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

 
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<div id= "page_title"><h1>Phosphate Sensor - <i>phoAp</i></h1>
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<div id= "page_title"><h1>Phosphate Sensor - <i>P<sub>phoA</sub></i> , <i>P<sub>phoBR</sub></i></h1></div>
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                      <a href="https://2015.igem.org/Team:HKUST-Rice/Modeling"><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 - Modeling </p></a>
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                <div id="MYicon2">
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                      <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>p<sub>dcuS</sub></i>) </p></a>
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<h1>Introduction</h1>
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<h1><i>E. coli</i> that glows in paucity of phosphate - at a glance</h1>
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<p>Phosphorus 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. Phosphorus is, therefore, 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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<img  src="https://static.igem.org/mediawiki/2015/1/17/Team_HKUST-Rice_2015_phosphate_diagram_1.png "style="width:100%;">
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<img id="Kgraph" src="https://static.igem.org/mediawiki/2015/d/d4/Team_HKUST-Rice_2015_phosphate_comparison.png" style="width:120%; padding-left: -20%;">
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<p style="font-size:110%; padding-left:2%; padding-right: 2% ; height'90px';"><strong>A.</strong> <i>E. coli</i> engineered with <a href="http://parts.igem.org/Part:BBa_K1682013"target="_blank">BBa_K1682013</a> or <a href="http://parts.igem.org/Part:BBa_K1682015"target="_blank">BBa_K1682015</a> functions as a phosphate biosensor. High concentrations of phosphate indirectly represses <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i> and decreases the expression of GFP.</p>
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<p style="font-size:110%; padding-left:2;height:'90px'; padding-right: 2%"  ><strong>B.</strong> The phosphate sensing promoters <a href="http://parts.igem.org/Part:BBa_K1682013"target="_blank">BBa_K1682013</a> and <a href="http://parts.igem.org/Part:BBa_K1682015"target="_blank">BBa_K1682015</a> can both detect a gradient of phosphate concentrations. Its activities at 0 and 300 μM were reported in Relative Fluorescence Level (a.u.).</p>
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<p><ul style="text-align:left; font-size:1em; line-height= 1em; font-family: 'Helvetica Neue', Helvetica, sans-serif;">
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                                        <li>Phosphate associate with essential growth factors for plant root development as well as seed production.</li>
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<li>We improved on <i>P<sub>phoA</sub></i>, <i>P<sub>phoBR</sub></i>. The data provided dynamic range of these promoters.</li>
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</ul></p>
 
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<h1>Phosphate sensor Design</h1>
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<h1><i>P<sub>phoA</sub></i> , <i>P<sub>phoBR</sub></i> and our engineered phosphate sensor BBa_K1682013 and BBa_K1682015 - the full story</h1>
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                                    <div class="project_image">
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<p>Phosphorus is an essential plant macronutrient as it associates with various growth factors for plant root development as well as seed production. Deficiency in phosphorus will result in stunted plant growth. Our aim is to engineer a phosphate sensor in <i>Escherichia coli</i> and detect phosphate level in soil. To this end, we obtained different sequences for <i>P<sub>phoA</sub></i> or <i>P<sub>phoBR</sub></i> and fused it with <i>gfp</i> (<i>gfpmut3b</i>). The promoters are activated under low phosphate condition, so green fluorescence will be produced to show phosphate level in soil.
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                                        <p  class="subTitle">An effort to make iGEM a better community</p>
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<p><i>P<sub>phoA</sub></i> <a href="http://parts.igem.org/Part:BBa_K1139201"target="_blank">(BBa_K1139201)</a> is first characterized and BioBricked by <a href="https://2013.igem.org/Team:Tokyo_Tech/Experiment/phoA_Promoter_Assay"target="_blank">Tokyo Tech 2013 iGEM team</a>. <i>P<sub>phoBR</sub></i> <a href="http://parts.igem.org/Part:BBa_K737024"target="_blank">(BBa_K737024)</a> is first characterized and BioBricked by <a href="https://2012.igem.org/Team:OUC-China/Project/Sensor/ResultandDiscussion"target="_blank">OUC-China 2012 iGEM team</a>. To provide more characterization data on these two promoters, we improved these promoters by obtaining different sequences.</p>
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<hr class="para">
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<p  class="subTitle">Endogenous phosphate sensing system in <i>E. coli</i></p>
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<img style="width:80%;" src="https://static.igem.org/mediawiki/2015/e/ea/Team_HKUST-Rice_2015_phosphate_diagram_2.png">
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<p style="font-size:110%; padding-left:6%;"><strong>Figure 1. The Pho phophorous sensing system in <i>E. coli</i>.</strong></p>
 
 
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<p><i>phoAp</i> promoter (Hsieh, Y. J., & Wanner, B. L.,2010).is cross-regulated by <i>phoB</i> and <i>phoR</i>, and is usually repressed under high phosphate concentration. PhoR behaves as an activator as well as an inactivator for PhoB. When phosphate is limited, <i>phoR</i> will phosphorylate PhoB and the phosphorylated PhoB will directly activate the expression of <i>phoAp</i> promoter. In contrast, when there is phosphate, <i>phoR</i> will repress PhoB phosphorylation which in turns inactivates <i>phoAp</i> promoter.
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<p><i>E. coli</i> has multiple native phosphorus sensing and regulation systems that we could use in the construct. Among them, we chose the PhoR/PhoB two-component system (TCS). It contains a sensory histidine kinase PhoR and a partner DNA-binding response regulator PhoB. PhoR is activated under low phosphate concentration, which will then phosphorylate PhoB. The phospho-PhoB is then capable of activating expression of the Pho regulon genes, two of the examples are <i>phoA</i> and <i>phoBR</i>. In high phosphate concentration, phoR is turned into an inhibitory state, which interferes with phosphorylation of PhoB. PhoB is, thus, not capable of activating expression of <i>phoA</i> and <i>phoBR</i>.
 +
</p>
  
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<br><br>For the constructs design, we have ligated GFP generator to the <i>phoAp</i> promoter. As a result, when under high phosphate concentration, the green fluorescence intensity will be repressed, while under low phosphate concentration, the situation will be vice versa.
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<p style = "font-size:110%">*The above text is our summarized understanding on phosphate-sensing system using information from Hsieh & Wanner (2010). Please refer to our references section below for the reference cited.</p>
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<p  class="subTitle">Design and Testing of phosphate sensing Device</p>
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<h1>Experiment that we did</h1>
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<p>We have done a characterization on pSB1C3-<i>phoAp</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.
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<img style="width:80%" src="https://static.igem.org/mediawiki/2015/7/7f/Team_HKUST-Rice_2015_phosphate_c%2Br.png">
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<p style="font-size:110%; padding-left:6%;"><strong>Figure 2. Construction and Testing of <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i>.</strong> A) Constructs of <a href="http://parts.igem.org/Part:BBa_K1682013"target="_blank">BBa_K1682013</a> and <a href="http://parts.igem.org/Part:BBa_K1682015"target="_blank">BBa_K1682015</a>. <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i> ligated with <i>gfp</i> (<i>gfpmut3b</i>) B) Characterization of <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i> in M9 minimal medium. Promoter's activities were reported in Fluorescence per Biomass. Error bar present SEM from 3 biological replicates.</p></div>
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<p>To construct a phosphate-sensing device, we cloned the promoters <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i>, and fused it with a GFP reporter <a href="http://parts.igem.org/Part:BBa_I13504"target="_blank">BBa_I13504</a> in BioBrick RFC10 standard. The activity of promoter can then be reported by the fluorescence level under different phosphate concentrations.<br><br>
  
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<br><br><i>E. coli</i> strain DH10B was used, and the concentration of the characterization of <i>phoAp</i> promoter was from 0 to 300 µM phosphate, with an intervals of 50µM.
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The activities of <i>P<sub>phoA</sub></i> <a href="http://parts.igem.org/Part:BBa_K1682013"target="_blank">(BBa_K1682013)</a> and <i>P<sub>phoBR</sub></i> <a href="http://parts.igem.org/Part:BBa_K1682015"target="_blank">(BBa_K1682015)</a> in different phosphate concentrations were measured in Fluorescence per Biomass, following a modified protocol (see below) from <a href="https://2013.igem.org/Team:Tokyo_Tech/Experiment/phoA_Promoter_Assay"target="_blank"> Tokyo Tech 2013 iGEM team</a>. They were found to be falling in a dynamic range of 0 - 300 μM of phosphate. In their dynamic range, there is a 2.99 fold change in Fluorescence for <i>P<sub>phoA</sub></i> and a 1.88 fold change in Fluorescence for <i>P<sub>phoBR</sub></i>.
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<p style="font-size:200%"><u>Preparing test medium with different concentration of phosphate</u></p>
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</p>
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<p>We have 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 concentration of phosphate (0, 10, 30, 50, 100, 150, 200, 250, 300 µM) were made by mixing the 2 solution in the following ratio. </p>
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                                <table border="1" style="width:100%; font-size:150%">
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                                  <td><b>Final Phosphate concentration (μM)</b></td>
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<div class="project_row">
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                                  <td><b>M9 minimal medium added (μl)</b></td>
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<p  class="subTitle">Comparing activities of <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i></p>
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                                  <td><b>M9 minimal medium without phosphate (replaced by Tris) added (ml)</b></td>
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<div class="project_image">
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                                  <td><b>150 ng/μl Chloramphenicol Antibiotics added (μl)</b></td>
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<img id="Kgraph" src="https://static.igem.org/mediawiki/2015/0/09/Team_HKUST-Rice_2015_phosphate_compare.png">
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                                </tr>
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<p style="font-size:110%; padding-left:6%;"><strong>Figure 3. Comparison between <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i> GFP expression.</strong></p></div>
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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>
+
−
                                  <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>
+
−
                                  <td>150</td>
+
−
                                  <td>130.43</td>
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−
                                  <td>60</td>
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−
                                  <td>60</td>
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−
                                </tr>
+
−
                                    <tr>
+
−
                                  <td>200</td>
+
−
                                  <td>171.42</td>
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−
                                  <td>60</td>
+
−
                                  <td>60</td>
+
−
                                </tr>
+
−
                                </tr>
+
−
                                    <tr>
+
−
                                  <td>250</td>
+
−
                                  <td>216.26</td>
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−
                                  <td>60</td>
+
−
                                  <td>60</td>
+
−
                                </tr>
+
−
                                </tr>
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−
                                    <tr>
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−
                                  <td>300</td>
+
−
                                  <td>260.87</td>
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−
                                  <td>60</td>
+
−
                                  <td>60</td>
+
−
                                </tr>
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−
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      <p style="font-size:200%"><u><i>phoAp</i> promoter Characterization</u></p>
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<p>pSB1C3-<i>phoAp</i>-BBa_I13504, positive control and negative control 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 5ml 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 950μl of test medium with different concentrations of phosphate (containing Chloramphenicol) in the 96-well deep well plate and further incubate at 37<sup>o</sup>C until the OD<sub>600</sub> of the cells reaches the mid-log phase. The fluorescence output were 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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<p>We selected <i>P<sub>phoA</sub></i> for our construct. We compared the activity difference of two promoters for 0 and 300 μM of phosphate, as they are the minimum and maximum dynamic range suggested by <a href="https://2013.igem.org/Team:Tokyo_Tech/Experiment/phoA_Promoter_Assay"target="_blank"> Tokyo Tech 2013 iGEM team</a>. Having The fold change of <i>P<sub>phoA</sub></i> GFP is 2.99, which is greater than 1.88 of <i>P<sub>phoBR</sub></i>. Therefore, we chose <i>P<sub>phoA</sub></i> as it has a greater range, so it is supposed to have larger difference within a certain range.</p>
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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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<h2>Materials and Methods</h2>
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<p>Please refer to <a href ="https://2015.igem.org/Team:HKUST-Rice/Protocol">our protocol page for the materials and methods used in characterization.</a></p>
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<h2>References</h2>
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<p style="font-size:125%"> Hsieh, Y. J., & Wanner, B. L. (2010). Global regulation by the seven-component P i signaling system. <i>Current opinion in microbiology, 13</i>(2), 198-203.</p>
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{{HKUST-Rice Directory}}

Latest revision as of 16:44, 20 November 2015


Phosphate Sensor - PphoA , PphoBR

E. coli that glows in paucity of phosphate - at a glance

A. E. coli engineered with BBa_K1682013 or BBa_K1682015 functions as a phosphate biosensor. High concentrations of phosphate indirectly represses PphoA and PphoBR and decreases the expression of GFP.

B. The phosphate sensing promoters BBa_K1682013 and BBa_K1682015 can both detect a gradient of phosphate concentrations. Its activities at 0 and 300 μM were reported in Relative Fluorescence Level (a.u.).

  • Phosphate associate with essential growth factors for plant root development as well as seed production.
  • We improved on PphoA, PphoBR. The data provided dynamic range of these promoters.

PphoA , PphoBR and our engineered phosphate sensor BBa_K1682013 and BBa_K1682015 - the full story

Phosphorus is an essential plant macronutrient as it associates with various growth factors for plant root development as well as seed production. Deficiency in phosphorus will result in stunted plant growth. Our aim is to engineer a phosphate sensor in Escherichia coli and detect phosphate level in soil. To this end, we obtained different sequences for PphoA or PphoBR and fused it with gfp (gfpmut3b). The promoters are activated under low phosphate condition, so green fluorescence will be produced to show phosphate level in soil.

An effort to make iGEM a better community

PphoA (BBa_K1139201) is first characterized and BioBricked by Tokyo Tech 2013 iGEM team. PphoBR (BBa_K737024) is first characterized and BioBricked by OUC-China 2012 iGEM team. To provide more characterization data on these two promoters, we improved these promoters by obtaining different sequences.

Endogenous phosphate sensing system in E. coli

Figure 1. The Pho phophorous sensing system in E. coli.

E. coli has multiple native phosphorus sensing and regulation systems that we could use in the construct. Among them, we chose the PhoR/PhoB two-component system (TCS). It contains a sensory histidine kinase PhoR and a partner DNA-binding response regulator PhoB. PhoR is activated under low phosphate concentration, which will then phosphorylate PhoB. The phospho-PhoB is then capable of activating expression of the Pho regulon genes, two of the examples are phoA and phoBR. In high phosphate concentration, phoR is turned into an inhibitory state, which interferes with phosphorylation of PhoB. PhoB is, thus, not capable of activating expression of phoA and phoBR.

*The above text is our summarized understanding on phosphate-sensing system using information from Hsieh & Wanner (2010). Please refer to our references section below for the reference cited.

Design and Testing of phosphate sensing Device

Figure 2. Construction and Testing of PphoA and PphoBR. A) Constructs of BBa_K1682013 and BBa_K1682015. PphoA and PphoBR ligated with gfp (gfpmut3b) B) Characterization of PphoA and PphoBR in M9 minimal medium. Promoter's activities were reported in Fluorescence per Biomass. Error bar present SEM from 3 biological replicates.

To construct a phosphate-sensing device, we cloned the promoters PphoA and PphoBR, and fused it with a GFP reporter BBa_I13504 in BioBrick RFC10 standard. The activity of promoter can then be reported by the fluorescence level under different phosphate concentrations.

The activities of PphoA (BBa_K1682013) and PphoBR (BBa_K1682015) in different phosphate concentrations were measured in Fluorescence per Biomass, following a modified protocol (see below) from Tokyo Tech 2013 iGEM team. They were found to be falling in a dynamic range of 0 - 300 μM of phosphate. In their dynamic range, there is a 2.99 fold change in Fluorescence for PphoA and a 1.88 fold change in Fluorescence for PphoBR.

Comparing activities of PphoA and PphoBR

Figure 3. Comparison between PphoA and PphoBR GFP expression.

We selected PphoA for our construct. We compared the activity difference of two promoters for 0 and 300 μM of phosphate, as they are the minimum and maximum dynamic range suggested by Tokyo Tech 2013 iGEM team. Having The fold change of PphoA GFP is 2.99, which is greater than 1.88 of PphoBR. Therefore, we chose PphoA as it has a greater range, so it is supposed to have larger difference within a certain range.

Materials and Methods

Please refer to our protocol page for the materials and methods used in characterization.

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.