Difference between revisions of "Team:HKUST-Rice/Phosphate Sensor"
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| − | <div id= "page_title"><h1>Phosphate Sensor - P<sub>phoA</sub> , < | + | <div id= "page_title"><h1>Phosphate Sensor - P<sub>phoA</sub> , P<sub>phoBR</sub></h1> |
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| − | <p>P<sub>phoA</sub> and < | + | <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>. |
| − | <br><br>For the constructs design, we have ligated GFP generator to P<sub>phoA</sub> and < | + | <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> | </p> | ||
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<hr class="para"> | <hr class="para"> | ||
<h1>Experiment performed</h1> | <h1>Experiment performed</h1> | ||
| − | <p>We have done a characterization on P<sub>phoA</sub> and < | + | <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. |
<br><br>Please visit <a href="#Methods_2">Methods</a> for more details</p> | <br><br>Please visit <a href="#Methods_2">Methods</a> for more details</p> | ||
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| − | <p style="font-size:200%"><u>P<sub>phoA</sub>and < | + | <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>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> |
Revision as of 03:06, 3 September 2015
Phosphate Sensor - PphoA , PphoBR
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. Plants deficient in phosphorus are stunted in growth and often have an abnormal dark-green color.
Phosphate sensor Design
PphoA and PphoBR is cross-regulated by PphoBR 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 PphoA and PphoBR. In contrast, when there is phosphate, PhoR will repress PhoB phosphorylation which in turn inactivates the PphoA and PphoBR.
For the constructs design, we have ligated GFP generator to PphoA and PphoBR, 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 PphoA and PphoBR 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 3 characterization trials.
Please visit Methods for more details
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.
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.
Methods
Preparing test medium with different concentrations of phosphate
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:
60 μl of antibiotics was added in each test medium solution.
| Final phosphate concentration (μM) | M9 minimal medium (μl) | M9 minimal medium without phosphate (replaced by Tris) (ml) |
| 0 | 0.00 | 60 |
| 10 | 8.58 | 60 |
| 30 | 25.73 | 60 |
| 50 | 42.85 | 60 |
| 100 | 85.71 | 60 |
| 150 | 130.43 | 60 |
| 200 | 171.42 | 60 |
| 250 | 216.26 | 60 |
| 300 | 260.87 | 60 |
PphoAand PphoBR characterization
To characterize PphoA and PphoBR, positive and negative controls were first grown overnight in 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. 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 37oC until the OD600 of the cells reached the mid-log phase. The fluorescence output was then measured using EnVision multilabel reader.