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

 
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<div id= "page_title"><h1>Phosphate Sensor - <i>P<sub>phoA</sub></i> , <i>P<sub>phoBR</sub></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%"> Nitrate sensor (<i>p<sub>dcuS</sub></i>) </p></a>
 
<p style="color:#5570b0; font-size: 130%"> Nitrate sensor (<i>p<sub>dcuS</sub></i>) </p></a>
 
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<h1><i>E. coli</i> that glows in paucity of phosphate - at a glance</h1>
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<h1>Introduction</h1>
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<p>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.
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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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<h1>Phosphate sensor Mechanism</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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<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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<img style="width:50%; height:400px; float:right; padding-left:2%; margin-top:-40px" src="https://static.igem.org/mediawiki/2015/6/64/Team_HKUST-Rice_2015_phosphate_mechanism_2.PNG" alt="image caption">
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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, <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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<h1>Phosphate sensor Design</h1>
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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>
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<div class="des">
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<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>
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<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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<h1> Experiments performed</h1>
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<p>Characterization on the constructs  (<a href="http://parts.igem.org/Part:BBa_K1682013"target="_blank">BBa_K1682013</a>) was done using <a href="https://static.igem.org/mediawiki/2015/2/2d/Team_HKUST-Rice_2015_M9_Minimal_Medium_%28Tris%29.pdf"target="_blank">M9 minimal medium (without phosphate)</a>. 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 the 3 characterization results together.
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<br><br>Please visit <a href="https://static.igem.org/mediawiki/2015/4/45/Team_HKUST-Rice_2015_Phosphate_sensor_Experiment_Protocol.pdf"target="_blank">Phosphate sensor Experiment Protocol</a> for more details.</p></div>
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<h1>Results</h1>
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<p>
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After measuring the GFP signal intensity using an EnVision® multilabel reader, the fluorescence signal were represented in fluorescence divided by biomass.
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<img style="margin-left:-10%;width: 90%;" src="https://static.igem.org/mediawiki/2015/3/3e/Team_HKUST-Rice_2015_abrvvv.PNG" alt="image caption"></div>
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                                        <p  class="subTitle">An effort to make iGEM a better community</p>
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<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>
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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.
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.</p> </div>
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<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.
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<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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<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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<h1>Discussion</h1>
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<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>
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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>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>.
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<p style="font-size:110%"><strong>Figure 4. Comparison between <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>
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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>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.
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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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<p  class="subTitle">Design and Testing of phosphate sensing Device</p>
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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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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  class="subTitle">Comparing activities of <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i></p>
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<div class="project_image">
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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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<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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<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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<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.