Team:WashU StLouis/Project

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<h1> Project Overview </h1>
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<h1> Project Description </h1>
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<p> Our project is currently the first step in a much larger endeavor. </p>
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<img src="https://static.igem.org/mediawiki/2014/5/57/WUSTL_IGEM_Big_Picture.tiff" width="500px">
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<p> Our project is currently the first step in a much larger
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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.  
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endeavor.</p>
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<h1> Content</h1>
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<td style="vertical-align: top; width: 50%;"><img
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style="width: 100%;" alt="E. coli to Synechocystis to Chloroplast"
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src="https://static.igem.org/mediawiki/2014/1/1f/Washu_2014_project_overview1.jpg"
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align="left"></td>
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<td
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style="vertical-align: middle; text-align: justify; width: 50%;">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. <br>
 +
<br>
 +
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>spp.
 +
6803. <br>
 +
<br>
 +
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.</td>
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</tr>
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<tr>
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<td
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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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style="font-style: italic;">E. coli</span> with the <span
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style="font-style: italic;">nif</span> cluster from <span
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style="font-style: italic;">Cyanothece </span>51142, and ran an
 +
Acetylene Reduction Assay in order to test for the strains ability to
 +
fix nitrogen. For more information, please consult our<a
 +
href="https://2014.igem.org/Team:WashU_StLouis/Project/nif">
 +
Nitrogenase </a> page.</p>
 +
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>
 +
Be sure to check out our <a
 +
href="https://2014.igem.org/Team:WashU_StLouis/Project/collaboration">
 +
Collaboration </a> and <a
 +
href="https://2014.igem.org/Team:WashU_StLouis/Parts"> Parts </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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</tr>
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</tbody>
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</table>
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&nbsp;
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<p> </p>
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<table
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<tbody>
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<tr>
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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
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style="font-style: italic;">Cyanothece</span> sp. 51142 nif cluster<br>
 +
•&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>
 +
<table
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border="0" cellpadding="5" cellspacing="5">
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<tr>
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<td
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style="vertical-align: middle; width: 50%; text-align: center;"><img
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style="width: 100%;" alt="Engineered strains"
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src="https://static.igem.org/mediawiki/2014/7/7c/WashU_Engineered_Strains.jpg"
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align="left" hspace="5"><span style="font-weight: bold;"> </span>
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<center>
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<center>
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<div style="text-align: left;">Figure above: Engineered
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strains of <span style="font-style: italic;">E. coli</span> being
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flushed with argon gas to create anaerobic conditions<br>
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</div>
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</center>
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</center>
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</td>
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<td
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style="vertical-align: middle; width: 50%; text-align: justify;">Through
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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.</td>
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</tr>
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</tbody>
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</table>
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<center>
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<center>
<h1>References </h1>
<h1>References </h1>
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<p>
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</center>
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iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you though about your project and what works inspired you. </p>  
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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
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href="http://protist.i.hosei.ac.jp/PDB/images/Prokaryotes/Chroococcaceae/Synechocystis/sp_02.jpg"><i>1.
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<p> You can use these subtopics to further explain your project</p>
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Synechocystis</i> </a></p>
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<div style="text-align: left; margin-left: 40px;"> </div>
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<ol>
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<p style="text-align: left; margin-left: 40px;"> <a
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<li>Overall project summary</li>
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href="http://www.pitch.com/imager/explosive-diarrhea-anyone/b/original/2575052/269f/EcoliEM4.jpg"><i>2.
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<li>Project Details</li>
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E. coli</i> </a> </p>
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<li>Materials and Methods</li>
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<div style="text-align: left; margin-left: 40px;"> </div>
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<li>The Experiments</li>
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<div style="text-align: left; margin-left: 40px;"> <a
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<li>Results</li>
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href="http://www.motherearthnews.com/%7E/media/Images/MEN/Editorial/Articles/Magazine%20Articles/1971/05-01/How%20to%20Dry%20Sweet%20Corn/corn.jpg">3.
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<li>Data analysis</li>
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Corn </a></div>
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<li>Conclusions</li>
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<p> </p>
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</ol>
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</center>
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<p>
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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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<p>
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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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</p>
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<a href=https://2014.igem.org/Team:WashU_StLouis/Project/nif> Nif Cluster </a> |
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<a href=https://2014.igem.org/Team:WashU_StLouis/Project/light> Light Regulation </a> |
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-
<a href=https://2014.igem.org/Team:WashU_StLouis/Project/collaboration> Collaboration </a> |
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<a href=https://2014.igem.org/Team:WashU_StLouis/Project/HumanPractice> Human Practices </a>  
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{{:Team:WashU_StLouis/footer}}

Latest revision as of 22:18, 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 and Parts 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 strains
Figure above: Engineered strains of E. coli being flushed with argon gas to create anaerobic conditions
 Through 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.

References

The pictures used above were taken from the following sources:

1. Synechocystis

2. E. coli

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