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

 
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<h1><i>E. coli</i> that glows in adequacy of NO<sub>3</sub><sup>-</sup> - at a glance</h1>
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<h1>Nitrate as a Macro-nutrient</h1>
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<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. Lack of nitrogen will lead to stunted growth, yellowing of leaves, etc.
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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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<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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                                        <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>
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<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>
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<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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<h1>Nitrate sensor Mechanism</h1>
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<h1> An effort to make iGEM a better community</h1>
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<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>
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<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>
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                                <p class="subTitle">Endogenous nitrate sensing system in <i>E. coli</i></p>
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<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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<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 <i>et al.</i>, 1989; Li et al., 1987) and NsrR regulatory protein (Partridge <i>et al.</i>, 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></div>
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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>
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<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>
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<h1>Nitrate sensor construct</h1>
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<h1>Nitrate Sensor construct</h1>
 
                            
 
                            
 
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<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 style="font-size:110%"><strong>Figure 2. Construct for nitrate sensing. </strong> <i>P<sub>yeaR</sub></i> with GFP generator.</p></div>
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<p>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>
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<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>
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<h1>Experiment performed</h1>
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<h1>Experiment Performed</h1>
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<p>Two sets of characterization on <a href="http://parts.igem.org/Part:BBa_K381001"target="_blank"> pSB1C3-BBa_K381001</a> (<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.  
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<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.  
 
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 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>
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<p>Please visit <a href="https://static.igem.org/mediawiki/2015/6/6d/Team_HKUST-Rice_2015_2_YeaRp_Experiment_Protocol.pdf"target="_blank"><i>P<sub>yeaR</sub></i> Experiment Protocol</a> for more details.  </p>
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<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>
  
  
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<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.
 
<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.
  
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<p><b>Dynamic range Characterization of <i>P<sub>yeaR</sub></i>  in LB</b></P>
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<p><b>Dynamic range Characterization of <i>P<sub>yeaR</sub></i>  in LB and M9</b></P>
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<p style="font-size:110%"><strong>Figure 3. Characterization of <i>P<sub>yeaR</sub></i> in LB.</strong> 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. (a) showing characterization within 0-10 mM concentration of nitrate; (b) showing characterization within 0-50 mM concentration of nitrate.</p> </div>
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According to Figure 3a, the relative fluorescence level increases 7.21 folds between 0 mM and 10 mM concentrations of nitrate. A continuous upward slope was obtained from 0 mM to 6 mM nitrate concentration. This 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, which may have been the cause in the promoter behavior difference. 
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<br><br>After obtaining the results of <i>P<sub>yeaR</sub></i> response behavior within 0-10 mM nitrate concentration, another characterization on 0-50 mM nitrate concentration was performed to further examine the behavior of <i>P<sub>yeaR</sub></i>. According to Figure 3b, 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 confirmed to be between 0-10 mM</p>
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<p><b>Dynamic range Characterization of <i>P<sub>yeaR</sub></i> in M9</b></P>
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<p style="font-size:110%"><strong>Figure 4. Characterization of <i>P<sub>yeaR</sub></i> in M9 minimal medium.</strong> 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. (a) showing characterization within 0-500 μM concentration of nitrate; (b) showing characterization within 0-1000 μM concentration of nitrate.</p> </div>
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<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>
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<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>
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<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>
  
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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.--> 
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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>
  
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<p>According to Figure 4a, the relative fluorescence level increases 3.12 folds from 0 μM and 500 μM nitrate concentrations.
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<p>According to <strong>B</strong>,
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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 4b, 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 confirmed to be between 0-500 μM.</p>  
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<!--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>  
 
 
  
 
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<h1>Further Improvements</h1>
 
<h1>Further Improvements</h1>
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<p>Since we were concerned that endogenous nitrate would affect the sensitivity of the promoter, a method in reducing the endogenous noise was designed.  
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<p>Since endogenous nitrate would affect the sensitivity of the promoter, a method in reducing the endogenous noise was designed.  
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<br><br>With P<sub>araBAD</sub> as an inducible promoter, 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 accurate in sensing nitrate concentrations.</p>
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<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: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 style="font-size:180%"><b>Rationale</b></p>
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<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 will not be able 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.</p>
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<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.
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<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>
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<p style="font-size:180%"><b>Result</b></p>
 
<p style="font-size:180%"><b>Result</b></p>
 
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<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>
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<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>
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<p>According to Figure 6, with L-arabinose added, the curve shifts downwards. 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>
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<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>
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<h2>Materials and Methods</h2>
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<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>
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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.

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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.