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

 

<div id= "page_title"><h1>Phosphate Sensor - <i>phoBRp</i></h1>

<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.  

<img src="https://static.igem.org/mediawiki/2015/8/83/Team_HKUST-Rice_2015_Phosphate_mechanism_3a.PNG" alt="image caption">

<p><i>phoBRp</i> promoter is cross-regulated by <i>phoB</i> and <i>phoR</i>. 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 the expression of <i>phoBRp</i> promoter. In contrast, when there is phosphate, PhoR will repress PhoB phosphorylation which in turns inactivates <i>phoBRp</i> promoter.  

For the constructs design, we have ligated GFP generator to the <i>phoBRp</i> promoter. As a result, under high phosphate concentration, the green fluorescence intensity will be repressed, while under low phosphate concentration, green fluorescence will be expressed.

<h1>Experiment performed</h1>

<p>We characterized pSB1C3-<i>phoBRp</i>-BBa_I13504 using Luria Broth (LB) medium. Quantitative characterization on the promoter was done by measuring the fluorescence signal intensity using an EnVision multilabel reader.

<br><br><i>E. coli</i> strain DH10B was used, and the concentrations for the characterization of <i>phoBRp</i> was from 0 to 300 µM phosphate, with an intervals of 50 µM.

<p style="font-size:200%"><u>Preparing test medium with different concentrations of phosphate</u></p>

<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>

                                   <td><b>M9 minimal medium (μl)</b></td>

                                   <td><b>M9 minimal medium without phosphate (replaced by Tris) (ml)</b></td>

                                   <td><b>Chloramphenicol (150 ng/μl) (μl)</b></td>

       <p style="font-size:200%"><u><i>phoBRp</i> characterization</u></p>

<p>To characterize 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^oC. 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 37^oC until the OD₆₀₀ of the cells reached the mid-log phase. The fluorescence output was then measured using EnVision multilabel reader.  

 

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