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

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                 <div id="MYicon2">
 
                 <div id="MYicon2">
 
                       <a href="https://2015.igem.org/Team:HKUST-Rice/Nitrate_Sensor_PdcuS"><img src="https://static.igem.org/mediawiki/2015/7/7a/HKUST-Rice15_rightarrow.png">
 
                       <a href="https://2015.igem.org/Team:HKUST-Rice/Nitrate_Sensor_PdcuS"><img src="https://static.igem.org/mediawiki/2015/7/7a/HKUST-Rice15_rightarrow.png">
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<p style="color:#5570b0; font-size: 130%"> Nitrate sensor (<i>dcuSp</i>) </p></a>
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<p style="color:#5570b0; font-size: 130%"> Nitrate sensor (p<sub>dcuS</sub>) </p></a>
 
</div>
 
</div>
  
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<div class="project_row">
 
<div class="project_row">
 
<h1>Introduction</h1>
 
<h1>Introduction</h1>
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<p>Phosphorus is vital to plant growth and is found in every living plant cell. It is a component of the nucleic acid structure of plants, which regulates protein synthesis. Therefore, it is important in cell division and development of new tissue. Plants deficient in phosphorus are stunted in growth and often have an abnormal dark-green color.
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<p>Phosphorus plays an essential role in plant growth. It associate with various growth factors for root development and seed
−
</p>
+
production, etc. Deficiency in Phosphorus lead to stunted growth, yet symptoms are not obvious. Therefore, it
 +
is important to monitor the level of both the organic and inorganic phosphorus level in soil for healthy plant growth.
 +
                    </p>
 
 
 
 
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</div>
 
</div>
 
 
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<p>P<sub>phoA</sub> and P<sub>phoBR</sub> is cross-regulated by P<sub>phoBR</sub> and <i>phoR</i>, and is usually repressed under high phosphate concentrations. PhoR behaves as an activator as well as an inactivator for PhoB. When phosphate is limited, PhoR will phosphorylate PhoB and the phosphorylated PhoB will directly activate P<sub>phoA</sub> and P<sub>phoBR</sub>. In contrast, when there is phosphate, PhoR will repress PhoB phosphorylation which in turn inactivates the P<sub>phoA</sub> and P<sub>phoBR</sub>.  
+
<p><i>Escherichia coli</i> detects inorganic phosphate (P(i)) from the environment by the PhoR/PhoB
 +
two-component system. As illustrated in Figure.1, P<sub>phoA</sub> and P<sub>phoBR</sub> is cross-regulated by PhoB and PhoR.
 +
The sensory histidine kinase PhoR can behaves as an activator or inactivator for PhoB depending on different states (inhibition state, activation state, deactivation state).  
 +
When phosphate is limited, PhoR act as a phospho-donor for the autophosphorylation of PhoB.
 +
The phosphorylated PhoB will directly activate P<sub>phoA</sub> and P<sub>phoBR</sub>. In contrast, when there is phosphate, PhoR interferes with phosphorylation of PhoB
 +
which in turn inactivates the P<sub>phoA</sub> and P<sub>phoBR</sub>.  
  
−
<br><br>For the constructs design, we have ligated GFP generator to P<sub>phoA</sub> and P<sub>phoBR</sub>, respecticely. As a result, under high phosphate concentrations, the green fluorescence intensity will be repressed; while under low phosphate concentrations, green fluorescence will be expressed.
+
−
</p>
+
                   
 +
<br><br>With the phosphate (Pho) regulon from <i>E. coli</i>,it can be utilized for detecting phosphate level. P<sub>phoA</sub> and  
 +
P<sub>phoBR</sub> from <i>E. coli</i> strain DH10B were cloned and ligated with the GFP generator (<a>pSB1C3-BBa_I13504</a>) respectively.
 +
Under high phosphate concentrations, repression on the green fluorescence intensity is expected; while under low phosphate  
 +
concentrations, expression on green fluorescence is expected.</p>
 
 
 
</div>
 
</div>
 
<div class="project_row">
 
<div class="project_row">
 
<hr class="para">
 
<hr class="para">
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<h1>Experiment performed</h1>
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<h1>Results obtained</h1>
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<p>We have done a characterization on P<sub>phoA</sub> and P<sub>phoBR</sub> using M9 minimal medium (without phosphate). Quantitative characterization on the promoters were done by measuring the fluorescence signal intensity using an EnVision multilabel reader.<br><br>The results were obtained by combining 3 characterization trials.
+
<p>Characterization on the contructs  (<a>BBa_K1682013</a>) using <a>M9 minimal medium (without phosphate)</a> were performed. Quantitative  
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<br><br>Please visit <a href="#Methods_2">Methods</a> for more details</p>
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characterization on the promoters were done by measuring the fluorescence signal intensity using an EnVision multilabel reader.<br><br>The  
−
</div>
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results were obtained by combining the 3 characterization results together.</p>
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<div class="project_row">
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−
<hr class="para">
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<h1>Result obtained</h1>
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<p>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.
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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.</p>
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<div class="project_image">
 
<div class="project_image">
 
<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
 
<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
 
</div>
 
</div>
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<p>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.
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<p>Eam in alienum accusamus, et probo reque vix. Vivendum necessitatibus qui ad, no vis enim veniam perpetua. Eu pri habemus
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senserit, dicit tation expetenda usu et. Sea eu dolor deserunt dissentias, sed an oportere moderatius assueverit. Usu te tation
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gloriatur, vidit tollit utinam mea id.</p>
 
 
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<br><br><p>Please visit <a href="#Methods_2">Methods</a> for more details</p>
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<p id="Methods_2">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.</p>
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</div>
 
</div>
  
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<div class="project_row">
 
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<hr class="para">
 
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<h1>Methods</h1>
 
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<p style="font-size:200%"><u>Preparing test medium with different concentrations of phosphate</u></p>
 
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<p> M9 minimal medium (J. Sambrook & D.W. Russell, 2001) and M9 minimal medium with Tris replacing phosphate were prepared. Test medium with different concentrations of phosphate (0, 10, 30, 50, 100, 150, 200, 250, and 300 µM) were made by mixing the 2 solutions in the following ratio: <br>60 μl of antibiotics was added in each test medium solution.</p>
 
  
−
                                <table border="1" style="width:100%; font-size:150%">
 
−
                                <tr>
 
−
                                  <td><b>Final phosphate concentration (μM)</b></td>
 
−
                                  <td><b>M9 minimal medium (μl)</b></td>
 
−
                                  <td><b>M9 minimal medium without <br>phosphate (replaced by Tris) (ml)</b></td>
 
−
                                 
 
−
                                </tr>
 
−
                                <tr>
 
−
                                  <td>0</td>
 
−
                                  <td>0.00</td>
 
−
                                  <td>60</td>
 
−
                                 
 
−
                                </tr>
 
−
                                <tr>
 
−
                                  <td>10</td>
 
−
                                  <td>8.58</td>
 
−
                                  <td>60</td>
 
−
                                 
 
−
                                </tr>
 
−
                                <tr>
 
−
                                  <td>30</td>
 
−
                                  <td>25.73</td>
 
−
                                  <td>60</td>
 
−
                                 
 
−
                                </tr>
 
−
                                <tr>
 
−
                                  <td>50</td>
 
−
                                  <td>42.85</td>
 
−
                                  <td>60</td>
 
−
                                 
 
−
                                </tr>
 
−
                                <tr>
 
−
                                  <td>100</td>
 
−
                                  <td>85.71</td>
 
−
                                  <td>60</td>
 
−
                                 
 
−
                                </tr>
 
−
                                <tr>
 
−
                                  <td>150</td>
 
−
                                  <td>130.43</td>
 
−
                                  <td>60</td>
 
−
                                 
 
−
                                </tr>
 
−
                                    <tr>
 
−
                                  <td>200</td>
 
−
                                  <td>171.42</td>
 
−
                                  <td>60</td>
 
−
                                                                  </tr>
 
−
                                </tr>
 
−
                                    <tr>
 
−
                                  <td>250</td>
 
−
                                  <td>216.26</td>
 
−
                                  <td>60</td>
 
−
                                 
 
−
                                </tr>
 
−
                                </tr>
 
−
                                    <tr>
 
−
                                  <td>300</td>
 
−
                                  <td>260.87</td>
 
−
                                  <td>60</td>
 
−
                                 
 
−
                                </tr>
 
−
                                  </table>
 
−
      <p style="font-size:200%"><u>P<sub>phoA</sub>and P<sub>phoBR</sub> characterization</u></p>
 
−
<p>To characterize P<sub>phoA</sub> and P<sub>phoBR</sub>, positive and negative controls were first grown overnight in Luria Broth (LB) medium containing Chloramphenicol at 37<sup>o</sup>C. The bacteria were then washed twice with 3 ml M9 minimal medium without phosphate (replaced by Tris), containing Ampicillin. Then, the cells were resuspended in 5 ml M9 minimal medium without phosphate (replaced by Tris) to obtain a final  OD<sub>600</sub> of 4. The cell suspension were then added to the test medium with different concentrations of phosphate (containing Chloramphenicol) in a 96-well deep well plate and further incubated at 37<sup>o</sup>C until the OD<sub>600</sub> of the cells reached the mid-log phase. The fluorescence output was then measured using EnVision multilabel reader. </p>
 
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</div>
 
 
 
  

Revision as of 08:30, 3 September 2015


Phosphate Sensor - PphoA , PphoBR

Introduction

Phosphorus plays an essential role in plant growth. It associate with various growth factors for root development and seed production, etc. Deficiency in Phosphorus lead to stunted growth, yet symptoms are not obvious. Therefore, it is important to monitor the level of both the organic and inorganic phosphorus level in soil for healthy plant growth.

Phosphate sensor Design

image caption

Escherichia coli detects inorganic phosphate (P(i)) from the environment by the PhoR/PhoB two-component system. As illustrated in Figure.1, PphoA and PphoBR is cross-regulated by PhoB and PhoR. The sensory histidine kinase PhoR can behaves as an activator or inactivator for PhoB depending on different states (inhibition state, activation state, deactivation state). When phosphate is limited, PhoR act as a phospho-donor for the autophosphorylation of PhoB. The phosphorylated PhoB will directly activate PphoA and PphoBR. In contrast, when there is phosphate, PhoR interferes with phosphorylation of PhoB which in turn inactivates the PphoA and PphoBR.

With the phosphate (Pho) regulon from E. coli,it can be utilized for detecting phosphate level. PphoA and PphoBR from E. coli strain DH10B were cloned and ligated with the GFP generator (pSB1C3-BBa_I13504) respectively. Under high phosphate concentrations, repression on the green fluorescence intensity is expected; while under low phosphate concentrations, expression on green fluorescence is expected.

Results obtained

Characterization on the contructs (BBa_K1682013) using M9 minimal medium (without phosphate) were performed. Quantitative characterization on the promoters were done by measuring the fluorescence signal intensity using an EnVision multilabel reader.

The results were obtained by combining the 3 characterization results together.

image caption

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.



Please visit Methods for more details