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

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<div id= "page_title"><h1>Phosphate Sensor - <i>phoAp</i> , <i>phoBRp</i></h1>
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<div id="MYicon1">
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                      <a href="https://2015.igem.org/Team:HKUST-Rice/Potassium_Sensor"><img src="https://static.igem.org/mediawiki/2015/e/ea/HKUST-Rice15_leftarrow.png">
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<p style="color:#5570b0; font-size: 130%"> Potassium sensor </p></a>
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                      <a href="https://2015.igem.org/Team:HKUST-Rice/Nitrate_Sensor_PducS"><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>ducSp</i>) </p></a>
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<div class="project_content">
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<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. It is also associated with complex energy transformations in the plant. Plants deficient in phosphorus are stunted in growth and often have an abnormal dark-green color.
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<div class="project_row">
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<hr class="para">
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<h1>Phosphate sensor Design</h1>
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                                    <div class="project_image">
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<img src="https://static.igem.org/mediawiki/2015/f/f4/Team_HKUST-Rice_2015_PhoAp_and_phoBRp_mechanism.PNG" alt="image caption">
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<p><i>phoAp</i> and <i>phoBRp</i> is cross-regulated by <i>phoB</i> 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 <i>phoAp</i> and <i>phoBRp</i>. In contrast, when there is phosphate, PhoR will repress PhoB phosphorylation which in turn inactivates the <i>phoAp</i> and <i>phoBRp</i>.
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<br><br>For the constructs design, we have ligated GFP generator to <i>phoAp</i> and <i>phoBRp</i>, 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.
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</p>
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</div>
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<div class="project_row">
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<hr class="para">
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<h1>Experiment performed</h1>
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<p>We have done a characterization on pSB1C3-<i>phoAp</i>-BBa_I13504 and pSB1C3-<i>phoBRp</i>-BBa_I13504 using Luria Broth (LB) medium. Quantitative characterization on the promoters were done by measuring the fluorescence signal intensity using an EnVision multilabel reader.
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<br><br><i>E. coli</i> strain DH10B was used, and the concentrations for the characterization of <i>phoAp</i> and <i>phoBRp</i> were from 0 to 300 µM phosphate, with intervals of 50 µM.
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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>We prepared a solution of M9 minimal medium (J. Sambrook & D.W. Russell, 2001) and a solution of M9 minimal medium with Tris replacing phosphate. 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: </p>
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                                <table border="1" style="width:100%; font-size:150%">
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                                <tr>
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                                  <td><b>Final phosphate concentration (μM)</b></td>
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                                  <td><b>M9 minimal medium (μl)</b></td>
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                                  <td><b>M9 minimal medium without phosphate (replaced by Tris) (ml)</b></td>
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                                  <td><b>Chloramphenicol (150 ng/μl) (μl)</b></td>
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                                </tr>
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                                <tr>
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                                  <td>0</td>
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                                  <td>0.00</td>
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                                  <td>60</td>
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                                  <td>60</td>
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                                </tr>
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                                <tr>
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                                  <td>10</td>
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                                  <td>8.58</td>
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                                  <td>60</td>
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                                  <td>60</td>
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                                </tr>
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                                <tr>
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                                  <td>30</td>
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                                  <td>25.73</td>
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                                  <td>60</td>
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                                  <td>60</td>
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                                </tr>
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                                <tr>
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                                  <td>50</td>
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                                  <td>42.85</td>
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                                  <td>60</td>
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                                  <td>60</td>
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                                </tr>
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                                <tr>
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                                  <td>100</td>
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                                  <td>85.71</td>
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                                  <td>60</td>
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                                  <td>60</td>
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                                </tr>
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                                <tr>
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                                  <td>150</td>
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                                  <td>130.43</td>
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                                  <td>60</td>
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                                  <td>60</td>
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                                </tr>
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                                    <tr>
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                                  <td>200</td>
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                                  <td>171.42</td>
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                                  <td>60</td>
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                                  <td>60</td>
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                                </tr>
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                                </tr>
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                                    <tr>
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                                  <td>250</td>
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                                  <td>216.26</td>
 +
                                  <td>60</td>
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                                  <td>60</td>
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                                </tr>
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                                </tr>
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                                    <tr>
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                                  <td>300</td>
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                                  <td>260.87</td>
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                                  <td>60</td>
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                                  <td>60</td>
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                                </tr>
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                                  </table>
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      <p style="font-size:200%"><u><i>phoAp</i> and <i>phoBRp</i> characterization</u></p>
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<p>To characterize pSB1C3-<i>phoAp</i>-BBa_I13504 and pSB1C3-<i>phoBRp</i>-BBa_I13504, positive and negative controls were first grown overnight in 5 ml 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. 50 μl of the prepared cell suspension were then added into 950 μl of 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.
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<br><br>This result was obtained by combining 3 characterization trials.</p>
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</div>
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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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<div class="project_image">
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<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
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Revision as of 11:26, 30 August 2015


Phosphate Sensor - phoAp , phoBRp

Introduction

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. It is also associated with complex energy transformations in the plant. Plants deficient in phosphorus are stunted in growth and often have an abnormal dark-green color.

Phosphate sensor Design

image caption

phoAp and phoBRp is cross-regulated by phoB and phoR, 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 phoAp and phoBRp. In contrast, when there is phosphate, PhoR will repress PhoB phosphorylation which in turn inactivates the phoAp and phoBRp.

For the constructs design, we have ligated GFP generator to phoAp and phoBRp, 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.

Experiment performed

We have done a characterization on pSB1C3-phoAp-BBa_I13504 and pSB1C3-phoBRp-BBa_I13504 using Luria Broth (LB) medium. Quantitative characterization on the promoters were done by measuring the fluorescence signal intensity using an EnVision multilabel reader.

E. coli strain DH10B was used, and the concentrations for the characterization of phoAp and phoBRp were from 0 to 300 µM phosphate, with intervals of 50 µM.

Preparing test medium with different concentrations of phosphate

We prepared a solution of M9 minimal medium (J. Sambrook & D.W. Russell, 2001) and a solution of M9 minimal medium with Tris replacing phosphate. 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:

Final phosphate concentration (μM) M9 minimal medium (μl) M9 minimal medium without phosphate (replaced by Tris) (ml) Chloramphenicol (150 ng/μl) (μl)
0 0.00 60 60
10 8.58 60 60
30 25.73 60 60
50 42.85 60 60
100 85.71 60 60
150 130.43 60 60
200 171.42 60 60
250 216.26 60 60
300 260.87 60 60

phoAp and phoBRp characterization

To characterize pSB1C3-phoAp-BBa_I13504 and pSB1C3-phoBRp-BBa_I13504, positive and negative controls were first grown overnight in 5 ml Luria Broth (LB) medium containing Chloramphenicol at 37oC. 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 OD600 of 4. 50 μl of the prepared cell suspension were then added into 950 μl of test medium with different concentrations of phosphate (containing Chloramphenicol) in a 96-well deep well plate and further incubated at 37oC until the OD600 of the cells reached the mid-log phase. The fluorescence output was then measured using EnVision multilabel reader.

This result was obtained by combining 3 characterization trials.

Result obtained

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.

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.