M9 minimal medium.

*GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 charaterization data obtained in 3 different days. Error bars were presented in SEM.

According to A, a plateau was shown starting from the 10 mM concentration point, suggesting that 10 mM nitrate concentration is the saturation point of PyeaR and the dynamic range of PyeaR is shown to be between 0-10 mM in our study. The relative fluorescence level increases 7.21 folds between 0 mM and 10 mM concentrations of nitrate.

According to B, a plateau was shown starting from the 500 μM concentration point, suggesting that 500 μM nitrate concentration is the saturation point of PyeaR and the dynamic range of PyeaR is shown to be between 0-500 μM in our study. The relative fluorescence level increases 3.12 folds from 0 μM and 500 μM nitrate concentrations.

Materials and Methods

Please refer to our protocol page for the materials and methods used in characterization.

References

Li, S. F., & DeMoss, J. A. (1987). Promoter region of the nar operon of Escherichia coli: nucleotide sequence and transcription initiation signals.Journal of bacteriology, 169(10), 4614-4620.

Lin, H. Y., Bledsoe, P. J., & Stewart, V. (2007). Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen-responsive regulator Fnr in Escherichia coli K-12. Journal of bacteriology, 189(21), 7539-7548.

Nohno, T., Noji, S., Taniguchi, S., & Saito, T. (1989). The narX and narL genes encoding the nitrate-sensing regulators of Escherichia coli are homologous to a family of prokaryotic two-component regulatory genes. Nucleic acids research,17(8), 2947-2957.

Partridge, J. D., Bodenmiller, D. M., Humphrys, M. S., & Spiro, S. (2009). NsrR targets in the Escherichia coli genome: new insights into DNA sequence requirements for binding and a role for NsrR in the regulation of motility.Molecular microbiology, 73(4), 680-694.