Team:WashU StLouis/Project

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<h1> Project Overview </h1>
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<center><h1> Project Description </h1></center>
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<center> <p> Our project is currently the first step in a much larger endeavor. </p> <br>
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<p> Our project is currently the first step in a much larger
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<img src="https://static.igem.org/mediawiki/2014/1/1f/Washu_2014_project_overview1.jpg" width="700px" align="right"> </center>
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endeavor.</p>
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<p> We are attempting to take the <i> nif </i> cluster from a cyanobacteria and get it to function in <i> E. coli </i> while simultaneously attempting to create a transcriptional regulation system that turns off in the light and turns on in the dark. In doing such, we hope to create a system for nitrogen fixation that operates exclusively in the absence of light in preparation for transformation into a photosynthetic system. After we come to a greater understanding of how the system works and perfect it, we can move on to working in a more complex organism, such as a cyanobacteria like <i> Synecosystis </i> 6803. The end goal is to create plants that can fix their own nitrogen by moving from the cyanobacteria into the chloroplast of the plant. Endosymbiotic theory postulates that cyanobacteria are the ancestors to chloroplasts, so this is the natural progression. </p>
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<p> For more information on our attempt to transform <i> E. coli </i> with the <i> nif </i> cluster, please visit the <a href=https://2014.igem.org/Team:WashU_StLouis/Project/nif> Nitrogenase </a> page. </p> <br>
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<p> For more information on our attempt to create a light-sensitive transcription system, please visit the <a href=https://2014.igem.org/Team:WashU_StLouis/Project/light> Light Regulation </a> page. </p> <br>
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<p> Be sure to check out our <a href=https://2014.igem.org/Team:WashU_StLouis/Project/collaboration> Collaboration </a>, <a href=https://2014.igem.org/Team:WashU_StLouis/Parts> Parts </a>, and <a href=https://2014.igem.org/Team:WashU_StLouis/Modeling> Modeling </a> pages, as well! </p>
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<td
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style="vertical-align: middle; text-align: justify; width: 50%;">We
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are attempting to take the <i> nif </i> cluster from a
 +
cyanobacteria and get it to function in <i> E. coli </i> while
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simultaneously attempting to create a transcriptional regulation system
 +
that turns off in the light and turns on in the dark. <br>
<br>
<br>
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<center><h1> Content</h1></center>
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In doing such, we
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<p> You can use these subtopics to further explain your project</p>
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hope to create a system for nitrogen fixation that operates exclusively
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in the absence of light in preparation for transformation into a
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<ol>
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photosynthetic system. After we come to a greater understanding of how
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<li>Overall project summary</li>
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the system works and perfect it, we can move on to working in a more
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<li>Project Details</li>
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complex organism, such as a cyanobacteria like <i> Synecosystis </i>spp.
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<li>Materials and Methods</li>
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6803. <br>
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<li>The Experiments</li>
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<br>
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<li>Results</li>
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The end goal is to create plants that can fix their own nitrogen
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<li>Data analysis</li>
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by moving from the cyanobacteria into the chloroplast of the plant.
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<li>Conclusions</li>
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Endosymbiotic theory postulates that cyanobacteria are the ancestors to
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</ol>
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chloroplasts, so this is the natural progression.</td>
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</tr>
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<p>
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<tr>
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It's important for teams to describe all the creativity that goes into an iGEM project, along with all the great ideas your team will come up with over the course of your work.  
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</p>
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style="vertical-align: middle; width: 50%; text-align: justify;">
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<p>This summer, we were successful in transforming <span
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<p>
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style="font-style: italic;">E. coli</span> with the <span
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It's also important to clearly describe your achievements so that judges will know what you tried to do and where you succeeded. Please write your project page such that what you achieved is easy to distinguish from what you attempted.  
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style="font-style: italic;">nif</span> cluster from <span
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</p>
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style="font-style: italic;">Cyanothece </span>51142, and ran an
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Acetylene Reduction Assay in order to test for the strains ability to
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<center><h1>References </h1> </center>
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fix nitrogen. For more information, please consult our<a
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<p>The pictures used above were taken from the following sources: </p> <p> <a href=http://protist.i.hosei.ac.jp/PDB/images/Prokaryotes/Chroococcaceae/Synechocystis/sp_02.jpg> http://protist.i.hosei.ac.jp/PDB/images/Prokaryotes/Chroococcaceae/Synechocystis/sp_02.jpg </a></p> <p> <a href=http://www.pitch.com/imager/explosive-diarrhea-anyone/b/original/2575052/269f/EcoliEM4.jpg> http://www.pitch.com/imager/explosive-diarrhea-anyone/b/original/2575052/269f/EcoliEM4.jpg </a> </p> <p> <a href=http://www.motherearthnews.com/~/media/Images/MEN/Editorial/Articles/Magazine%20Articles/1971/05-01/How%20to%20Dry%20Sweet%20Corn/corn.jpg> http://www.motherearthnews.com/~/media/Images/MEN/Editorial/Articles/Magazine%20Articles/1971/05-01/How%20to%20Dry%20Sweet%20Corn/corn.jpg </a> </p>
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href="https://2014.igem.org/Team:WashU_StLouis/Project/nif">
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Nitrogenase </a> page.</p>
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This summer, we were also successful in cloning a light regulated
 +
repressor system in order to turn OFF transcription of genes in the
 +
presence of light. For more information on our methods and background,
 +
please visit the <a
 +
href="https://2014.igem.org/Team:WashU_StLouis/Project/light">Light
 +
Regulation </a> page. <br>
 +
<br>
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Be sure to check out our <a
 +
href="https://2014.igem.org/Team:WashU_StLouis/Project/collaboration">
 +
Collaboration </a>, <a
 +
href="https://2014.igem.org/Team:WashU_StLouis/Parts"> Parts </a>, and
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<a href="https://2014.igem.org/Team:WashU_StLouis/Modeling">
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Modeling </a> pages, as well!</td>
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<td
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style="vertical-align: middle; width: 50%; text-align: justify;"><img
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style="width: 100%;" alt="WashU project"
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src="https://static.igem.org/mediawiki/2014/4/4e/WashU_Project_Overview.png"></td>
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&nbsp;
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<p> </p>
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<td style="vertical-align: top; text-align: center;">Organism<br>
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</td>
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<td style="vertical-align: top; text-align: center;">Ease
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of Engineering<br>
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</td>
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<td style="vertical-align: top; text-align: center;">Photosynthetic<br>
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</td>
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<td style="vertical-align: top; text-align: center;">Crop
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Plant<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top; text-align: center;"><span
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style="font-style: italic;">E. coli</span><br>
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</td>
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<td style="vertical-align: top; text-align: center;">✓✓<br>
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</td>
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<td style="vertical-align: top; text-align: center;">✕<br>
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</td>
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<td style="vertical-align: top; text-align: center;">✕<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top; text-align: center;">S. 6803<br>
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</td>
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<td style="vertical-align: top; text-align: center;">✓</td>
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<td style="vertical-align: top; text-align: center;">✓</td>
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<td style="vertical-align: top; text-align: center;">✕<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top; text-align: center;">Chloroplast<br>
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</td>
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<td style="vertical-align: top; text-align: center;">✕<br>
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</td>
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<td style="vertical-align: top; text-align: center;">✓</td>
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<td style="vertical-align: top; text-align: center;">✓</td>
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</tr>
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</tbody>
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</table>
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<br>
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Our teams project goals were to:<br>
 +
•&nbsp;&nbsp;&nbsp; Determine the optimal conditions for culturing<span
 +
style="font-style: italic;"> E. coli</span> strains containing the <span
 +
style="font-style: italic;">Cyanothece</span> sp. 51142 nif cluster<br>
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•&nbsp;&nbsp;&nbsp; Select the best strains for further testing<br>
 +
•&nbsp;&nbsp;&nbsp; Create a light repressed gene regulatory mechanism<br>
 +
•&nbsp;&nbsp;&nbsp; Compare fold change of light induction with new
 +
hybrid promoter<br>
 +
<br>
 +
<img style="width: 50%;" alt="Engineered strains"
 +
src="https://static.igem.org/mediawiki/2014/7/7c/WashU_Engineered_Strains.jpg"
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align="left" hspace="5">Through our experiments, we concluded that:<br>
 +
•&nbsp;&nbsp;&nbsp; Of the five E. coli strains tested, JM109 and
 +
WM1788 showed strongest nitrogenase activity. <br>
 +
•&nbsp;&nbsp;&nbsp; The linear relationship between nitrogen fixation
 +
activity and time matches that seen in nature. <br>
 +
•&nbsp;&nbsp;&nbsp; Optimal conditions: glucose as carbon-source,
 +
glutamate as nitrogen-source, LB as inoculating media, minimal M9 as
 +
testing media for GC assay, anaerobic environment at&nbsp;&nbsp; 30 °C
 +
for overnight preparation before acetylene reduction assay. <br>
 +
•&nbsp;&nbsp;&nbsp; Troubleshoot a faulty reporter mechanism<br>
 +
•&nbsp;&nbsp;&nbsp; Created a hybrid promoter<br>
 +
•&nbsp;&nbsp;&nbsp; Ran light experiments that showed discernable fold
 +
change in on and off states with appropriate amounts of aTc.<br>
 +
<br>
 +
In future, we intend to:<br>
 +
•&nbsp;&nbsp;&nbsp; Alter conditions to increase activity in JM109 and
 +
WM1788 <br>
 +
•&nbsp;&nbsp;&nbsp; Determine a minimal nif cluster<br>
 +
•&nbsp;&nbsp;&nbsp; Directly check and optimize light sensitive
 +
promoters<br>
 +
•&nbsp;&nbsp;&nbsp; Adjust the leakiness of the light sensor system to
 +
not need aTc<br>
 +
•&nbsp;&nbsp;&nbsp; Swap out the reporter protein with the nif cluster
 +
to get both systems working in conjunction.<br>
 +
•&nbsp;&nbsp;&nbsp; Transition into cyanobacteria by transferring the
 +
genes with the nif cluster back into Synechocystis S. 6803.<br>
 +
<br>
 +
<br>
 +
<br>
 +
<span style="font-weight: bold;"><br>
 +
</span>
<center>
<center>
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<a href=https://2014.igem.org/Team:WashU_StLouis/Project/nif> Nitrogenase </a> |
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<center>
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<a href=https://2014.igem.org/Team:WashU_StLouis/Project/light> Light Regulation </a> |
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<br>
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<a href=https://2014.igem.org/Team:WashU_StLouis/Project/collaboration> Collaboration </a> |
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<div style="text-align: left;">Figure above: Engineered strains
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<a href=https://2014.igem.org/Team:WashU_StLouis/Parts> Parts </a> |
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of <span style="font-style: italic;">E. coli</span> being flushed with
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<a href=https://2014.igem.org/Team:WashU_StLouis/Modeling> Modeling </a>
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argon gas<br>
 +
</div>
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<h1>References </h1>
</center>
</center>
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<p style="text-align: left;">The pictures used above were taken
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from the following sources: </p>
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<div style="text-align: left;"> </div>
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<p style="text-align: left; margin-left: 40px;"> <a
 +
href="http://protist.i.hosei.ac.jp/PDB/images/Prokaryotes/Chroococcaceae/Synechocystis/sp_02.jpg"><i>1.
 +
Synechocystis</i> </a></p>
 +
<div style="text-align: left; margin-left: 40px;"> </div>
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<p style="text-align: left; margin-left: 40px;"> <a
 +
href="http://www.pitch.com/imager/explosive-diarrhea-anyone/b/original/2575052/269f/EcoliEM4.jpg"><i>2.
 +
E. coli</i> </a> </p>
 +
<div style="text-align: left; margin-left: 40px;"> </div>
 +
<div style="text-align: left; margin-left: 40px;"> <a
 +
href="http://www.motherearthnews.com/%7E/media/Images/MEN/Editorial/Articles/Magazine%20Articles/1971/05-01/How%20to%20Dry%20Sweet%20Corn/corn.jpg">3.
 +
Corn </a></div>
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<p> </p>
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</center>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;"><br>
</td>
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Revision as of 17:13, 17 October 2014



Project Overview

Our project is currently the first step in a much larger endeavor.

E. coli to Synechocystis to Chloroplast We are attempting to take the nif cluster from a cyanobacteria and get it to function in E. coli while simultaneously attempting to create a transcriptional regulation system that turns off in the light and turns on in the dark.

In doing such, we hope to create a system for nitrogen fixation that operates exclusively in the absence of light in preparation for transformation into a photosynthetic system. After we come to a greater understanding of how the system works and perfect it, we can move on to working in a more complex organism, such as a cyanobacteria like Synecosystis spp. 6803.

The end goal is to create plants that can fix their own nitrogen by moving from the cyanobacteria into the chloroplast of the plant. Endosymbiotic theory postulates that cyanobacteria are the ancestors to chloroplasts, so this is the natural progression.

This summer, we were successful in transforming E. coli with the nif cluster from Cyanothece 51142, and ran an Acetylene Reduction Assay in order to test for the strains ability to fix nitrogen. For more information, please consult our Nitrogenase page.

This summer, we were also successful in cloning a light regulated repressor system in order to turn OFF transcription of genes in the presence of light. For more information on our methods and background, please visit the Light Regulation page.

Be sure to check out our Collaboration , Parts , and Modeling pages, as well!		 WashU project
 

Organism
 Ease of Engineering
 Photosynthetic
 Crop Plant
E. coli
 ✓✓
 ✕
 ✕
S. 6803
 ✓ ✓ ✕
Chloroplast
 ✕
 ✓ ✓

Our teams project goals were to:
•    Determine the optimal conditions for culturing E. coli strains containing the Cyanothece sp. 51142 nif cluster
•    Select the best strains for further testing
•    Create a light repressed gene regulatory mechanism
•    Compare fold change of light induction with new hybrid promoter

Engineered strainsThrough our experiments, we concluded that:
•    Of the five E. coli strains tested, JM109 and WM1788 showed strongest nitrogenase activity.
•    The linear relationship between nitrogen fixation activity and time matches that seen in nature.
•    Optimal conditions: glucose as carbon-source, glutamate as nitrogen-source, LB as inoculating media, minimal M9 as testing media for GC assay, anaerobic environment at   30 °C for overnight preparation before acetylene reduction assay.
•    Troubleshoot a faulty reporter mechanism
•    Created a hybrid promoter
•    Ran light experiments that showed discernable fold change in on and off states with appropriate amounts of aTc.

In future, we intend to:
•    Alter conditions to increase activity in JM109 and WM1788
•    Determine a minimal nif cluster
•    Directly check and optimize light sensitive promoters
•    Adjust the leakiness of the light sensor system to not need aTc
•    Swap out the reporter protein with the nif cluster to get both systems working in conjunction.
•    Transition into cyanobacteria by transferring the genes with the nif cluster back into Synechocystis S. 6803.

Figure above: Engineered strains of E. coli being flushed with argon gas

References

The pictures used above were taken from the following sources:

1. Synechocystis

2. E. coli


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