Difference between revisions of "Team:ICT-Mumbai/Results"

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{{ICT-Mumbai}}
 
 
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<title>ICT-Mumbai 2017</title>
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<h1>Results</h1>
 
  
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<p>Here you can describe the results of your project and your future plans. </p>
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body,h1,h2,h3,h4,h5,h6 {font-family: "Lato", sans-serif;}
  
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<h5>What should this page contain?</h5>
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<ul>
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    height: 100%;
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<li> Clearly and objectively describe the results of your work.</li>
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<li> Future plans for the project. </li>
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}
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<li> Considerations for replicating the experiments. </li>
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<h5>You should also describe what your results mean: </h5>
 
  
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<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
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<li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li>
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<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
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.bgimg-1 {
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    background-image: url("https://static.igem.org/mediawiki/2017/b/ba/ICT-Mumbai_homepage_image.png");
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    min-height: 500px;
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background-image: url("https://static.igem.org/mediawiki/2017/4/44/ICT-Mumbai_homepage_dyeodorant_image.png");
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<h5> Project Achievements </h5>
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
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<li>A list of linked bullet points of the successful results during your project</li>
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<body>
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
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      <i class="fa fa-bars"></i>
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    </a>
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  <a href="https://2017.igem.org/Team:ICT-Mumbai" class="w3-bar-item w3-button w3-hide-small"><i class="fa fa-home" aria-hidden="true"></i>
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Home</a>
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    Project</button></a>
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        <a href="https://2017.igem.org/Team:ICT-Mumbai/Background" class="w3-bar-item w3-button">Background</a>
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        <a href="https://2017.igem.org/Team:ICT-Mumbai/Description" class="w3-bar-item w3-button">Description</a>
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        <a href="https://2017.igem.org/Team:ICT-Mumbai/Design" class="w3-bar-item w3-button">Design</a>
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        <a href="https://2017.igem.org/Team:ICT-Mumbai/Protocols" class="w3-bar-item w3-button">Protocols</a>
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        <a href="https://2017.igem.org/Team:ICT-Mumbai/Notebook"class="w3-bar-item w3-button">Notebook</a>
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        <a href="https://2017.igem.org/Team:ICT-Mumbai/InterLab"class="w3-bar-item w3-button">Interlab</a>
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        <a href="https://2017.igem.org/Team:ICT-Mumbai/Results"class="w3-bar-item w3-button">Results</a>
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</div>
 
</div>
 
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<h5>Inspiration</h5>
 
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<p>See how other teams presented their results.</p>
 
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<ul>
 
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
 
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
 
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
 
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</ul>
 
  
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<button class="w3-button" class="w3-bar-item w3-button w3-hide-small"><i class="fa fa-users" aria-hidden="true"></i>Team</button></a>
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<div class="w3-dropdown-content w3-bar-block w3-animate-opacity">
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        <a href="https://2017.igem.org/Team:ICT-Mumbai/Team"class="w3-bar-item w3-button">Team</a>
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        <a href="https://2017.igem.org/Team:ICT-Mumbai/Collaborations" class="w3-bar-item w3-button">Collaborations</a>
 
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<a href="https://2017.igem.org/Team:ICT-Mumbai/Parts" button class="w3-button" class="w3-bar-item w3-button w3-hide-small"><i class="fa fa-cogs" aria-hidden="true"></i>  Part</a>
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<a href="https://2017.igem.org/Team:ICT-Mumbai/Safety" class="w3-bar-item w3-button w3-hide-small"><i class="fa fa-flag" aria-hidden="true"></i>
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Safety</a>
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<a href="https://2017.igem.org/Team:ICT-Mumbai/HP/Gold_Integrated" class="w3-bar-item w3-button w3-hide-small"><i class="fa fa-handshake-o" aria-hidden="true"></i>
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Human Practices</a>
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<a href="https://2017.igem.org/Team:ICT-Mumbai/Attributions" class="w3-bar-item w3-button w3-hide-small"><i class="fa fa-user-o" aria-hidden="true"></i>Attributions</a>
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    <a href="#about" class="w3-bar-item w3-button" onclick="toggleFunction()">ABOUT</a>
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    <a href="#portfolio" class="w3-bar-item w3-button" onclick="toggleFunction()">PORTFOLIO</a>
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    <a href="#contact" class="w3-bar-item w3-button" onclick="toggleFunction()">CONTACT</a>
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    <a href="#" class="w3-bar-item w3-button">SEARCH</a>
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<!-- Container (About Section) -->
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  <h3 class="w3-center" style="color:#000080">The big picture</h3>
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<p style="height:70 px; font-family:'Lato'; font-size:20px; colour:lightgrey;">As described in <a href="https://2017.igem.org/Team:ICT-Mumbai/Description">Description</a>, we wish to construct Escherichia coli cells that can assimilate ammonia and convert it into an innocuous substance. As the first step, we decided to overexpress the native enzyme glutamine synthetase, which condenses ammonia with glutamate to form glutamine.</p>
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<div class="w3-content w3-container w3-padding-64" id="about">
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  <h3 class="w3-center" style="color:#000080">Cloning <i>glnA</i> in pET43.1b</h3>
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<p style="height:70 px;font-family:'Lato'; font-size:20px; colour:lightgrey;">The <i>glnA</i> gene codes for the enzyme glutamine synthetase. We amplified <i>glnA</i> from the <i>E. coli</i> MG1655 genome and cloned it in plasmid pET43.1b between the NdeI and HindIII sites to form plasmid pET43-glnA.</p>
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<div class="w3-content w3-container w3-padding-64" id="about">
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  <h4 class="w3-center" style="color:#000080">Co-expression of <i>indC</i></h4>
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<p style="height:70 px;  font-family:'Lato';  font-size:20px; colour:lightgrey;">The gene <i>indC</i> codes for blue pigment synthase (Bps), which converts glutamine to the blue-colored compound indigoidine. Plasmid pRB5, which was obtained as a kind gift from Team Heidelberg, carries the <i>indC</i> gene under control of a <i>lacI</i>-regulated promoter.</p>
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<p style="height:70 px;  font-family:'Lato';  font-size:20px; colour:lightgrey;">We decided to co-transform plasmids pET43-glnA and pRB5 in <i>E. coli</i> BL21(DE3). However, we realized that both these plasmids have the same origin of replication (from pMB1), and would therefore not be maintained in a single cell. To circumvent this problem, we decided to clone <i>glnA</i> in a plasmid that has a different origin of replication.</p>
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<div class="w3-content w3-container w3-padding-64" id="about">
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  <h5 class="w3-center" style="color:#000080">Cloning <i>glnA</i> in pSEVA234</h5>
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<p style="height:70 px;  font-family:'Lato';  font-size:20px; colour:lightgrey;">Plasmid pSEVA234 has an <i>oriT</i> origin of replication, and therefore will be able to co-exist with plasmids that have a pMB1 origin of replication. To clone <i>glnA</i> in pSEVA234, pET43-glnA was digested with Psp5II and XhoI to release the <i>lacI-T7 promoter-glnA</i> fragment. This was blunted and cloned in SmaI-digested pSEVA234 to form plasmid pSEVA234-glnA.</p>
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<div class="w3-content w3-container w3-padding-64" id="about">
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  <h5 class="w3-center" style="color:#000080">Biobricking <i>ychH</i> promoter</h5>
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</style>
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<p style="height:70 px;  font-family:'Lato';  font-size:20px; colour:lightgrey;">As described in <a href="https://2017.igem.org/Team:ICT-Mumbai/Parts">Parts</a>, we wish to express enzymes for ammonia synthesis using a constitutive promoter that is active under nutrient starvation conditions. For this purpose, we chose the promoter of the <i>ychH</i> gene. We amplified a fragment containing the <i>ychH</i> promoter from the <i>E. coli</i> MG1655 genome using primers Fwd_BB_ychH_prom
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(5’-GTTTCTTCGAATTCGCGGCCGCTTCTAGAGgtttttttgtcctgagtgtgtacataac) and Rev_BB_ychH_prom
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(5’-GAAGAAACCTGCAGCGGCCGCTACTAGTAtcacctccggaactttctg), which contain the prefix and suffix, respectively, of a BioBrick. The BioBricked <i>ychH</i> promoter, along with the native RBS, was cloned in EcoRI- and PstI-digested pSB1C3 and submitted to the iGEM Parts Registry as Part BBa_K2479000.
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</p>
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<div class="w3-content w3-container w3-padding-64" id="about">
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  <h5 class="w3-center" style="color:#000080">Getting E. coli to work</h5>
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<style>
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<p style="height:70 px;  font-family:'Lato';  font-size:20px; colour:lightgrey;">The enzyme glutamine synthetase requires ATP as a cofactor, and releases protons on catalyzing the formation of glutamine from ammonium and glutamate. Hence, ATP will have to be continuously supplied to keep on assimilating ammonium. This can be achieved by pumping out the protons formed using proteorhodopsin, which is a light-powered proton pump. The resulting proton gradient can then drive ATP synthase to form ATP, required for glutamine synthetase activity.
 +
</p>
 +
  
  
  
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<p style="height:70 px;  font-family:'Lato';  font-size:20px; colour:lightgrey;">A schematic representation of our concept is depicted below:
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</p>
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<br>
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<img src="https://static.igem.org/mediawiki/2017/8/8e/ICT-Mumbai_description_image.jpeg" height="100%" width="100%">
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</body>
 
</html>
 
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Revision as of 16:04, 1 November 2017

ICT-Mumbai 2017

The big picture

As described in Description, we wish to construct Escherichia coli cells that can assimilate ammonia and convert it into an innocuous substance. As the first step, we decided to overexpress the native enzyme glutamine synthetase, which condenses ammonia with glutamate to form glutamine.

Cloning glnA in pET43.1b

The glnA gene codes for the enzyme glutamine synthetase. We amplified glnA from the E. coli MG1655 genome and cloned it in plasmid pET43.1b between the NdeI and HindIII sites to form plasmid pET43-glnA.

Co-expression of indC

The gene indC codes for blue pigment synthase (Bps), which converts glutamine to the blue-colored compound indigoidine. Plasmid pRB5, which was obtained as a kind gift from Team Heidelberg, carries the indC gene under control of a lacI-regulated promoter.

We decided to co-transform plasmids pET43-glnA and pRB5 in E. coli BL21(DE3). However, we realized that both these plasmids have the same origin of replication (from pMB1), and would therefore not be maintained in a single cell. To circumvent this problem, we decided to clone glnA in a plasmid that has a different origin of replication.

Cloning glnA in pSEVA234

Plasmid pSEVA234 has an oriT origin of replication, and therefore will be able to co-exist with plasmids that have a pMB1 origin of replication. To clone glnA in pSEVA234, pET43-glnA was digested with Psp5II and XhoI to release the lacI-T7 promoter-glnA fragment. This was blunted and cloned in SmaI-digested pSEVA234 to form plasmid pSEVA234-glnA.

Biobricking ychH promoter

As described in Parts, we wish to express enzymes for ammonia synthesis using a constitutive promoter that is active under nutrient starvation conditions. For this purpose, we chose the promoter of the ychH gene. We amplified a fragment containing the ychH promoter from the E. coli MG1655 genome using primers Fwd_BB_ychH_prom (5’-GTTTCTTCGAATTCGCGGCCGCTTCTAGAGgtttttttgtcctgagtgtgtacataac) and Rev_BB_ychH_prom (5’-GAAGAAACCTGCAGCGGCCGCTACTAGTAtcacctccggaactttctg), which contain the prefix and suffix, respectively, of a BioBrick. The BioBricked ychH promoter, along with the native RBS, was cloned in EcoRI- and PstI-digested pSB1C3 and submitted to the iGEM Parts Registry as Part BBa_K2479000.

Getting E. coli to work

The enzyme glutamine synthetase requires ATP as a cofactor, and releases protons on catalyzing the formation of glutamine from ammonium and glutamate. Hence, ATP will have to be continuously supplied to keep on assimilating ammonium. This can be achieved by pumping out the protons formed using proteorhodopsin, which is a light-powered proton pump. The resulting proton gradient can then drive ATP synthase to form ATP, required for glutamine synthetase activity.

A schematic representation of our concept is depicted below: