Team:Peking/Project/BioSensors/DmpR

From 2013.igem.org

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<ul id="navigationbar">
<ul id="navigationbar">
<li id="PKU_navbar_Home" class="Navbar_Item">
<li id="PKU_navbar_Home" class="Navbar_Item">
 +
                       
<a href="https://2013.igem.org/Team:Peking">Home</a>
<a href="https://2013.igem.org/Team:Peking">Home</a>
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<li id="PKU_navbar_Team" class="Navbar_Item">
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<a href="https://2013.igem.org/Team:Peking/Team">Team</a>
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<ul id="Team_Sublist">
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<li><a href="https://2013.igem.org/Team:Peking/Team/Members">Members</a></li>
<li><a href="https://2013.igem.org/Team:Peking/Team/Members">Members</a></li>
<li><a href="https://2013.igem.org/Team:Peking/Team/Notebook">Notebook</a></li>
<li><a href="https://2013.igem.org/Team:Peking/Team/Notebook">Notebook</a></li>
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<a href="https://2013.igem.org/Team:Peking/Project">Project</a>
<a href="https://2013.igem.org/Team:Peking/Project">Project</a>
<ul id="Project_Sublist">
<ul id="Project_Sublist">
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                                 <li><a href="https://2013.igem.org/Team:Peking/Project/AutoSensorMining">Auto Sensor Mining</a></li>
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                                <div class="BackgroundofSublist"></div>
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                                 <li><a href="https://2013.igem.org/Team:Peking/Project/SensorMining">Biosensor Mining</a></li>
<li><a href="https://2013.igem.org/Team:Peking/Project/BioSensors">Biosensors</a></li>
<li><a href="https://2013.igem.org/Team:Peking/Project/BioSensors">Biosensors</a></li>
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<li><a href="https://2013.igem.org/Team:Peking/Project/Plugins">Plug-ins</a></li>
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<li><a href="https://2013.igem.org/Team:Peking/Project/Plugins">Adaptors</a></li>
<li><a href="https://2013.igem.org/Team:Peking/Project/BandpassFilter">Band-pass Filter</a></li>
<li><a href="https://2013.igem.org/Team:Peking/Project/BandpassFilter">Band-pass Filter</a></li>
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                                 <li><a href="https://2013.igem.org/Team:Peking/Project/Application">Application</a></li>
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                                 <li><a href="https://2013.igem.org/Team:Peking/Project/Devices">Devices</a></li>
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</li>
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<li id="PKU_navbar_Model" class="Navbar_Item">
<li id="PKU_navbar_Model" class="Navbar_Item">
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<a href="https://2013.igem.org/Team:Peking/Model">Model</a>
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<a href="">Model</a>
<ul id="Model_Sublist">
<ul id="Model_Sublist">
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                                <li><a href="https://2013.igem.org/Team:Peking/Model">Band-pass Filter</a></li>
 +
                                <li><a href="https://2013.igem.org/Team:Peking/ModelforFinetuning">Biosensor Fine-tuning</a></li>
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                         <li id="PKU_navbar_HumanPractice" class="Navbar_Item" style="width:90px">
                         <li id="PKU_navbar_HumanPractice" class="Navbar_Item" style="width:90px">
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<a href="https://2013.igem.org/Team:Peking/HumanPractice">Data page</a>
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<a href="">Data page</a>
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<ul id="DataPage_Sublist">
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                                 <li><a href="https://2013.igem.org/Team:Peking/DataPage/Parts">Parts</a></li>
                                 <li><a href="https://2013.igem.org/Team:Peking/DataPage/Parts">Parts</a></li>
<li><a href="https://2013.igem.org/Team:Peking/DataPage/JudgingCriteria">Judging Criteria</a></li>
<li><a href="https://2013.igem.org/Team:Peking/DataPage/JudgingCriteria">Judging Criteria</a></li>
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<li id="PKU_navbar_HumanPractice" class="Navbar_Item" style="width:120px">
<li id="PKU_navbar_HumanPractice" class="Navbar_Item" style="width:120px">
<a href="https://2013.igem.org/Team:Peking/HumanPractice">Human Practice</a>
<a href="https://2013.igem.org/Team:Peking/HumanPractice">Human Practice</a>
-
<ul id="HumanPractice_Sublist">
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<ul id="HumanPractice_Sublist">
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                                 <li><a href="https://2013.igem.org/Team:Peking/HumanPractice/Questionnaire">Questionnaire</a></li>
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                                <div class="BackgroundofSublist"></div>
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<li><a href="https://2013.igem.org/Team:Peking/HumanPractice/FactoryVisit">Factory Visit</a></li>
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                                 <li><a href="https://2013.igem.org/Team:Peking/HumanPractice/Questionnaire">Questionnaire Survey</a></li>
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                                 <li><a href="https://2013.igem.org/Team:Peking/HumanPractice/iGEMWorkshop">iGEM Workshop</a></li>
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<li><a href="https://2013.igem.org/Team:Peking/HumanPractice/FactoryVisit">Visit and Interview</a></li>
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<li><a href="https://2013.igem.org/Team:Peking/HumanPractice/ModeliGEM">Model iGEM</a></li>
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                                 <li><a href="https://2013.igem.org/Team:Peking/HumanPractice/ModeliGEM">Practical Analysis</a></li>
 +
<li><a href="https://2013.igem.org/Team:Peking/HumanPractice/iGEMWorkshop">Team Communication</a></li>
 +
 
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</ul>
</li>
</li>
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         <a href="https://igem.org/Team_Wikis?year=2013"><img id="iGEM_logo" src="https://static.igem.org/mediawiki/igem.org/4/48/Peking_igemlogo.jpg"/></a>
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         <a href="https://2013.igem.org/Main_Page"><img id="iGEM_logo" src="https://static.igem.org/mediawiki/igem.org/4/48/Peking_igemlogo.jpg"/></a>
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<div id="ProjectTitle">
<div id="ProjectTitle">
<h1 id="ProjectName">Biosensors</h1>
<h1 id="ProjectName">Biosensors</h1>
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                <h1 id="ProjectSubname">A FAST, EASY AND ACCURATE METHOD TO DETECT TOXIC AROMATIC COMPOUNDS</h1>
 
                 <img src="https://static.igem.org/mediawiki/igem.org/9/96/Peking2013-biosensortitile-zyh.jpg"/>
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</div>
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                     <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/XylR">XylR</a><li>
                     <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/XylR">XylR</a><li>
                     <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/HbpR">HbpR</a><li>
                     <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/HbpR">HbpR</a><li>
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                     <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/HcaR#Mileston1">HcaR</a><li>
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                     <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/HcaR">HcaR</a><li>
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                     <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/HcaR#Mileston2">HpaR</a><li>
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                     <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/HpaR">HpaR</a><li>
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                     <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/HcaR#Mileston3">PaaX</a><li>
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                     <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/HpaR#ContentHpaR4">PaaX</a><li>
                     <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/DmpR">DmpR</a><li>
                     <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/DmpR">DmpR</a><li>
                     <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/NahR">NahR</a><li>
                     <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/NahR">NahR</a><li>
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<img id="DmpRFigure1" src="https://static.igem.org/mediawiki/igem.org/5/5f/Peking2013_DmpR_Figure1.png"/>
<img id="DmpRFigure1" src="https://static.igem.org/mediawiki/igem.org/5/5f/Peking2013_DmpR_Figure1.png"/>
<img id="DmpRFigure2" src="https://static.igem.org/mediawiki/igem.org/7/78/Peking2013_DmpR_Figure2.png"/>
<img id="DmpRFigure2" src="https://static.igem.org/mediawiki/igem.org/7/78/Peking2013_DmpR_Figure2.png"/>
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<img id="DmpRFigure3" src="https://static.igem.org/mediawiki/igem.org/b/b3/Peking2013_DmpR_Figure3.png"/>
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<img id="DmpRFigure3" src="https://static.igem.org/mediawiki/2013/5/57/Peking2013_DmpRfigure3.png"/>
<img id="DmpRFigure4" src="https://static.igem.org/mediawiki/igem.org/3/3b/Peking2013_DmpR_Figure4.png"/>
<img id="DmpRFigure4" src="https://static.igem.org/mediawiki/igem.org/3/3b/Peking2013_DmpR_Figure4.png"/>
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<img id="DmpRFigure5" src="https://static.igem.org/mediawiki/igem.org/6/6f/Peking2013_DmpRFig5.png"/>
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<img id="DmpRFigure5" src="https://static.igem.org/mediawiki/2013/thumb/6/6f/Peking2013_DmpRFig5.png/800px-Peking2013_DmpRFig5.png"/>
<img id="DmpRFigure6" src="https://static.igem.org/mediawiki/igem.org/1/1e/Peking2013_DmpR_Figure6.png"/>
<img id="DmpRFigure6" src="https://static.igem.org/mediawiki/igem.org/1/1e/Peking2013_DmpR_Figure6.png"/>
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<img id="DmpRFigure7" src="https://static.igem.org/mediawiki/igem.org/7/7c/Peking2013_DmpR_Figure7.jpg"/>
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<img id="DmpRFigure8" src="https://static.igem.org/mediawiki/igem.org/7/7b/Peking2013_DmpR_Figure8.png"/>
<img id="DmpRFigure8" src="https://static.igem.org/mediawiki/igem.org/7/7b/Peking2013_DmpR_Figure8.png"/>
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<img id="DmpRFigure9" src="https://static.igem.org/mediawiki/igem.org/0/0d/Peking2013_DmpR_Figure9.png"/>
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<img id="DmpRFigure9" src="https://static.igem.org/mediawiki/2013/8/8f/Peking2013_DmpR_Figure8.PNG"/>
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<img id="DmpRFigure11" src="https://static.igem.org/mediawiki/2013/4/46/Peking2013_DmpRFigure11.png"/>
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<p id="ContentDmpR1">
<p id="ContentDmpR1">
-
DmpR from Pseudomonas sp.CF600 <SUP>[2][4-8]</SUP> is a sigma54-dependent transcriptional factor that tightly controls the expression of the dmp operon (dmpKLMNOPQBCDEFGHI) (<B>Fig.1</B> ).This operon carries genes encoding enzymes for the degradation of (methyl)phenols to pyruvate and acetyl-CoA<SUP>[1]</SUP>.(<B>Fig.2</B>)  
+
DmpR <a href="https://2013.igem.org/Team:Peking/Project/SensorMining">bioinformatically mined</a> from <I>Pseudomonas sp</I>.CF600 <a href="#ReferenceDmpR"><sup>[1-6]</sup></a> is a &sigma;<sup>54</sup>-dependent transcriptional factor that tightly controls the expression of the <I>dmp</I> operon (<I>dmpKLMNOPQBCDEFGHI</I>) (<B>Fig. 1</B>). This operon carries genes encoding enzymes for the degradation of (methyl) phenols into pyruvate and acetyl-CoA<a href="#ReferenceDmpR"><sup>[7]</sup></a> (<B>Fig. 2</B>).
</p>
</p>
<p id="ContentDmpR2">
<p id="ContentDmpR2">
-
DmpR is a transcriptional activator of Po promoter which controls ON/OFF expression of dmp operon. It binds to Po promoter as tetramer on two diverted UAS sequence (Upstream Activating Sequence). The transcription initiation of dmp operon also requires IHF factor (Integration Host Factor), which has two binding sites on Po promoter and enhance the transcription efficiency. (<B>Fig.3</B>)
+
The cognate promoter of DmpR is <I>Po</I> promoter. The DmpR protein binds to <I>Po</I> promoter as hexamer on two distinct UAS (Upstream Activating Sequence). The transcription initiation of <I>dmp</I> operon also requires IHF (Integration Host Factor), which has two binding sites in <I>Po</I> promoter and enhances the transcription efficiency (<B>Fig. 3</B>).
</p>
</p>
<p id="ContentDmpR3">
<p id="ContentDmpR3">
-
DmpR protein consists of four domains (<B>Fig.4</B>): A domain is the signal reception domain, which undergoes conformational change when exposed to proper inducers, including phenol, 2-chlorophenol, 2,4-dichlorophenol, methyl-phenols and other substituted phenols <SUP>[3][5]</SUP>.B domain is a linker, mutations of which change the interaction between A domain and C domain, regulating the relative spatial position of them. C domain is the transcriptional activation domain. D domain contains a helix-turn-helix motif, which is capable of binding DNA sequence on Po promoter <SUP>[2]</SUP>.
+
DmpR protein consists of four domains (<B>Fig. 4</B>): Domain A is the effector-sensing domain, which undergoes conformational change when exposed to proper inducers, including phenol, 2-chlorophenol, 2,4-dichlorophenol, methyl-phenols and other substituted phenols <a href="#ReferenceDmpR"><sup>[3][8]</sup></a>. Domain B is a linker domain where mutations would modulate the interactions between Domain A and Domain C. Domain C is the transcriptional activation domain. Domain D contains a helix-turn-helix motif, which is responsible for the DNA binding at <I>Po</I> promoter <a href="#ReferenceDmpR"><sup>[1]</sup></a>.
</p>
</p>
<p id="ContentDmpR4">
<p id="ContentDmpR4">
-
The mechanism of Po promoter activation consists of four steps, DmpR dimer formation, DmpR tetramer formation, DNA bending and recruit of RNAP (<B>Fig.5</B>). With the cooperation of IHF, transcription from Po promoter initiates.  
+
The mechanism of <I>Po</I> promoter activation consists of four steps, DmpR dimerization, DmpR hexamer formation, DNA bending and RNAP recruitment (<B>Fig. 5</B>). Ater the 4 steps, with the help of IHF, transcription from <I>Po</I> promoter initiates thereby.  
</p>
</p>
<p id="ContentDmpR5">
<p id="ContentDmpR5">
-
A random mutation of DmpR A domain with capacity to detect phenolic molecules was selected. People found that the mutant Q10R strongly enhanced the response to phenol and substituted ones, and mutant D116V suggested that the aspartate at position 116 acted to restrict the effector range of wild-type DmpR.
+
We collected and analyzed all of the information about DmpR. See <b>Table 1</b> for the comprehensive summary of DmpR mutants and accompanied novel aromatics-sensing characteristics, which provides a rich repertoire for the synthetic biologists to customize the aromatics-sensing characteristics of DmpR protein.  
</p>
</p>
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<p id="ContentDmpR6">
<p id="ContentDmpR6">
-
A lot of work have been done about DmpR, but there is no general method for testing the induction ratio, and different works obtained different induction ratio. Our team obtained DmpR from Professor V. Shingler and the synthesized promotor Po sequence from GeneScript. Plasmid containing Pr-DmpR was double transformed with plasmid containing the inducible promoter Po and reporter gene sfGFP (<B>Fig.6</B>). Similar to other sensors, plasmid with RBS BBa_B0032 before sfGFP was chosen for its relatively higher induction ratio during primary test for RBS library (data not shown) (<B>Fig.6</B>).
+
Peking iGEM has adopted DmpR to build a biosensor circuit (<b>Fig. 6</b>). Plasmid carrying <I>Pr</I>-DmpR was co-transformed with the plasmid containing the inducible promoter <I>Po</I> and reporter gene sfGFP (<B>Fig. 6</B>). Similar to other biosensors, plasmid with RBS BBa_B0032 preceding sfGFP was chosen due to its relatively higher induction ratio during primary test for the RBS library.
</p>
</p>
<p id="ContentDmpR7">
<p id="ContentDmpR7">
-
We tested the DmpR using almost every protocol mentioned in the previous work and our general method. Comparison of these different protocols is listed in Table 2.
+
We evaluated the performance of DmpR using our own protocols and almost every protocol mentioned in the previous studies (for more details about these three protocols, Test Protocol 1-3, <a href="https://2013.igem.org/Team:Peking/Team/Notebook/Protocols#Content1">Click Here</a>). Results showed that the <a href="https://2013.igem.org/Team:Peking/Team/Notebook/Protocols#Content2"> Test Protocol 3</a> works the best, with which we tested the ON-OFF ratios of all the 78 aromatic compounds. 7 compounds out of the 78 showed significant induction ratios (>2) (<B>Fig. 7</B>): They are Phl, 2-MePhl, 2-ClPhl, 3-ClPhl, Cat, 4-NtPhl and 2-APhl (<a href="https://static.igem.org/mediawiki/igem.org/2/24/Peking2013_Chemicals_V3%2B.pdf">Click Here</a> for the full names of the aromatic compounds). The presence of phenol is consistent with previous studies. The other 6 compounds, however, have not been reported in previous works.
</p>
</p>
-
 
-
<p id="ContentDmpR8">
 
-
 
-
The normalized fluorescence intensity (Fluorescence / OD600) of biosensor without or with inducers of the three protocols were compared (<B>Fig.7</B>). Results showed that protocol 3 generated the best result. It is possibly because that induction during the end of the plateau phase facilitated the stable expression of regulator DmpR and the adding of fresh LB medium was conducive to the rapid expression of sfGFP under induction.
 
-
 
-
        </p>
 
-
 
-
<p id="ContentDmpR9">
 
-
 
-
We then tested the on-off ratio of all of the 78 aromatics using the protocol 3. DmpR stain showed low basal expression level of sfGFP and 7 compounds showed observably induction ratio (>2) (<B>Fig.8</B>), namely Phl, 2-MePhl, 2-ClPhl, 3-ClPhl, Cat, 4-NtPhl and 2-APhl (To see more information of the compounds, click here ).
 
-
 
-
        </p>
 
<p id="ContentDmpR10">
<p id="ContentDmpR10">
-
After finding the compounds with showed observably induction ratio, we tested the dose response curve of each compound via test protocol 3 (<B>Fig.9</B>).  
+
To provide more detailed information about the aromatics-sensing profile, we carefully examined the individual dose-response curves of the 7 compounds via <a href="https://2013.igem.org/Team:Peking/Team/Notebook/Protocols#Content2"> Test Protocol 3</a> (<B>Fig. 8</B>).  
</p>
</p>
<p id="ContentDmpR11">
<p id="ContentDmpR11">
-
In summary, we found a robust and convenient protocol to test Dmp and DmpR functions as a robust sensor for phenol and its derivative.  
+
In summary, we have successfully constructed the DmpR biosensing circuit and fine-tuned it guided by our experience obtained from the building of other biosensors. The aromatics-sensing profile of DmpR biosensor is considerably narrow (<b>Fig. 7</b>), making it a robust and convenient biosensor for the presence of phenol and its derivatives.
</p>
</p>
          
          
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<B>Figure 1.</B>Dmp operon. Dmp operon carries genes encoding enzymes for the degradation of (methyl-)phenols to pyruvate and acetyl-CoA,the intermediates of TCA Cycle. The operon is positively controlled by dmpR gene product,resulting in expression of catabolic enzymes when inducer like phenol is present.
+
<B>Figure. 1.</B> The schematic structure of <I>dmp</I> operon. <I>Dmp</I> operon carries genes encoding enzymes for the degradation of (methyl-)phenols to pyruvate and acetyl-CoA, the intermediates of TCA Cycle. The operon is positively controlled by the <I>dmpR</I> gene product, resulting in expression of catabolic enzymes when inducers like phenol are present.
</p>
</p>
<p id="FigureDmpR2">
<p id="FigureDmpR2">
-
<B>Figure 2. </B>The catabolic pathway of phenol controlled by dmp operon.Metabolic enzymes along the pathway are represented in numbers.1 through 8:1,phenol hydroxylase(PH);2,catechol 2,3-dioxygenase(C23O);3,2-hydroxymuconic semialdehyde hydrolase(2HMSH);4,2-hydroxymuconic semialdehyde dehydrogenase(2HMSD);5,4-oxalocrotonate isomerase (4OI);6,4-oxalocrotonate decarboxylase(4OD);7,2-oxopent-4-cnoate hydeatase(OEH);8,4-hydroxy-2-2oxovalerate aldolase(HOA).
+
<B>Figure. 2.</B> The catabolic pathway of phenol controlled by the <I>dmp</I> operon. Metabolic enzymes along the pathway are (from Step 1 to Step 8): 1, phenol hydroxylase (PH) ; 2, catechol 2, 3-dioxygenase (C23O); 3, 2-hydroxymuconic semialdehyde hydrolase (2HMSH); 4, 2-hydroxymuconic semialdehyde dehydrogenase (2HMSD) ;5, 4-oxalocrotonate isomerase (4OI); 6, 4-oxalocrotonate decarboxylase (4OD) ;7, 2-oxopent-4-cnoate hydeatase (OEH); 8, 4-hydroxy-2-2oxovalerate aldolase (HOA).
</p>
</p>
<p id="FigureDmpR3">
<p id="FigureDmpR3">
-
<B>Figure 3. </B>Po promoter structure. The UAS of this promoter marked in green is combined of two parts in contrast direction to which DmpR binds. The box with yellow background represents IHF binding sites. The box with pink background represents σ54 binding site with -24 region and -12 region marked in red. The G with right angle represents +1 site.
+
<B>Figure. 3.</B> The schematic structure of <I>Po</I> promoter. The UASs of this promoter shaded in green are dyad sequences to which the DmpR protein binds. The yellow shaded boxes denote IHF binding sites. The pink shaded box represents &sigma;<sup>54</sup> binding site with -24 region and -12 motifs highlighted in red. The highlighted nucleotide G represents the transcription start (+1) site.
</p>
</p>
<p id="FigureDmpR4">
<p id="FigureDmpR4">
-
<B>Figure 4.</B> Structure of DmpR protein. From N terminal to C terminal are domain A, domain B, domain C, domain D.
+
<B>Figure. 4.</B> The schematic structure of DmpR protein. From N-terminal to C-terminal are Domain A, Domain B, Domain C, Domain D.  
</p>
</p>
<p id="FigureDmpR5">
<p id="FigureDmpR5">
-
<B>Figure 5.</B> Schematic mode of the activation of DmpR regulator (A) The inactive regular dimer binds to its inducer, which results in a protein conformation change. (B)Binding of ATP triggers multimerization of the dimers to a hexamer (or haptamer).(C) ATP hydrolysis coupled to correct interaction with RNA polymerase triggers transcription activation. (D) Dissociation of the hexamer to a dimer on ATP hydrolysis and dissociation of the inducer <SUP>[8]</SUP>.
+
<B>Figure. 5.</B> The mechanism of transcription activation by DmpR. (<b>a</b>) The inactive dimer binds to its inducer, which results in a protein conformational change. (<b>b</b>) Binding of ATP triggers multimerization of the dimers to hexamers (or haptamer). (<b>c</b>) ATP hydrolysis coupled with RNA polymerase recruitment triggers transcription activation. (<b>d</b>) Dissociation of the hexamers into dimers after ATP hydrolysis <a href="#ReferenceDmpR"><sup>[6]</sup></a>.
</p>
</p>
<p id="FigureDmpR6">
<p id="FigureDmpR6">
-
<B>Figure 6. </B>The structure of the DmpR biosensor circuit. DmpR is constitutively expressed and functions to regulate the transcription of sfGFP gene via promoter Po. As for the RBS of sfGFP, BBa_B0032 was chosen due to its better performance compared to RBS of other transcriptional strengths.
+
<B>Figure. 6.</B>The schematic diagram for the DmpR biosensor circuit. DmpR is constitutively expressed and functions to regulate the transcription of sfGFP gene via promoter <I>Po</I>. As for the RBS of sfGFP, BBa_B0032 was decided due to its better performance compared to RBS of other transcriptional strengths.
</p>
</p>
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<p id="FigureDmpR7">
 
-
</B>¤Protocol 1:</B> Overnight culture of the biosensor DmpR were diluted 100-fold into LB medium. Add appropriate phenol derivative into 200-μL samples. Exposure to phenolic molecules was for 12h with shaking at 30℃.Then the sample was measured by Flow Cytometer and Microplate Reader.
 
-
<br/><B>¤¤Protocol 2:</B> Overnight culture of the biosensor DmpR were diluted 100-fold into LB medium. When the OD 600 of the cells reached between 0.2-0.3 as measured on a spectrophotometer, add appropriate phenol derivative into 200-μL samples. Exposure to phenolic molecules was for 6h with shaking at 30℃.Then the sample was measured by Flow Cytometer and Microplate Reader.<SUP>[3]</SUP>
 
-
<br/><B>¤¤¤Protocol 3:</B> Overnight culture of the biosensor DmpR were diluted 100-fold into LB medium. When the OD 600 of the cells reached between 0.8-1.0 as measured on a spectrophotometer. 2mL samples were pelleted by centrifugation and immediately suspended in 2 mL of fresh LB medium containing the appropriate phenol derivative. Exposure to phenolic molecules was for 4h with shaking at 30℃.Then the sample was measured by Flow Cytometer and Microplate Reader.<SUP>[8]</SUP>
 
-
<br/><B>* </B>We also used M9 minimal instead of LB medium in the protocols and elongate the induction time as E.coil grows much more slowly in M9 minimal.
 
-
<br/><B>** </B>For more details about these three protocols, click here.
 
-
</p>
 
-
<p id="FigureDmpR8">
+
 
-
<B>Figure 7.</B> The result of three protocols measured by Microplate Reader when 1000μM of phenol was added. The induction ratios of three protocols are 1.69, 1.57 and 4.41, which meant that protocol 3 generated the best result.
+
-
</p>
+
<p id="FigureDmpR9">
<p id="FigureDmpR9">
-
<B>Figure 8.</B> Response of DmpR biosensor to various aromatics. (For the full name of the compounds, CLICK HERE(hyperlink is needed here)). (a)The induction ratio column in the ON-OFF test. DmpR could respond to 7 out of 78 aromatics with the induction ratio over 2. (b) The detection range of DmpR biosensor is profiled in dark cyan at the aromatics spectrum. The structure formula of typical inducer is listed around the cycle spectrum, near its chemical formula.
+
<B>Figure. 7.</B>  
 +
<a href="https://2013.igem.org/Team:Peking/Team/Notebook/Protocols#Content3">ON/OFF test</a> to evaluate the induction ratios of all aromatic compounds in the aromatics spectrum (For the full names of the compounds, <a href="https://static.igem.org/mediawiki/igem.org/2/24/Peking2013_Chemicals_V3%2B.pdf">Click Here</a>). (<b>a</b>) The induction ratios of all 78 aromatic species for the DmpR biosensor. The DmpR biosensor could respond to 7 out of the 78 aromatics with the induction ratio higher than 2. (<b>b</b>) The aromatics-sensing profile of DmpR biosensor. The aromatic species that can elicit strong responses of DmpR biosensor are highlighted in cyan in the aromatics spectrum. The structure formula of typical inducer is also presented around the spectrum. The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to object inducers by the basal fluorescence intensity of the biosensor itself.
 +
 
</p>
</p>
<p id="FigureDmpR10">
<p id="FigureDmpR10">
-
<B>Figure 9.</B> Dose-response curve of DmpR biosensor. The biosensor was test under the concentration of 10μM, 30μM, 100μM, 300μM, 1000μM.
+
<B>Figure. 8.</B> Dose-response curves of DmpR biosensor induced by 7 strong inducers (phenol, its homologs and derivatives). The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to inducers by the basal fluorescence intensity of the biosensor. For the full names of the compounds, <a href="https://static.igem.org/mediawiki/igem.org/2/24/Peking2013_Chemicals_V3%2B.pdf">Click Here</a> .
</p>
</p>
        
        
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<B>REFERENCE:</B>
<B>REFERENCE:</B>
<br/>
<br/>
-
[1]. SHINGLER, V.; POWLOWSKI, J.; MARKLUND, U. Nucleotide sequence and functional analysis of the complete phenol/3, 4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600. Journal of bacteriology, (1992), 174.3: 711-724.
+
 
 +
[1]. SHINGLER, V.; PAVEL, H. Direct regulation of the ATPase activity of the transcriptional activator DmpR by aromatic compounds. <I>Molecular microbiology</I>, (1995), 17.3: 505-513.  
<br/>
<br/>
-
[2]. SHINGLER, V.; PAVEL, H. Direct regulation of the ATPase activity of the transcriptional activator DmpR by aromatic compounds. Molecular microbiology, (1995), 17.3: 505-513.  
+
[2]. SHINGLER, Victoria; MOORE, Terry. Sensing of aromatic compounds by the DmpR transcriptional activator of phenol-catabolizing <I>Pseudomonas sp.</I> strain CF600. <I>Journal of bacteriology</I>, (1994), 176.6: 1555-1560.
<br/>
<br/>
-
[3]. GUPTA, Saurabh, et al. An Effective Strategy for a Whole-Cell Biosensor Based on Putative Effector Interaction Site of the Regulatory DmpR Protein. PloS one, (2012), 7.8: e43527.  
+
[3]. SZE, Chun Chau; LAURIE, Andrew D.; SHINGLER, Victoria. In Vivo and In Vitro Effects of Integration Host Factor at the DmpR-Regulated &sigma;<sup>54</sup>-Dependent Po Promoter. <I>Journal of bacteriology</I>, (2001), 183.9: 2842-2851.
<br/>
<br/>
-
[4]. SHINGLER, Victoria; MOORE, Terry. Sensing of aromatic compounds by the DmpR transcriptional activator of phenol-catabolizing Pseudomonas sp. strain CF600. Journal of bacteriology, (1994), 176.6: 1555-1560.
+
[4]. SARAND, Inga, et al. Role of the DmpR-mediated regulatory circuit in bacterial biodegradation properties in methylphenol-amended soils. <I>Applied and environmental microbiology</I>, (2001), 67.1: 162-171.  
<br/>
<br/>
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[5]. SZE, Chun Chau; LAURIE, Andrew D.; SHINGLER, Victoria. In Vivo and In Vitro Effects of Integration Host Factor at the DmpR-Regulated ς54-Dependent Po Promoter. Journal of bacteriology, (2001), 183.9: 2842-2851.
+
[5].WISE, Arlene A.; KUSKE, Cheryl R. Generation of novel bacterial regulatory proteins that detect priority pollutant phenols. <I>Applied and environmental microbiology</I>, (2000), 66.1: 163-169.  
<br/>
<br/>
-
[6]. SARAND, Inga, et al. Role of the DmpR-mediated regulatory circuit in bacterial biodegradation properties in methylphenol-amended soils. Applied and environmental microbiology, (2001), 67.1: 162-171.  
+
[6]. TROPEL, David; VAN DER MEER, Jan Roelof. Bacterial transcriptional regulators for degradation pathways of aromatic compounds. <I>Microbiology and Molecular Biology Reviews</I>, (2004), 68.3: 474-500.
 +
</br>
 +
[7]. SHINGLER, V.; POWLOWSKI, J.; MARKLUND, U. Nucleotide sequence and functional analysis of the complete phenol/3, 4-dimethylphenol catabolic pathway of <I>Pseudomonas sp</I>. strain CF600. <I>Journal of bacteriology</I>, (1992), 174.3: 711-724.
<br/>
<br/>
-
[7].WISE, Arlene A.; KUSKE, Cheryl R. Generation of novel bacterial regulatory proteins that detect priority pollutant phenols. Applied and environmental microbiology, (2000), 66.1: 163-169.  
+
[8]. GUPTA, Saurabh, et al. An Effective Strategy for a Whole-Cell Biosensor Based on Putative Effector Interaction Site of the Regulatory DmpR Protein. <I>PloS one</I>, (2012), 7.8: e43527.  
<br/>
<br/>
-
[8]. TROPEL, David; VAN DER MEER, Jan Roelof. Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiology and Molecular Biology Reviews, (2004), 68.3: 474-500.
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<h1 id="DmpRTableTitle"><B> Table 1 .Various analogs that activate DmpR</B></h1>
 
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<table border="1" id="DmpRTable">
 
-
<tr>
 
-
  <th>Amino acid substitution</th>
 
-
  <th>Inducers</th>
 
-
  <th>Reference</th>
 
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</tr>
 
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<tr>
 
-
  <td>Gln<sup>10</sup><Code>&rarr;</Code>Arg</td>
 
-
  <td>Phenol, 2,4-Dichlorophenol,<br/>
 
-
4-Chloro-3-methylphenol,<br/>
 
-
2,4-Methylphenol,2-Chlorophenol
 
-
</td>
 
-
  <td>KUSKE <I>et al</I> <sup>[7]</sup></td>
 
-
</tr>
 
-
<tr>
 
-
  <td>Lys<sup>6</sup><Code>&rarr;</Code>Glu<br/>Phe<sup>42</sup><Code>&rarr;</Code>Ser</td>
 
-
  <td>Phenol, 2,4-Dichlorophenol,<br/>
 
-
2-Dimethylphenol,2-Nitrophenol
 
-
</td>
 
-
  <td>KUSKE <I>et al</I> <sup>[7]</sup></td>
 
-
</tr>
 
-
<tr>
 
-
  <td>Gln<sup>10</sup><Code>&rarr;</Code>Arg<br/>Lys<sup>42</sup><Code>&rarr;</Code>Met</td>
 
-
  <td>Phenol, 2,4-Dichlorophenol,<br/>
 
-
2-Chlorophenol,2-Dimethylphenol,<br/>
 
-
2-Nitrophenol
 
-
</td>
 
-
  <td>KUSKE <I>et al</I> <sup>[7]</sup></td>
 
-
</tr>
 
-
<tr>
 
-
  <td>As<sup>116</sup><Code>&rarr;</Code>Val</td>
 
-
  <td>Phenol</td>
 
-
  <td>KUSKE <I>et al</I> <sup>[7]</sup></td>
 
-
</tr>
 
-
</table>
+
 
  </div>
  </div>

Latest revision as of 18:15, 28 October 2013

Biosensors

DmpR

Mechanism

Build Our Own Sensor!

DmpR bioinformatically mined from Pseudomonas sp.CF600 [1-6] is a σ54-dependent transcriptional factor that tightly controls the expression of the dmp operon (dmpKLMNOPQBCDEFGHI) (Fig. 1). This operon carries genes encoding enzymes for the degradation of (methyl) phenols into pyruvate and acetyl-CoA[7] (Fig. 2).

The cognate promoter of DmpR is Po promoter. The DmpR protein binds to Po promoter as hexamer on two distinct UAS (Upstream Activating Sequence). The transcription initiation of dmp operon also requires IHF (Integration Host Factor), which has two binding sites in Po promoter and enhances the transcription efficiency (Fig. 3).

DmpR protein consists of four domains (Fig. 4): Domain A is the effector-sensing domain, which undergoes conformational change when exposed to proper inducers, including phenol, 2-chlorophenol, 2,4-dichlorophenol, methyl-phenols and other substituted phenols [3][8]. Domain B is a linker domain where mutations would modulate the interactions between Domain A and Domain C. Domain C is the transcriptional activation domain. Domain D contains a helix-turn-helix motif, which is responsible for the DNA binding at Po promoter [1].

The mechanism of Po promoter activation consists of four steps, DmpR dimerization, DmpR hexamer formation, DNA bending and RNAP recruitment (Fig. 5). Ater the 4 steps, with the help of IHF, transcription from Po promoter initiates thereby.

We collected and analyzed all of the information about DmpR. See Table 1 for the comprehensive summary of DmpR mutants and accompanied novel aromatics-sensing characteristics, which provides a rich repertoire for the synthetic biologists to customize the aromatics-sensing characteristics of DmpR protein.

Peking iGEM has adopted DmpR to build a biosensor circuit (Fig. 6). Plasmid carrying Pr-DmpR was co-transformed with the plasmid containing the inducible promoter Po and reporter gene sfGFP (Fig. 6). Similar to other biosensors, plasmid with RBS BBa_B0032 preceding sfGFP was chosen due to its relatively higher induction ratio during primary test for the RBS library.

We evaluated the performance of DmpR using our own protocols and almost every protocol mentioned in the previous studies (for more details about these three protocols, Test Protocol 1-3, Click Here). Results showed that the Test Protocol 3 works the best, with which we tested the ON-OFF ratios of all the 78 aromatic compounds. 7 compounds out of the 78 showed significant induction ratios (>2) (Fig. 7): They are Phl, 2-MePhl, 2-ClPhl, 3-ClPhl, Cat, 4-NtPhl and 2-APhl (Click Here for the full names of the aromatic compounds). The presence of phenol is consistent with previous studies. The other 6 compounds, however, have not been reported in previous works.

To provide more detailed information about the aromatics-sensing profile, we carefully examined the individual dose-response curves of the 7 compounds via Test Protocol 3 (Fig. 8).

In summary, we have successfully constructed the DmpR biosensing circuit and fine-tuned it guided by our experience obtained from the building of other biosensors. The aromatics-sensing profile of DmpR biosensor is considerably narrow (Fig. 7), making it a robust and convenient biosensor for the presence of phenol and its derivatives.

Figure. 1. The schematic structure of dmp operon. Dmp operon carries genes encoding enzymes for the degradation of (methyl-)phenols to pyruvate and acetyl-CoA, the intermediates of TCA Cycle. The operon is positively controlled by the dmpR gene product, resulting in expression of catabolic enzymes when inducers like phenol are present.

Figure. 2. The catabolic pathway of phenol controlled by the dmp operon. Metabolic enzymes along the pathway are (from Step 1 to Step 8): 1, phenol hydroxylase (PH) ; 2, catechol 2, 3-dioxygenase (C23O); 3, 2-hydroxymuconic semialdehyde hydrolase (2HMSH); 4, 2-hydroxymuconic semialdehyde dehydrogenase (2HMSD) ;5, 4-oxalocrotonate isomerase (4OI); 6, 4-oxalocrotonate decarboxylase (4OD) ;7, 2-oxopent-4-cnoate hydeatase (OEH); 8, 4-hydroxy-2-2oxovalerate aldolase (HOA).

Figure. 3. The schematic structure of Po promoter. The UASs of this promoter shaded in green are dyad sequences to which the DmpR protein binds. The yellow shaded boxes denote IHF binding sites. The pink shaded box represents σ54 binding site with -24 region and -12 motifs highlighted in red. The highlighted nucleotide G represents the transcription start (+1) site.

Figure. 4. The schematic structure of DmpR protein. From N-terminal to C-terminal are Domain A, Domain B, Domain C, Domain D.

Figure. 5. The mechanism of transcription activation by DmpR. (a) The inactive dimer binds to its inducer, which results in a protein conformational change. (b) Binding of ATP triggers multimerization of the dimers to hexamers (or haptamer). (c) ATP hydrolysis coupled with RNA polymerase recruitment triggers transcription activation. (d) Dissociation of the hexamers into dimers after ATP hydrolysis [6].

Figure. 6.The schematic diagram for the DmpR biosensor circuit. DmpR is constitutively expressed and functions to regulate the transcription of sfGFP gene via promoter Po. As for the RBS of sfGFP, BBa_B0032 was decided due to its better performance compared to RBS of other transcriptional strengths.

Figure. 7. ON/OFF test to evaluate the induction ratios of all aromatic compounds in the aromatics spectrum (For the full names of the compounds, Click Here). (a) The induction ratios of all 78 aromatic species for the DmpR biosensor. The DmpR biosensor could respond to 7 out of the 78 aromatics with the induction ratio higher than 2. (b) The aromatics-sensing profile of DmpR biosensor. The aromatic species that can elicit strong responses of DmpR biosensor are highlighted in cyan in the aromatics spectrum. The structure formula of typical inducer is also presented around the spectrum. The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to object inducers by the basal fluorescence intensity of the biosensor itself.

Figure. 8. Dose-response curves of DmpR biosensor induced by 7 strong inducers (phenol, its homologs and derivatives). The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to inducers by the basal fluorescence intensity of the biosensor. For the full names of the compounds, Click Here .

REFERENCE:
[1]. SHINGLER, V.; PAVEL, H. Direct regulation of the ATPase activity of the transcriptional activator DmpR by aromatic compounds. Molecular microbiology, (1995), 17.3: 505-513.
[2]. SHINGLER, Victoria; MOORE, Terry. Sensing of aromatic compounds by the DmpR transcriptional activator of phenol-catabolizing Pseudomonas sp. strain CF600. Journal of bacteriology, (1994), 176.6: 1555-1560.
[3]. SZE, Chun Chau; LAURIE, Andrew D.; SHINGLER, Victoria. In Vivo and In Vitro Effects of Integration Host Factor at the DmpR-Regulated σ54-Dependent Po Promoter. Journal of bacteriology, (2001), 183.9: 2842-2851.
[4]. SARAND, Inga, et al. Role of the DmpR-mediated regulatory circuit in bacterial biodegradation properties in methylphenol-amended soils. Applied and environmental microbiology, (2001), 67.1: 162-171.
[5].WISE, Arlene A.; KUSKE, Cheryl R. Generation of novel bacterial regulatory proteins that detect priority pollutant phenols. Applied and environmental microbiology, (2000), 66.1: 163-169.
[6]. TROPEL, David; VAN DER MEER, Jan Roelof. Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiology and Molecular Biology Reviews, (2004), 68.3: 474-500.
[7]. SHINGLER, V.; POWLOWSKI, J.; MARKLUND, U. Nucleotide sequence and functional analysis of the complete phenol/3, 4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600. Journal of bacteriology, (1992), 174.3: 711-724.
[8]. GUPTA, Saurabh, et al. An Effective Strategy for a Whole-Cell Biosensor Based on Putative Effector Interaction Site of the Regulatory DmpR Protein. PloS one, (2012), 7.8: e43527.