Difference between revisions of "Team:HKUST-Rice/Phosphate Sensor"
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| − | <div id= "page_title"><h1>Phosphate Sensor - P<sub>phoA</sub> , P<sub>phoBR</sub></h1> | + | <div id= "page_title"><h1>Phosphate Sensor - <i>P<sub>phoA</sub></i> , <i>P<sub>phoBR</sub></i></h1> |
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| − | <p><i>Escherichia coli </i>(<i>E. coli</i>) detects inorganic phosphate (P(i)) from the environment by the PhoR/PhoB two-component system (Hsieh & Wanner, 2010). 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 behaves either 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 both P<sub>phoA</sub> and P<sub>phoBR</sub>. </p> | + | <p><i>Escherichia coli </i>(<i>E. coli</i>) detects inorganic phosphate (P(i)) from the environment by the PhoR/PhoB two-component system (Hsieh & Wanner, 2010). As illustrated in Figure 1, <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i> is cross-regulated by PhoB and PhoR. The sensory histidine kinase PhoR behaves either 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 <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i>. In contrast, when there is phosphate, PhoR interferes with phosphorylation of PhoB which in turn inactivates both <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i>. </p> |
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<img style="width:45%; height:400px; float:left; padding-right:10px; margin-top:-10px" src="https://static.igem.org/mediawiki/2015/a/ae/Team_HKUST-Rice_2015_phosphatecc.PNG" alt="image caption"><br><br></div> | <img style="width:45%; height:400px; float:left; padding-right:10px; margin-top:-10px" src="https://static.igem.org/mediawiki/2015/a/ae/Team_HKUST-Rice_2015_phosphatecc.PNG" alt="image caption"><br><br></div> | ||
<div class="des"> | <div class="des"> | ||
| − | <p style="font-size:110%; float:left; padding-top:0px;"><strong>Figure 2. Phosphate sensor constructs.</strong> (a) P<sub>phoA</sub> with GFP generator and (b) P<sub>phoBR</sub> with GFP generator.</strong></p> </div><br><br> | + | <p style="font-size:110%; float:left; padding-top:0px;"><strong>Figure 2. Phosphate sensor constructs.</strong> (a) <i>P<sub>phoA</sub></i> with GFP generator and (b) <i>P<sub>phoBR</sub></i> with GFP generator.</strong></p> </div><br><br> |
| − | <p>With the phosphate (<i>pho</i>) 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 was cloned and ligated with the GFP generator (<a href="http://parts.igem.org/Part:BBa_I13504"target="_blank">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> | + | <p>With the phosphate (<i>pho</i>) regulon from <i>E. coli</i>, it can be utilized for detecting phosphate level. <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i> from <i>E. coli</i> strain DH10B was cloned and ligated with the GFP generator (<a href="http://parts.igem.org/Part:BBa_I13504"target="_blank">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> |
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<img style="margin-left:-10%;width: 80%;" src="https://static.igem.org/mediawiki/2015/f/ff/Team_HKUST-Rice_2015_phospateresult.PNG" alt="image caption"></div> | <img style="margin-left:-10%;width: 80%;" src="https://static.igem.org/mediawiki/2015/f/ff/Team_HKUST-Rice_2015_phospateresult.PNG" alt="image caption"></div> | ||
<div class="des"> | <div class="des"> | ||
| − | <p style="font-size:110%"><strong>Figure 3. Characterization of P<sub>phoA</sub> and P<sub>phoBR</sub> in M9 minimal medium.</strong> | + | <p style="font-size:110%"><strong>Figure 3. Characterization of <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i> in M9 minimal medium.</strong> |
| − | GFP expression of pSB1C3-P<sub>phoA</sub>-BBa_I13504 and pSB1C3-P<sub>phoBR</sub>-BBa_I13504 in 0, 10, 30, 50, 100, 150, 200 and 300 µM phosphate concentration are shown. Error bars were presented in SEM. | + | GFP expression of pSB1C3-<i>P<sub>phoA</sub></i>-BBa_I13504 and pSB1C3-<i>P<sub>phoBR</sub></i>-BBa_I13504 in 0, 10, 30, 50, 100, 150, 200 and 300 µM phosphate concentration are shown. Error bars were presented in SEM. |
.</p> </div> | .</p> </div> | ||
| − | <p>As shown in Figure 3a, P<sub>phoA</sub> is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 2.99 folds between 0 to 200μM concentration of phosphate. Furthermore, a plateau is observed starting from the 200 μM phosphate concentration point, suggesting that the dynamic range of P<sub>phoA</sub> is from 0-200 μM of phosphate. | + | <p>As shown in Figure 3a, <i>P<sub>phoA</sub></i> is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 2.99 folds between 0 to 200μM concentration of phosphate. Furthermore, a plateau is observed starting from the 200 μM phosphate concentration point, suggesting that the dynamic range of <i>P<sub>phoA</sub></i> is from 0-200 μM of phosphate. |
| − | <br><br>As shown in Figure 3b, P<sub>phoBR</sub> is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 1.88 folds between 0 to 250 μM concentration of phosphate. Furthermore, a plateau is observed starting from the 250μM of phosphate concentration point, suggesting that the dynamic range of P<sub>phoBR</sub> is from 0-250μM of phosphate. | + | <br><br>As shown in Figure 3b, <i>P<sub>phoBR</sub></i> is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 1.88 folds between 0 to 250 μM concentration of phosphate. Furthermore, a plateau is observed starting from the 250μM of phosphate concentration point, suggesting that the dynamic range of <i>P<sub>phoBR</sub></i> is from 0-250μM of phosphate. |
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<hr class="para"> | <hr class="para"> | ||
<h1>Discussion</h1> | <h1>Discussion</h1> | ||
| − | <p>In order to select a better phosphate sensor, the fold change of P<sub>phoA</sub> and P<sub>phoBR</sub> were being compared.</p> | + | <p>In order to select a better phosphate sensor, the fold change of <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i> were being compared.</p> |
<div class="project_image"> | <div class="project_image"> | ||
<img style="width:auto;height:300px" src="https://static.igem.org/mediawiki/2015/9/95/Team_HKUST-Rice_2015_Phosphate_comparsion.PNG" alt="image caption"> | <img style="width:auto;height:300px" src="https://static.igem.org/mediawiki/2015/9/95/Team_HKUST-Rice_2015_Phosphate_comparsion.PNG" alt="image caption"> | ||
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| − | <p style="font-size:110%"><strong>Figure 4. Fold change of P<sub>phoA</sub> and P<sub>phoBR</sub> GFP expression.</strong>The fold change of P<sub>phoA</sub> and P<sub>phoBR</sub> GFP expression between 0 and 250 μM of phosphate. For P<sub>phoA</sub>, the relative fluorescence intensity of 0 to 250 μM of phosphate is about 2.99 folds. For P<sub>phoBR</sub>, it is about 1.88 folds. Error bars were presented in SEM.</p></div> | + | <p style="font-size:110%"><strong>Figure 4. Fold change of <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i> GFP expression.</strong>The fold change of <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i> GFP expression between 0 and 250 μM of phosphate. For <i>P<sub>phoA</sub></i>, the relative fluorescence intensity of 0 to 250 μM of phosphate is about 2.99 folds. For <i>P<sub>phoBR</sub></i>, it is about 1.88 folds. Error bars were presented in SEM.</p></div> |
</div> | </div> | ||
| − | <p>The fold change of P<sub>phoA</sub> GFP expression is greater than that of P<sub>phoBR</sub> between 0 and 250μM of phosphate. Since the GFP expression gradient of P<sub>phoA</sub> construct is more significant, it is better to use P<sub>phoA</sub> to differentiate various phosphate concentration in the soil. | + | <p>The fold change of <i>P<sub>phoA</sub></i> GFP expression is greater than that of <i>P<sub>phoBR</sub></i> between 0 and 250μM of phosphate. Since the GFP expression gradient of <i>P<sub>phoA</sub></i> construct is more significant, it is better to use <i>P<sub>phoA</sub></i> to differentiate various phosphate concentration in the soil. |
</div> | </div> | ||
Revision as of 11:03, 15 September 2015
Phosphate Sensor - PphoA , PphoBR
Introduction
Phosphorus plays an essential role in plant growth. It associates with various growth factors for root development, seed production, etc. Deficiency in phosphorus leads to stunted plant growth, yet the symptoms are not obvious. Therefore, it is important to monitor the levels of phosphate in the soil for healthy plant growth.
Phosphate sensor Mechanism
Escherichia coli (E. coli) detects inorganic phosphate (P(i)) from the environment by the PhoR/PhoB two-component system (Hsieh & Wanner, 2010). As illustrated in Figure 1, PphoA and PphoBR is cross-regulated by PhoB and PhoR. The sensory histidine kinase PhoR behaves either 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 both PphoA and PphoBR.
Phosphate sensor Design
Figure 2. Phosphate sensor constructs. (a) PphoA with GFP generator and (b) PphoBR with GFP generator.
With the phosphate (pho) regulon from E. coli, it can be utilized for detecting phosphate level. PphoA and PphoBR from E. coli strain DH10B was 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.
Experiments performed
Characterization on the constructs (BBa_K1682013) was done using M9 minimal medium (without phosphate). 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.
Please visit Phosphate sensor Experiment Protocol for more details.
Results
After measuring the GFP signal intensity using an EnVision® multilabel reader, the data were processed in Relative Fluorescence Unit (RFU) (in OD600) against phosphate concentration.
Figure 3. Characterization of PphoA and PphoBR in M9 minimal medium. GFP expression of pSB1C3-PphoA-BBa_I13504 and pSB1C3-PphoBR-BBa_I13504 in 0, 10, 30, 50, 100, 150, 200 and 300 µM phosphate concentration are shown. Error bars were presented in SEM. .
As shown in Figure 3a, PphoA is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 2.99 folds between 0 to 200μM concentration of phosphate. Furthermore, a plateau is observed starting from the 200 μM phosphate concentration point, suggesting that the dynamic range of PphoA is from 0-200 μM of phosphate.
As shown in Figure 3b, PphoBR is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 1.88 folds between 0 to 250 μM concentration of phosphate. Furthermore, a plateau is observed starting from the 250μM of phosphate concentration point, suggesting that the dynamic range of PphoBR is from 0-250μM of phosphate.
Discussion
In order to select a better phosphate sensor, the fold change of PphoA and PphoBR were being compared.
Figure 4. Fold change of PphoA and PphoBR GFP expression.The fold change of PphoA and PphoBR GFP expression between 0 and 250 μM of phosphate. For PphoA, the relative fluorescence intensity of 0 to 250 μM of phosphate is about 2.99 folds. For PphoBR, it is about 1.88 folds. Error bars were presented in SEM.
The fold change of PphoA GFP expression is greater than that of PphoBR between 0 and 250μM of phosphate. Since the GFP expression gradient of PphoA construct is more significant, it is better to use PphoA to differentiate various phosphate concentration in the soil.
References
Hsieh, Y. J., & Wanner, B. L. (2010). Global regulation by the seven-component P i signaling system. Current opinion in microbiology, 13(2), 198-203.