http://2010.igem.org/wiki/index.php?title=Special:Contributions/Liszabruder&feed=atom&limit=50&target=Liszabruder&year=&month=2010.igem.org - User contributions [en]2024-03-28T15:10:55ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Lethbridge/CollaborationTeam:Lethbridge/Collaboration2010-10-27T23:28:50Z<p>Liszabruder: </p>
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</a><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Art"><br />
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</a><br />
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<center><br />
Check out these important project links!<br />
</center><br />
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</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Results"><br />
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</a><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><br />
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<BLOCKQUOTE><br />
<br />
=<font color="white">Collaborations=<br />
<br />
We definitely enjoy working with other iGEM teams and learning about their projects, ideas and people. We have done multiple surveys as well as sent or exchanged BioBrick parts with other teams. This includes:<br />
<br />
==<font color="white">Team Northwestern==<br />
<br />
<html><a href="https://2010.igem.org/Team:Northwestern" target="new"><font color="#00DC00">Northwestern</font></a></html> approached us early in the iGEM season inquiring about our <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00"> magnetic nanoparticle project</font></a></html>. We sent them our Mms6 expression construct in hopes that we could work together on the project. The expression of the protein is toxic to the cell and neither team has successfully expressed this protein at a sufficient concentration to date.<br />
<br />
==<font color="white">Team Calgary==<br />
<br />
<html><a href="https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary</font></a></html> is a team we have communicated with extensively throughout the season. In an attempt to pinpoint the expression of the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00"> Mms6 protein</font></a></html> we have sent them the coding sequence for the protein and they have sent us their expression-test construct, which is their project this season. This was an attempt to both determine our Mms6 expression issue as well as test their construct.<br />
<br />
==<font color="white">Team METU Turkey Software==<br />
<br />
We filled out the <html><a href=" https://2010.igem.org./Team:METU_Turkey_Software" target="new"><font color="#00DC00">METU Turkey Software</font></a></html> survey that was based upon creating a standard for parts entry on the <html><a href="http://partsregistry.org/Main_Page" target="new"><font color="#00DC00"> Registry of Standard Parts</font></a></html>. <br />
<br />
==<font color="white">Team Hong Kong CUHK==<br />
<br />
The survey from <html><a href="https://2010.igem.org./Team:Hong_Kong-CUHK" target="new"><font color="#00DC00"> Hong Kong CUHK</font></a></html> was based on information security and inquirers about the public's awareness of information protection and the development of bacteria-based encryption.<br />
<br />
==<font color="white">Team Mexico UNAM CINVESTAV==<br />
<br />
The survey from <html><a href="https://2010.igem.org/Team:Mexico-UNAM-CINVESTAV/Home" target="new"><font color="#00DC00">Mexico UNAM CINVESTAV</font></a></html> asked about the teams as well as questions about our knowledge of patents and our feelings towards them. The survey results can be found <html><a href="https://2010.igem.org/Team:Mexico-UNAM-CINVESTAV/Human_Practices/Survey" target="new"><font color="#00DC00">here</font></a></html>.<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/CollaborationTeam:Lethbridge/Collaboration2010-10-27T23:26:19Z<p>Liszabruder: </p>
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</a><br />
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</a><br />
</th><br />
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<img src="https://static.igem.org/mediawiki/2010/8/84/UofLPartsSubmittedToTheRegistrybutton.jpg" width="80"/><br />
</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Modeling"><br />
<img src="https://static.igem.org/mediawiki/2010/e/e1/UofLModelingbutton.jpg" width="80"/><br />
</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><br />
<img src="https://static.igem.org/mediawiki/2010/2/26/UofLEthicsbutton.jpg" width="80"/><br />
</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Safety"><br />
<img src="https://static.igem.org/mediawiki/2010/0/00/UofLSafetybutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Art"><br />
<img src="https://static.igem.org/mediawiki/2010/0/0a/UofLArt.jpg" width="80"/><br />
</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/News"><br />
<img src="https://static.igem.org/mediawiki/2010/c/c3/UofLNewsButton.jpg" width="80"/><br />
</a><br />
</th><br />
</table><br />
</body><br />
</html><br />
<hr><br />
<center><br />
Check out these important project links!<br />
</center><br />
<html><br />
<body><br />
<center><br />
<table border="0" width="20%" style="background-color:#000000"><br />
<tr><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Judging"><br />
<img src="https://static.igem.org/mediawiki/2010/f/fd/UofLjudgingbutton.jpg" width="60"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Results"><br />
<img src="https://static.igem.org/mediawiki/2010/3/3f/UofLresultsbutton.jpg" width="60"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><br />
<img src="https://static.igem.org/mediawiki/2010/b/b2/UofLcollaborationbutton.png" width="80"/><br />
</a><br />
</th><br />
<br />
<tr><br />
</table><br />
</center><br />
</body><br />
</html><br />
<hr><br />
<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Collaborations=<br />
<br />
We definitely enjoy working with other iGEM teams and learning about their projects, ideas and people. We have done multiple surveys as well as sent or exchanged BioBrick parts with other teams. This includes:<br />
<br />
==<font color="white">Team METU Turkey Software==<br />
<br />
We filled out the <html><a href=" https://2010.igem.org./Team:METU_Turkey_Software" target="new"><font color="#00DC00">METU Turkey Software</font></a></html> survey that was based upon creating a standard for parts entry on the <html><a href="http://partsregistry.org/Main_Page" target="new"><font color="#00DC00"> Registry of Standard Parts</font></a></html>. <br />
<br />
==<font color="white">Team Hong Kong CUHK==<br />
<br />
The survey from <html><a href="https://2010.igem.org./Team:Hong_Kong-CUHK" target="new"><font color="#00DC00"> Hong Kong CUHK</font></a></html> was based on information security and inquirers about the public's awareness of information protection and the development of bacteria-based encryption.<br />
<br />
==<font color="white">Team Mexico UNAM CINVESTAV==<br />
<br />
The survey from <html><a href="https://2010.igem.org/Team:Mexico-UNAM-CINVESTAV/Home" target="new"><font color="#00DC00">Mexico UNAM CINVESTAV</font></a></html> asked about the teams as well as questions about our knowledge of patents and our feelings towards them. The survey results can be found <html><a href="https://2010.igem.org/Team:Mexico-UNAM-CINVESTAV/Human_Practices/Survey" target="new"><font color="#00DC00">here</font></a></html>.<br />
<br />
==<font color="white">Team Northwestern==<br />
<br />
<html><a href="https://2010.igem.org/Team:Northwestern" target="new"><font color="#00DC00">Northwestern</font></a></html> approached us early in the iGEM season inquiring about our <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00"> magnetic nanoparticle project</font></a></html>. We sent them our Mms6 expression construct in hopes that we could work together on the project. The expression of the protein is toxic to the cell and neither team has successfully expressed this protein at a sufficient concentration to date.<br />
<br />
==<font color="white">Team Calgary==<br />
<br />
<html><a href="https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary</font></a></html> is a team we have communicated with extensively throughout the season. In an attempt to pinpoint the expression of the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00"> Mms6 protein</font></a></html> we have sent them the coding sequence for the protein and they have sent us their expression-test construct, which is their project this season. This was an attempt to both determine our Mms6 expression issue as well as test their construct.<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/JudgingTeam:Lethbridge/Judging2010-10-27T23:23:41Z<p>Liszabruder: /* Bronze Criteria */</p>
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</a><br />
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</th><br />
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</a><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Project"><br />
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</a><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work"><br />
<img src="https://static.igem.org/mediawiki/2010/7/73/UofLNotebookbutton.jpg" width="80"/><br />
</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Parts"><br />
<img src="https://static.igem.org/mediawiki/2010/8/84/UofLPartsSubmittedToTheRegistrybutton.jpg" width="80"/><br />
</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Modeling"><br />
<img src="https://static.igem.org/mediawiki/2010/e/e1/UofLModelingbutton.jpg" width="80"/><br />
</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><br />
<img src="https://static.igem.org/mediawiki/2010/2/26/UofLEthicsbutton.jpg" width="80"/><br />
</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Safety"><br />
<img src="https://static.igem.org/mediawiki/2010/0/00/UofLSafetybutton.jpg" width="80"/><br />
</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Art"><br />
<img src="https://static.igem.org/mediawiki/2010/0/0a/UofLArt.jpg" width="80"/><br />
</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/News"><br />
<img src="https://static.igem.org/mediawiki/2010/c/c3/UofLNewsButton.jpg" width="80"/><br />
</a><br />
</th><br />
</table><br />
</body><br />
</html><br />
<hr><br />
<center><br />
Check out these important project links!<br />
</center><br />
<html><br />
<body><br />
<center><br />
<table border="0" width="20%" style="background-color:#000000"><br />
<tr><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Judging"><br />
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</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Results"><br />
<img src="https://static.igem.org/mediawiki/2010/3/3f/UofLresultsbutton.jpg" width="60"/><br />
</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><br />
<img src="https://static.igem.org/mediawiki/2010/b/b2/UofLcollaborationbutton.png" width="60"/><br />
</a><br />
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<hr><br />
<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Judging=<br />
<br />
This page is to help everyone find the information that they need quickly and easily. Happy browsing!<br />
<br />
=<font color="#FFD700">Gold Criteria=<br />
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<th align="left"><font color="#FFD700"><br />
We took a closer look at the previously submitted xylE construct. You can take a look at what we did on our <a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Existing_Parts"><font color="#00DC00"> Characterized Existing Parts</font></a> page, our <a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a> page and on the registry for part <a href="http://partsregistry.org/Part:BBa_K118021" target="new"><font color="#00DC00"> BBa_K118021</font></a>.<br />
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<th align="left"><font color="FFD700"><br />
Take a look at our <a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><font color="#00DC00"> collaborations</font></a>!<br />
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</body><br />
</html><br />
<br />
=<font color="#C0C0C0">Silver Criteria=<br />
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They worked!! Take a look at some of our new BioBrick parts that we made and characterized! You can find them on our <a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Parts"><font color="#00DC00"> Characterized Parts</font></a> page and our <a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a> page. These pages also have the link to the parts on the Registry.<br />
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=<font color="#8C7853">Bronze Criteria=<br />
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We had a SPECTACULAR summer!! Check out our <a href="https://2010.igem.org/Team:Lethbridge/Art"><font color="#00DC00"> Artsmith</font></a> and <a href="https://2010.igem.org/Team:Lethbridge/News"><font color="#00DC00"> Publicity</font></a> pages!<br />
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<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
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We are no doubt having fun at the Jamboree! <br />
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<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
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Come and check out our poster and presentation! We love talking about our project and hearing alternate ways to approach it.<br />
<br />
</th><br />
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</tr><br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
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Definitely registered for the Jamboree.<br />
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</th><br />
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</tr><br />
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<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
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Submitted the iGEM 2010 Judging form.<br />
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<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
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Have a look at our team's <a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00"> project description</font></a>. We have four subsections to our project; <a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00"> catechol degradation</font></a>, <a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> compartmentalization</font></a>, <a href="https://2010.igem.org/Team:Lethbridge/Project/DNA_Degradation"><font color="#00DC00"> DNA degradation</font></a> and <a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00"> magnetic nanoparticles</font></a>.<br />
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<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
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Take a look at the <a href="https://2010.igem.org/Team:Lethbridge/Parts"><font color="#00DC00"> list of parts </font></a>we have designed and the 9 that we submitted.<br />
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<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
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We have submitted 9 new BioBricks to the registry along with entering their information.<br />
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</th><br />
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</tr><br />
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</table><br />
</body><br />
</html><br />
<br />
=<font color="white">Attribution and Contributions=<br />
The <html><a href="https://2010.igem.org/Team:Lethbridge/Team"><font color="#00DC00"> Lethbridge iGEM team</font></a></html> declare there is no scientific overlap between our project and the projects of the <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Graduate_Students"><font color="#00DC00"> advisors</font></a></html> or <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Supervisors"><font color="#00DC00"> instructors</font></a></html>. The <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Undergraduate_Students"><font color="#00DC00"> undergraduates</font></a></html>, in order to apply for the <html><a href="https://2010.igem.org/Oil_Sands" target="new"><font color="#00DC00"> Oil Sands Initiative Grant</font></a></html>, independently determined the benefits of using the catechol-2,3-dioxygenase for bioremediation of the tailings ponds. We were ecstatic to receive the grant in May 2010. The characterization and development of the <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00"> project</font></a></html> in the laboratory was done by the undergraduates with the supervision of the advisors and instructors as necessary. All the information found within our wiki was done by the team's undergraduates with the exception of the <html><a href="https://2010.igem.org/Team:Lethbridge/Modeling"><font color="#00DC00"> Modelling</font></a></html> which was carried out by the advisors and the <html><a href="https://2010.igem.org/Team:Lethbridge/Art"><font color="#00DC00"> Artsmith</font></a></html> which was done primarily by or in collaboration with <html><a href="https://2010.igem.org/Team:Lethbridge/Team#A._William_Smith"><font color="#00DC00"> Anonymous Smith</font></a></html> from our new media department at the University of Lethbridge.<br />
<br />
=<font color="white">Other Awesome Stuff!=<br />
<br />
==<font color="white">Important Team Parts==<br />
<br />
Take a look at part <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00">BBa_K331030</font></a></html>, <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00">BBa_K331031</font></a></html>, and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>. They are a few of the parts that we have characterize that you can find on our <html><a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Parts"><font color="#00DC00"> Characterized Parts</font></a></html> and <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> pages.<br />
<br><br />
<br><br />
<br />
==<font color="white">Human Practice==<br />
===<font color="white">“Study the past, if you would divine the future” – Confucius===<br />
<br />
Scientific study is generally perceived as the development of new ideas and novel data, but underlying this is the fact that scientific advancement is made by building formerly known information on top of new innovation. Indeed, without the discovery of the cell, synthetic biology would never exist.<br />
<br><br><br />
In the same way that scientific advancements can be made by looking at prior invention, the Lethbridge iGEM Team believes that synthetic biology ethical advancements can also be made by looking at ethical concerns of the past. Due to the fact that synthetic biology is such a new science, we are in the position to dictate ethical rules that should be implemented as new discoveries are made. <br />
<br><br><br />
Lethbridge iGEM Team has chosen to look at significant scientific discoveries of the past and analyze them from ethical, environmental, economic, legal and social standpoints. Learning how ethics has been dealt with (or should have been dealt with!) in the past can significantly shape the direction of ethical development in the field of synthetic biology. <br />
<br><br><br />
Through our analysis of cloning, antibiotics, the steam engine, internet and nuclear power, the Lethbridge iGEM Team will “divine the future” of ethics and its relationship with the newly developing field of synthetic biology.<br />
<br><br />
<br><br />
Click <html><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><font color="#00DC00"> here</font></a></html> for more ethics information!<br />
<br><br />
<br><br />
==<font color="white">Safety==<br />
<br />
What has our team looked at to ensure the lab, the public and the environment are safe? Take a look at our <html><a href="https://2010.igem.org/Team:Lethbridge/Safety"><font color="#00DC00"> Safety</font></a></html> page!<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/JudgingTeam:Lethbridge/Judging2010-10-27T23:23:02Z<p>Liszabruder: /* Silver Criteria */</p>
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<a href="https://2010.igem.org/Team:Lethbridge"><br />
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</a><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Team"><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><br />
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<img src="https://static.igem.org/mediawiki/2010/0/00/UofLSafetybutton.jpg" width="80"/><br />
</a><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Art"><br />
<img src="https://static.igem.org/mediawiki/2010/0/0a/UofLArt.jpg" width="80"/><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/News"><br />
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<center><br />
Check out these important project links!<br />
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</a><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Results"><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><br />
<img src="https://static.igem.org/mediawiki/2010/b/b2/UofLcollaborationbutton.png" width="60"/><br />
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<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Judging=<br />
<br />
This page is to help everyone find the information that they need quickly and easily. Happy browsing!<br />
<br />
=<font color="#FFD700">Gold Criteria=<br />
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<table border="0" cellpadding="8" width="90%" style="background-color:#000000"><br />
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We took a closer look at the previously submitted xylE construct. You can take a look at what we did on our <a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Existing_Parts"><font color="#00DC00"> Characterized Existing Parts</font></a> page, our <a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a> page and on the registry for part <a href="http://partsregistry.org/Part:BBa_K118021" target="new"><font color="#00DC00"> BBa_K118021</font></a>.<br />
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<img src="https://static.igem.org/mediawiki/2010/f/f1/UofLgoldcheck.jpg"/><br />
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Take a look at our <a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><font color="#00DC00"> collaborations</font></a>!<br />
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<br />
=<font color="#C0C0C0">Silver Criteria=<br />
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They worked!! Take a look at some of our new BioBrick parts that we made and characterized! You can find them on our <a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Parts"><font color="#00DC00"> Characterized Parts</font></a> page and our <a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a> page. These pages also have the link to the parts on the Registry.<br />
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=<font color="8C7853">Bronze Criteria=<br />
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<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
We had a SPECTACULAR summer!! Check out our <a href="https://2010.igem.org/Team:Lethbridge/Art"><font color="#00DC00"> Artsmith</font></a> and <a href="https://2010.igem.org/Team:Lethbridge/News"><font color="#00DC00"> Publicity</font></a> pages!<br />
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<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
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We are no doubt having fun at the Jamboree! <br />
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<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
Come and check out our poster and presentation! We love talking about our project and hearing alternate ways to approach it.<br />
<br />
</th><br />
<br />
</tr><br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
Definitely registered for the Jamboree.<br />
<br />
</th><br />
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</tr><br />
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<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
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Submitted the iGEM 2010 Judging form.<br />
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<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
Have a look at our team's <a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00"> project description</font></a>. We have four subsections to our project; <a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00"> catechol degradation</font></a>, <a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> compartmentalization</font></a>, <a href="https://2010.igem.org/Team:Lethbridge/Project/DNA_Degradation"><font color="#00DC00"> DNA degradation</font></a> and <a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00"> magnetic nanoparticles</font></a>.<br />
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Take a look at the <a href="https://2010.igem.org/Team:Lethbridge/Parts"><font color="#00DC00"> list of parts </font></a>we have designed and the 9 that we submitted.<br />
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We have submitted 9 new BioBricks to the registry along with entering their information.<br />
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</tr><br />
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</table><br />
</body><br />
</html><br />
<br />
=<font color="white">Attribution and Contributions=<br />
The <html><a href="https://2010.igem.org/Team:Lethbridge/Team"><font color="#00DC00"> Lethbridge iGEM team</font></a></html> declare there is no scientific overlap between our project and the projects of the <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Graduate_Students"><font color="#00DC00"> advisors</font></a></html> or <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Supervisors"><font color="#00DC00"> instructors</font></a></html>. The <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Undergraduate_Students"><font color="#00DC00"> undergraduates</font></a></html>, in order to apply for the <html><a href="https://2010.igem.org/Oil_Sands" target="new"><font color="#00DC00"> Oil Sands Initiative Grant</font></a></html>, independently determined the benefits of using the catechol-2,3-dioxygenase for bioremediation of the tailings ponds. We were ecstatic to receive the grant in May 2010. The characterization and development of the <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00"> project</font></a></html> in the laboratory was done by the undergraduates with the supervision of the advisors and instructors as necessary. All the information found within our wiki was done by the team's undergraduates with the exception of the <html><a href="https://2010.igem.org/Team:Lethbridge/Modeling"><font color="#00DC00"> Modelling</font></a></html> which was carried out by the advisors and the <html><a href="https://2010.igem.org/Team:Lethbridge/Art"><font color="#00DC00"> Artsmith</font></a></html> which was done primarily by or in collaboration with <html><a href="https://2010.igem.org/Team:Lethbridge/Team#A._William_Smith"><font color="#00DC00"> Anonymous Smith</font></a></html> from our new media department at the University of Lethbridge.<br />
<br />
=<font color="white">Other Awesome Stuff!=<br />
<br />
==<font color="white">Important Team Parts==<br />
<br />
Take a look at part <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00">BBa_K331030</font></a></html>, <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00">BBa_K331031</font></a></html>, and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>. They are a few of the parts that we have characterize that you can find on our <html><a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Parts"><font color="#00DC00"> Characterized Parts</font></a></html> and <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> pages.<br />
<br><br />
<br><br />
<br />
==<font color="white">Human Practice==<br />
===<font color="white">“Study the past, if you would divine the future” – Confucius===<br />
<br />
Scientific study is generally perceived as the development of new ideas and novel data, but underlying this is the fact that scientific advancement is made by building formerly known information on top of new innovation. Indeed, without the discovery of the cell, synthetic biology would never exist.<br />
<br><br><br />
In the same way that scientific advancements can be made by looking at prior invention, the Lethbridge iGEM Team believes that synthetic biology ethical advancements can also be made by looking at ethical concerns of the past. Due to the fact that synthetic biology is such a new science, we are in the position to dictate ethical rules that should be implemented as new discoveries are made. <br />
<br><br><br />
Lethbridge iGEM Team has chosen to look at significant scientific discoveries of the past and analyze them from ethical, environmental, economic, legal and social standpoints. Learning how ethics has been dealt with (or should have been dealt with!) in the past can significantly shape the direction of ethical development in the field of synthetic biology. <br />
<br><br><br />
Through our analysis of cloning, antibiotics, the steam engine, internet and nuclear power, the Lethbridge iGEM Team will “divine the future” of ethics and its relationship with the newly developing field of synthetic biology.<br />
<br><br />
<br><br />
Click <html><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><font color="#00DC00"> here</font></a></html> for more ethics information!<br />
<br><br />
<br><br />
==<font color="white">Safety==<br />
<br />
What has our team looked at to ensure the lab, the public and the environment are safe? Take a look at our <html><a href="https://2010.igem.org/Team:Lethbridge/Safety"><font color="#00DC00"> Safety</font></a></html> page!<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/JudgingTeam:Lethbridge/Judging2010-10-27T23:22:15Z<p>Liszabruder: /* Gold Criteria */</p>
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</a><br />
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<hr><br />
<center><br />
Check out these important project links!<br />
</center><br />
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<center><br />
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<hr><br />
<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Judging=<br />
<br />
This page is to help everyone find the information that they need quickly and easily. Happy browsing!<br />
<br />
=<font color="#FFD700">Gold Criteria=<br />
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We took a closer look at the previously submitted xylE construct. You can take a look at what we did on our <a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Existing_Parts"><font color="#00DC00"> Characterized Existing Parts</font></a> page, our <a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a> page and on the registry for part <a href="http://partsregistry.org/Part:BBa_K118021" target="new"><font color="#00DC00"> BBa_K118021</font></a>.<br />
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Take a look at our <a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><font color="#00DC00"> collaborations</font></a>!<br />
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=<font color="C0C0C0">Silver Criteria=<br />
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They worked!! Take a look at some of our new BioBrick parts that we made and characterized! You can find them on our <a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Parts"><font color="#00DC00"> Characterized Parts</font></a> page and our <a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a> page. These pages also have the link to the parts on the Registry.<br />
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=<font color="8C7853">Bronze Criteria=<br />
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We had a SPECTACULAR summer!! Check out our <a href="https://2010.igem.org/Team:Lethbridge/Art"><font color="#00DC00"> Artsmith</font></a> and <a href="https://2010.igem.org/Team:Lethbridge/News"><font color="#00DC00"> Publicity</font></a> pages!<br />
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We are no doubt having fun at the Jamboree! <br />
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Come and check out our poster and presentation! We love talking about our project and hearing alternate ways to approach it.<br />
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Definitely registered for the Jamboree.<br />
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Submitted the iGEM 2010 Judging form.<br />
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Have a look at our team's <a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00"> project description</font></a>. We have four subsections to our project; <a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00"> catechol degradation</font></a>, <a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> compartmentalization</font></a>, <a href="https://2010.igem.org/Team:Lethbridge/Project/DNA_Degradation"><font color="#00DC00"> DNA degradation</font></a> and <a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00"> magnetic nanoparticles</font></a>.<br />
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Take a look at the <a href="https://2010.igem.org/Team:Lethbridge/Parts"><font color="#00DC00"> list of parts </font></a>we have designed and the 9 that we submitted.<br />
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We have submitted 9 new BioBricks to the registry along with entering their information.<br />
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<br />
=<font color="white">Attribution and Contributions=<br />
The <html><a href="https://2010.igem.org/Team:Lethbridge/Team"><font color="#00DC00"> Lethbridge iGEM team</font></a></html> declare there is no scientific overlap between our project and the projects of the <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Graduate_Students"><font color="#00DC00"> advisors</font></a></html> or <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Supervisors"><font color="#00DC00"> instructors</font></a></html>. The <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Undergraduate_Students"><font color="#00DC00"> undergraduates</font></a></html>, in order to apply for the <html><a href="https://2010.igem.org/Oil_Sands" target="new"><font color="#00DC00"> Oil Sands Initiative Grant</font></a></html>, independently determined the benefits of using the catechol-2,3-dioxygenase for bioremediation of the tailings ponds. We were ecstatic to receive the grant in May 2010. The characterization and development of the <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00"> project</font></a></html> in the laboratory was done by the undergraduates with the supervision of the advisors and instructors as necessary. All the information found within our wiki was done by the team's undergraduates with the exception of the <html><a href="https://2010.igem.org/Team:Lethbridge/Modeling"><font color="#00DC00"> Modelling</font></a></html> which was carried out by the advisors and the <html><a href="https://2010.igem.org/Team:Lethbridge/Art"><font color="#00DC00"> Artsmith</font></a></html> which was done primarily by or in collaboration with <html><a href="https://2010.igem.org/Team:Lethbridge/Team#A._William_Smith"><font color="#00DC00"> Anonymous Smith</font></a></html> from our new media department at the University of Lethbridge.<br />
<br />
=<font color="white">Other Awesome Stuff!=<br />
<br />
==<font color="white">Important Team Parts==<br />
<br />
Take a look at part <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00">BBa_K331030</font></a></html>, <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00">BBa_K331031</font></a></html>, and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>. They are a few of the parts that we have characterize that you can find on our <html><a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Parts"><font color="#00DC00"> Characterized Parts</font></a></html> and <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> pages.<br />
<br><br />
<br><br />
<br />
==<font color="white">Human Practice==<br />
===<font color="white">“Study the past, if you would divine the future” – Confucius===<br />
<br />
Scientific study is generally perceived as the development of new ideas and novel data, but underlying this is the fact that scientific advancement is made by building formerly known information on top of new innovation. Indeed, without the discovery of the cell, synthetic biology would never exist.<br />
<br><br><br />
In the same way that scientific advancements can be made by looking at prior invention, the Lethbridge iGEM Team believes that synthetic biology ethical advancements can also be made by looking at ethical concerns of the past. Due to the fact that synthetic biology is such a new science, we are in the position to dictate ethical rules that should be implemented as new discoveries are made. <br />
<br><br><br />
Lethbridge iGEM Team has chosen to look at significant scientific discoveries of the past and analyze them from ethical, environmental, economic, legal and social standpoints. Learning how ethics has been dealt with (or should have been dealt with!) in the past can significantly shape the direction of ethical development in the field of synthetic biology. <br />
<br><br><br />
Through our analysis of cloning, antibiotics, the steam engine, internet and nuclear power, the Lethbridge iGEM Team will “divine the future” of ethics and its relationship with the newly developing field of synthetic biology.<br />
<br><br />
<br><br />
Click <html><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><font color="#00DC00"> here</font></a></html> for more ethics information!<br />
<br><br />
<br><br />
==<font color="white">Safety==<br />
<br />
What has our team looked at to ensure the lab, the public and the environment are safe? Take a look at our <html><a href="https://2010.igem.org/Team:Lethbridge/Safety"><font color="#00DC00"> Safety</font></a></html> page!<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T23:07:56Z<p>Liszabruder: /* Results of the Bradford Assay */</p>
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<a href="https://2010.igem.org/Team:Lethbridge"><br />
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</a><br />
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<img src="https://static.igem.org/mediawiki/2010/0/0d/UofLTeam.jpg" width="80"/><br />
</a><br />
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<img src="https://static.igem.org/mediawiki/2010/8/8d/UofLProjectbutton.jpg" width="80"/><br />
</a><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work"><br />
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<img src="https://static.igem.org/mediawiki/2010/8/84/UofLPartsSubmittedToTheRegistrybutton.jpg" width="80"/><br />
</a><br />
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<img src="https://static.igem.org/mediawiki/2010/e/e1/UofLModelingbutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><br />
<img src="https://static.igem.org/mediawiki/2010/2/26/UofLEthicsbutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Safety"><br />
<img src="https://static.igem.org/mediawiki/2010/0/00/UofLSafetybutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Art"><br />
<img src="https://static.igem.org/mediawiki/2010/0/0a/UofLArt.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/News"><br />
<img src="https://static.igem.org/mediawiki/2010/c/c3/UofLNewsButton.jpg" width="80"/><br />
</a><br />
</th><br />
</table><br />
</body><br />
</html><br />
<hr><br />
<center><br />
Check out these important project links!<br />
</center><br />
<html><br />
<body><br />
<center><br />
<table border="0" width="20%" style="background-color:#000000"><br />
<tr><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Judging"><br />
<img src="https://static.igem.org/mediawiki/2010/f/fd/UofLjudgingbutton.jpg" width="60"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Results"><br />
<img src="https://static.igem.org/mediawiki/2010/3/3f/UofLresultsbutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><br />
<img src="https://static.igem.org/mediawiki/2010/b/b2/UofLcollaborationbutton.png" width="60"/><br />
</a><br />
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<hr><br />
<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
==<font color="white">Characterization of xylE==<br />
<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/Part:BBa_K118021" target="new"><font color="#00DC00" size="+1">BBa_K118021</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The addition of the catechol to a solution of xylE will result in the production of 2-HMS.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K118021" target="new"><font color="#00DC00">BBa_K118021</font></a></html> was transformed into <i>Escherichia coli</i> DH5α (E. coli) cells using our transformation protocol. We confirmed that the cells hosted the plasmid containing the BBa_118021 part and we begun experiments to characterize catechol degredation. <br />
<br><br><br />
In our first experiment we grew a 5 mL culture of our engineered <i>E. coli cells</i> in M9 minimal media overnight. The cells were spun down at 14000 rfc for 2 minutes. We raised the catechol concentration of this solution to 100 mM, the solution immediately turned bright yellow.<br />
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<font color="white">Figure 1. Left: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting the pUC19 plasmid. Right: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting part BBa_118021. The yellow colour suggests the production of 2-HMS.<br />
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In our second experiment we wanted to measure the absorbance of 2-hydroxymuconate semialdehyde over time. We grew a 5 mL culture of our engineered <i>E. coli</i> cells in M9 media overnight. 1 mL of the cell solution was removed for analysis. The cell solution was raised to 100 mM catachol. Immediately the formation of 2-HMS was tracked. The formation of 2-HMS can easily be tracked, as it absorbs light at 375 nm.<br />
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<font color="white">Figure 2. Production of 2-HMS over time.<br />
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xylE contains an iron molecule in its active site. Our hypothesis was that the iron molecule is oxidized after converting a single catechol molecule to 2-HMS; rendering the xylE inactive. We also want to test how xylE would behave in vitro rather than <i>in vivo</i>.<br />
<br><br><br />
To test this hypothesis we grew <i>E. coli</i> containing BBa_K118021 in 500 mL of Lysogeny broth (LB) media with ampicillin. <i>E.coli</i> containing the pUC19 plasmid were also grown in 500 mL of Lysogeny broth (LB) media with ampicillin to act as a negative control through out the course experiment. The cells were grown to a final optical density of 4.55 AU (measured at 600 nm). The cells were first spun down at 3800 rcf for 5 minutes. The supernatant was decanted and the cells were re-suspended in 40 mL M9 minimal media and incubated with 10 mg of lysozyme for 10 minutes. This cell suspension was spun down at 10000 rcf for 30 minutes. The supernatant was taken off and spun at 30000 rcf (S30) for 1 hour. The supernatant of the S30 samples was divided. Half the samples were flash frozen in liquid nitrogen and stored at -80<sup>o</sup>C. The second half was spun at 100000 rcf for 45 minutes. <br />
<br><br><br />
In order to measure the 2-HMS production we needed to determine the concentration of protein per volume of sedimentation material. To determine the concentration of the S30 and S100 xylE sedimentation a Bradford assay was conducted using a standard curve of BSA. Concentrations of the negative controls (pUC 19) for each of the S30 and S100 samples were also determined. Absorbance readings were taken at a 595 nm wavelength and concentration reported in µg/mL.<br />
<br><br><br />
<br />
===<font color="white">Results of the Bradford Assay===<br />
<hr><br />
We determined the concentrations of S30, S30 negative control, S100, and S100 negative control to be 779 µg/mL, 104.5 µg/mL, 519.3 µg/mL, and 100.1 µg/mL respectively.<br />
<br><br><br />
The different volumes of S30 and S100 extracts of xylE cells and pUC19 cells were incubated in a 0.05 mM catechol solution and the production of 2-HMS was observed over time. This observation was measured by recording absorbance readings at 375 nm over a ten minute period immediately after the introduction of catechol. To measure the absorbance of 2-HMS the S30 and S100 samples had to be diluted. The samples were diluted with 20 mM Tris pH 8.0 and also with double distilled water. <br />
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===<font color="white">Results===<br />
<hr><br />
We did not observe a difference in the catechol production in samples containing Tris vs. samples containing d<sub>2</sub>H<sub>2</sub>O. <font color="red" size="+2">Justin and Adam to fill in!</font><br />
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<font color="white">Figure 3. <font color="red" size="+2">Justin and Adam to fill in!</font><br />
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===<font color="white">Conclusion===<br />
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<font color="red" size="+2">Justin and Adam to fill in!</font><br />
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=<font color="white">Compartmentalization Parts=<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathways within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
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==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
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===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
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===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
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===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
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===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
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===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
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==<font color="white">Characterization of C-terminal Oligo Arginine Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
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===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
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===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
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===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
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===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
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===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
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===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
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===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
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<br><br />
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=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
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==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
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===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
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===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
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===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
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===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br />
[[image:UofLmms6growth.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/CollaborationTeam:Lethbridge/Collaboration2010-10-27T23:06:30Z<p>Liszabruder: </p>
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<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Collaborations=<br />
<br />
We definitely enjoy working with other iGEM teams and learning about their projects, ideas and people. We have done multiple surveys as well as sent or exchanged BioBrick parts with other teams. This includes:<br />
<br />
==<font color="white">Team Northwestern==<br />
<br />
<html><a href="https://2010.igem.org/Team:Northwestern" target="new"><font color="#00DC00">Northwestern</font></a></html> approached us early in the iGEM season inquiring about our <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00"> magnetic nanoparticle project</font></a></html>. We sent them our Mms6 expression construct in hopes that we could work together on the project. The expression of the protein is toxic to the cell and neither team has successfully expressed this protein at a sufficient concentration to date.<br />
<br />
==<font color="white">Team Calgary==<br />
<br />
<html><a href="https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary</font></a></html> is a team we have communicated with extensively throughout the season. In an attempt to pinpoint the expression of the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00"> Mms6 protein</font></a></html> we have sent them the coding sequence for the protein and they have sent us their expression-test construct, which is their project this season. This was an attempt to both determine our Mms6 expression issue as well as test their construct.<br />
<br />
==<font color="white">Team METU Turkey Software==<br />
<br />
We filled out the <html><a href=" https://2010.igem.org./Team:METU_Turkey_Software" target="new"><font color="#00DC00">METU Turkey Software</font></a></html> survey that was based upon creating a standard for parts entry on the <html><a href="http://partsregistry.org/Main_Page" target="new"><font color="#00DC00"> Registry of Standard Parts</font></a></html>. <br />
<br />
==<font color="white">Team Hong Kong CUHK==<br />
<br />
The survey from <html><a href="https://2010.igem.org./Team:Hong_Kong-CUHK" target="new"><font color="#00DC00"> Hong Kong CUHK</font></a></html> was based on information security and inquirers about the public's awareness of information protection and the development of bacteria-based encryption.<br />
<br />
==<font color="white">Team Mexico UNAM CINVESTAV==<br />
<br />
The survey from <html><a href="https://2010.igem.org/Team:Mexico-UNAM-CINVESTAV/Home" target="new"><font color="#00DC00">Mexico UNAM CINVESTAV</font></a></html> asked about the teams as well as questions about our knowledge of patents and our feelings towards them. The survey results can be found <html><a href="https://2010.igem.org/Team:Mexico-UNAM-CINVESTAV/Human_Practices/Survey" target="new"><font color="#00DC00">here</font></a></html>.<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T22:14:12Z<p>Liszabruder: /* Characterization of Cyan Fluorescent Protein */</p>
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<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
==<font color="white">Characterization of xylE==<br />
<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/Part:BBa_K118021" target="new"><font color="#00DC00" size="+1">BBa_K118021</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The addition of the catechol to a solution of xylE will result in the production of 2-HMS.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K118021" target="new"><font color="#00DC00">BBa_K118021</font></a></html> was transformed into <i>Escherichia coli</i> DH5α (E. coli) cells using our transformation protocol. We confirmed that the cells hosted the plasmid containing the BBa_118021 part and we begun experiments to characterize catechol degredation. <br />
<br><br><br />
In our first experiment we grew a 5 mL culture of our engineered <i>E. coli cells</i> in M9 minimal media overnight. The cells were spun down at 14000 rfc for 2 minutes. We raised the catechol concentration of this solution to 100 mM, the solution immediately turned bright yellow.<br />
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<p><br />
<font color="white">Figure 1. Left: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting the pUC19 plasmid. Right: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting part BBa_118021. The yellow colour suggests the production of 2-HMS.<br />
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In our second experiment we wanted to measure the absorbance of 2-hydroxymuconate semialdehyde over time. We grew a 5 mL culture of our engineered <i>E. coli</i> cells in M9 media overnight. 1 mL of the cell solution was removed for analysis. The cell solution was raised to 100 mM catachol. Immediately the formation of 2-HMS was tracked. The formation of 2-HMS can easily be tracked, as it absorbs light at 375 nm.<br />
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<font color="white">Figure 2. Production of 2-HMS over time.<br />
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xylE contains an iron molecule in its active site. Our hypothesis was that the iron molecule is oxidized after converting a single catechol molecule to 2-HMS; rendering the xylE inactive. We also want to test how xylE would behave in vitro rather than <i>in vivo</i>.<br />
<br><br><br />
To test this hypothesis we grew <i>E. coli</i> containing BBa_K118021 in 500 mL of Lysogeny broth (LB) media with ampicillin. <i>E.coli</i> containing the pUC19 plasmid were also grown in 500 mL of Lysogeny broth (LB) media with ampicillin to act as a negative control through out the course experiment. The cells were grown to a final optical density of 4.55 AU (measured at 600 nm). The cells were first spun down at 3800 rcf for 5 minutes. The supernatant was decanted and the cells were re-suspended in 40 mL M9 minimal media and incubated with 10 mg of lysozyme for 10 minutes. This cell suspension was spun down at 10000 rcf for 30 minutes. The supernatant was taken off and spun at 30000 rcf (S30) for 1 hour. The supernatant of the S30 samples was divided. Half the samples were flash frozen in liquid nitrogen and stored at -80<sup>o</sup>C. The second half was spun at 100000 rcf for 45 minutes. <br />
<br><br><br />
In order to measure the 2-HMS production we needed to determine the concentration of protein per volume of sedimentation material. To determine the concentration of the S30 and S100 xylE sedimentation a Bradford assay was conducted using a standard curve of BSA. Concentrations of the negative controls (pUC 19) for each of the S30 and S100 samples were also determined. Absorbance readings were taken at a 595 nm wavelength and concentration reported in µg/mL.<br />
<br><br><br />
<br />
===<font color="white">Results of the Bradford Assay===<br />
<hr><br />
We determined the concentrations of S30, S30 negative control, S100, and S100 negative control to be 779 µg/mL, 104.5 µg/mL, 519.3 µg/mL, and 100.1 µg/mL respectively.<br />
<br><br><br />
The different volumes of S30 and S100 extracts of xylE cells and pUC19 cells were incubated in a 0.05 mM catechol solution and the production of 2-HMS was observed over time. This observation was measured by recording absorbance readings at 375 nm over a ten minute period immediately after the introduction of catechol. To measure the absorbance of 2-HMS the S30 and S100 samples had to be diluted. The samples were diluted with 20 mM Tris pH 8.0 and also with d<sub>2</sub>H<sub>2</sub>O. <br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
We did not observe a difference in the catechol production in samples containing Tris vs. samples containing d<sub>2</sub>H<sub>2</sub>O. <font color="red" size="+2">Justin and Adam to fill in!</font><br />
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<font color="white">Figure 3. <font color="red" size="+2">Justin and Adam to fill in!</font><br />
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===<font color="white">Conclusion===<br />
<hr><br />
<font color="red" size="+2">Justin and Adam to fill in!</font><br />
<br><br><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathways within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
<br />
==<font color="white">Characterization of C-terminal Oligo Arginine Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br />
<br><br />
<br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br />
[[image:UofLmms6growth.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/Parts/Characterized_Existing_PartsTeam:Lethbridge/Parts/Characterized Existing Parts2010-10-27T22:11:47Z<p>Liszabruder: /* Characterized Parts */</p>
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<font color="white">Check out the parts we have submitted and the parts we have characterized! You can also access our <a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results page here</font></a>!<br />
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<BLOCKQUOTE><br />
=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
==<font color="white">Characterized Parts</font>==<br />
<br />
You can view <html><a href="http://partsregistry.org/Part:BBa_K118021" target="new"><font color="#00DC00">BBa_K118021</font></a></html> on the registry and find the experimental data on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Placement_of_Oligoarginine_Tail_on_Proteins"><font color="#00DC00">Results</font></a></html> page.<br />
<br><br><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathways within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so the system can be optimized. These parts we have received directly from the Registry and were further characterized for future use in characterization of the microcompartment system.<br />
<br />
==<font color="white">N-terminal Arginine Fusion Vector==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Placement_of_Oligoarginine_Tail_on_Proteins"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
==<font color="white">C-terminal Arginine Fusion Vector==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00">BBa_K249005</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Placement_of_Oligoarginine_Tail_on_Proteins"><font color="#00DC00">Results</font></a></html> page.<br />
<br><br><br />
<br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br />
==<font color="white">Mms6 (Nanoparticles)==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Characterization_of_Mms6"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T21:35:51Z<p>Liszabruder: /* Compartmentalization Parts */</p>
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<BLOCKQUOTE><br />
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=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
==<font color="white">Characterization of xylE==<br />
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===<font color="white">Characterized Parts</font>===<br />
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<html><a href="http://partsregistry.org/Part:BBa_K118021" target="new"><font color="#00DC00" size="+1">BBa_K118021</font></a></html><br />
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===<font color="white">Hypothesis</font>===<br />
<hr><br />
The addition of the catechol to a solution of xylE will result in the production of 2-HMS.<br />
<br><br><br />
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===<font color="white">Method</font>===<br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K118021" target="new"><font color="#00DC00">BBa_K118021</font></a></html> was transformed into <i>Escherichia coli</i> DH5α (E. coli) cells using our transformation protocol. We confirmed that the cells hosted the plasmid containing the BBa_118021 part and we begun experiments to characterize catechol degredation. <br />
<br><br><br />
In our first experiment we grew a 5 mL culture of our engineered <i>E. coli cells</i> in M9 minimal media overnight. The cells were spun down at 14000 rfc for 2 minutes. We raised the catechol concentration of this solution to 100 mM, the solution immediately turned bright yellow.<br />
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<font color="white">Figure 1. Left: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting the pUC19 plasmid. Right: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting part BBa_118021. The yellow colour suggests the production of 2-HMS.<br />
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In our second experiment we wanted to measure the absorbance of 2-hydroxymuconate semialdehyde over time. We grew a 5 mL culture of our engineered <i>E. coli</i> cells in M9 media overnight. 1 mL of the cell solution was removed for analysis. The cell solution was raised to 100 mM catachol. Immediately the formation of 2-HMS was tracked. The formation of 2-HMS can easily be tracked, as it absorbs light at 375 nm.<br />
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<font color="white">Figure 2. Production of 2-HMS over time.<br />
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xylE contains an iron molecule in its active site. Our hypothesis was that the iron molecule is oxidized after converting a single catechol molecule to 2-HMS; rendering the xylE inactive. We also want to test how xylE would behave in vitro rather than <i>in vivo</i>.<br />
<br><br><br />
To test this hypothesis we grew <i>E. coli</i> containing BBa_K118021 in 500 mL of Lysogeny broth (LB) media with ampicillin. <i>E.coli</i> containing the pUC19 plasmid were also grown in 500 mL of Lysogeny broth (LB) media with ampicillin to act as a negative control through out the course experiment. The cells were grown to a final optical density of 4.55 AU (measured at 600 nm). The cells were first spun down at 3800 rcf for 5 minutes. The supernatant was decanted and the cells were re-suspended in 40 mL M9 minimal media and incubated with 10 mg of lysozyme for 10 minutes. This cell suspension was spun down at 10000 rcf for 30 minutes. The supernatant was taken off and spun at 30000 rcf (S30) for 1 hour. The supernatant of the S30 samples was divided. Half the samples were flash frozen in liquid nitrogen and stored at -80<sup>o</sup>C. The second half was spun at 100000 rcf for 45 minutes. <br />
<br><br><br />
In order to measure the 2-HMS production we needed to determine the concentration of protein per volume of sedimentation material. To determine the concentration of the S30 and S100 xylE sedimentation a Bradford assay was conducted using a standard curve of BSA. Concentrations of the negative controls (pUC 19) for each of the S30 and S100 samples were also determined. Absorbance readings were taken at a 595 nm wavelength and concentration reported in µg/mL.<br />
<br><br><br />
<br />
===<font color="white">Results of the Bradford Assay===<br />
<hr><br />
We determined the concentrations of S30, S30 negative control, S100, and S100 negative control to be 779 µg/mL, 104.5 µg/mL, 519.3 µg/mL, and 100.1 µg/mL respectively.<br />
<br><br><br />
The different volumes of S30 and S100 extracts of xylE cells and pUC19 cells were incubated in a 0.05 mM catechol solution and the production of 2-HMS was observed over time. This observation was measured by recording absorbance readings at 375 nm over a ten minute period immediately after the introduction of catechol. To measure the absorbance of 2-HMS the S30 and S100 samples had to be diluted. The samples were diluted with 20 mM Tris pH 8.0 and also with d<sub>2</sub>H<sub>2</sub>O. <br />
<br><br><br />
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===<font color="white">Results===<br />
<hr><br />
We did not observe a difference in the catechol production in samples containing Tris vs. samples containing d<sub>2</sub>H<sub>2</sub>O. <font color="red" size="+2">Justin and Adam to fill in!</font><br />
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<font color="white">Figure 3. <font color="red" size="+2">Justin and Adam to fill in!</font><br />
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===<font color="white">Conclusion===<br />
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<font color="red" size="+2">Justin and Adam to fill in!</font><br />
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=<font color="white">Compartmentalization Parts=<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathways within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
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==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
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===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
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===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
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<br><br />
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==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
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===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
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===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
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===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
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===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
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===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br />
<br><br />
<br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
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===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
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===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br />
[[image:UofLmms6growth.jpg|450px|center]]<br />
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===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T21:35:13Z<p>Liszabruder: /* Method */</p>
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Check out these important project links!<br />
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<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
==<font color="white">Characterization of xylE==<br />
<br />
===<font color="white">Characterized Parts</font>===<br />
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<html><a href="http://partsregistry.org/Part:BBa_K118021" target="new"><font color="#00DC00" size="+1">BBa_K118021</font></a></html><br />
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===<font color="white">Hypothesis</font>===<br />
<hr><br />
The addition of the catechol to a solution of xylE will result in the production of 2-HMS.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K118021" target="new"><font color="#00DC00">BBa_K118021</font></a></html> was transformed into <i>Escherichia coli</i> DH5α (E. coli) cells using our transformation protocol. We confirmed that the cells hosted the plasmid containing the BBa_118021 part and we begun experiments to characterize catechol degredation. <br />
<br><br><br />
In our first experiment we grew a 5 mL culture of our engineered <i>E. coli cells</i> in M9 minimal media overnight. The cells were spun down at 14000 rfc for 2 minutes. We raised the catechol concentration of this solution to 100 mM, the solution immediately turned bright yellow.<br />
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<p><br />
<font color="white">Figure 1. Left: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting the pUC19 plasmid. Right: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting part BBa_118021. The yellow colour suggests the production of 2-HMS.<br />
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In our second experiment we wanted to measure the absorbance of 2-hydroxymuconate semialdehyde over time. We grew a 5 mL culture of our engineered <i>E. coli</i> cells in M9 media overnight. 1 mL of the cell solution was removed for analysis. The cell solution was raised to 100 mM catachol. Immediately the formation of 2-HMS was tracked. The formation of 2-HMS can easily be tracked, as it absorbs light at 375 nm.<br />
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<font color="white">Figure 2. Production of 2-HMS over time.<br />
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xylE contains an iron molecule in its active site. Our hypothesis was that the iron molecule is oxidized after converting a single catechol molecule to 2-HMS; rendering the xylE inactive. We also want to test how xylE would behave in vitro rather than <i>in vivo</i>.<br />
<br><br><br />
To test this hypothesis we grew <i>E. coli</i> containing BBa_K118021 in 500 mL of Lysogeny broth (LB) media with ampicillin. <i>E.coli</i> containing the pUC19 plasmid were also grown in 500 mL of Lysogeny broth (LB) media with ampicillin to act as a negative control through out the course experiment. The cells were grown to a final optical density of 4.55 AU (measured at 600 nm). The cells were first spun down at 3800 rcf for 5 minutes. The supernatant was decanted and the cells were re-suspended in 40 mL M9 minimal media and incubated with 10 mg of lysozyme for 10 minutes. This cell suspension was spun down at 10000 rcf for 30 minutes. The supernatant was taken off and spun at 30000 rcf (S30) for 1 hour. The supernatant of the S30 samples was divided. Half the samples were flash frozen in liquid nitrogen and stored at -80<sup>o</sup>C. The second half was spun at 100000 rcf for 45 minutes. <br />
<br><br><br />
In order to measure the 2-HMS production we needed to determine the concentration of protein per volume of sedimentation material. To determine the concentration of the S30 and S100 xylE sedimentation a Bradford assay was conducted using a standard curve of BSA. Concentrations of the negative controls (pUC 19) for each of the S30 and S100 samples were also determined. Absorbance readings were taken at a 595 nm wavelength and concentration reported in µg/mL.<br />
<br><br><br />
<br />
===<font color="white">Results of the Bradford Assay===<br />
<hr><br />
We determined the concentrations of S30, S30 negative control, S100, and S100 negative control to be 779 µg/mL, 104.5 µg/mL, 519.3 µg/mL, and 100.1 µg/mL respectively.<br />
<br><br><br />
The different volumes of S30 and S100 extracts of xylE cells and pUC19 cells were incubated in a 0.05 mM catechol solution and the production of 2-HMS was observed over time. This observation was measured by recording absorbance readings at 375 nm over a ten minute period immediately after the introduction of catechol. To measure the absorbance of 2-HMS the S30 and S100 samples had to be diluted. The samples were diluted with 20 mM Tris pH 8.0 and also with d<sub>2</sub>H<sub>2</sub>O. <br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
We did not observe a difference in the catechol production in samples containing Tris vs. samples containing d<sub>2</sub>H<sub>2</sub>O. <font color="red" size="+2">Justin and Adam to fill in!</font><br />
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<font color="white">Figure 3. <font color="red" size="+2">Justin and Adam to fill in!</font><br />
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===<font color="white">Conclusion===<br />
<hr><br />
<font color="red" size="+2">Justin and Adam to fill in!</font><br />
<br><br><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
<br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br />
<br><br />
<br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br />
[[image:UofLmms6growth.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T21:34:42Z<p>Liszabruder: /* Characterized Parts */</p>
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Check out these important project links!<br />
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<BLOCKQUOTE><br />
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=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
==<font color="white">Characterization of xylE==<br />
<br />
===<font color="white">Characterized Parts</font>===<br />
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<html><a href="http://partsregistry.org/Part:BBa_K118021" target="new"><font color="#00DC00" size="+1">BBa_K118021</font></a></html><br />
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<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The addition of the catechol to a solution of xylE will result in the production of 2-HMS.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00">BBa_118021</font></a></html> was transformed into <i>Escherichia coli</i> DH5α (E. coli) cells using our transformation protocol. We confirmed that the cells hosted the plasmid containing the BBa_118021 part and we begun experiments to characterize catechol degredation. <br />
<br><br><br />
In our first experiment we grew a 5 mL culture of our engineered <i>E. coli cells</i> in M9 minimal media overnight. The cells were spun down at 14000 rfc for 2 minutes. We raised the catechol concentration of this solution to 100 mM, the solution immediately turned bright yellow.<br />
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<font color="white">Figure 1. Left: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting the pUC19 plasmid. Right: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting part BBa_118021. The yellow colour suggests the production of 2-HMS.<br />
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In our second experiment we wanted to measure the absorbance of 2-hydroxymuconate semialdehyde over time. We grew a 5 mL culture of our engineered <i>E. coli</i> cells in M9 media overnight. 1 mL of the cell solution was removed for analysis. The cell solution was raised to 100 mM catachol. Immediately the formation of 2-HMS was tracked. The formation of 2-HMS can easily be tracked, as it absorbs light at 375 nm.<br />
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<font color="white">Figure 2. Production of 2-HMS over time.<br />
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xylE contains an iron molecule in its active site. Our hypothesis was that the iron molecule is oxidized after converting a single catechol molecule to 2-HMS; rendering the xylE inactive. We also want to test how xylE would behave in vitro rather than <i>in vivo</i>.<br />
<br><br><br />
To test this hypothesis we grew <i>E. coli</i> containing BBa_K118021 in 500 mL of Lysogeny broth (LB) media with ampicillin. <i>E.coli</i> containing the pUC19 plasmid were also grown in 500 mL of Lysogeny broth (LB) media with ampicillin to act as a negative control through out the course experiment. The cells were grown to a final optical density of 4.55 AU (measured at 600 nm). The cells were first spun down at 3800 rcf for 5 minutes. The supernatant was decanted and the cells were re-suspended in 40 mL M9 minimal media and incubated with 10 mg of lysozyme for 10 minutes. This cell suspension was spun down at 10000 rcf for 30 minutes. The supernatant was taken off and spun at 30000 rcf (S30) for 1 hour. The supernatant of the S30 samples was divided. Half the samples were flash frozen in liquid nitrogen and stored at -80<sup>o</sup>C. The second half was spun at 100000 rcf for 45 minutes. <br />
<br><br><br />
In order to measure the 2-HMS production we needed to determine the concentration of protein per volume of sedimentation material. To determine the concentration of the S30 and S100 xylE sedimentation a Bradford assay was conducted using a standard curve of BSA. Concentrations of the negative controls (pUC 19) for each of the S30 and S100 samples were also determined. Absorbance readings were taken at a 595 nm wavelength and concentration reported in µg/mL.<br />
<br><br><br />
<br />
===<font color="white">Results of the Bradford Assay===<br />
<hr><br />
We determined the concentrations of S30, S30 negative control, S100, and S100 negative control to be 779 µg/mL, 104.5 µg/mL, 519.3 µg/mL, and 100.1 µg/mL respectively.<br />
<br><br><br />
The different volumes of S30 and S100 extracts of xylE cells and pUC19 cells were incubated in a 0.05 mM catechol solution and the production of 2-HMS was observed over time. This observation was measured by recording absorbance readings at 375 nm over a ten minute period immediately after the introduction of catechol. To measure the absorbance of 2-HMS the S30 and S100 samples had to be diluted. The samples were diluted with 20 mM Tris pH 8.0 and also with d<sub>2</sub>H<sub>2</sub>O. <br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
We did not observe a difference in the catechol production in samples containing Tris vs. samples containing d<sub>2</sub>H<sub>2</sub>O. <font color="red" size="+2">Justin and Adam to fill in!</font><br />
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<font color="white">Figure 3. <font color="red" size="+2">Justin and Adam to fill in!</font><br />
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===<font color="white">Conclusion===<br />
<hr><br />
<font color="red" size="+2">Justin and Adam to fill in!</font><br />
<br><br><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
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===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
<br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
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===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
<br><br />
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===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br />
<br><br />
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=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
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===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br />
[[image:UofLmms6growth.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T21:32:55Z<p>Liszabruder: /* Characterized Parts */</p>
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Check out these important project links!<br />
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<BLOCKQUOTE><br />
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=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
==<font color="white">Characterization of xylE==<br />
<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/Part:BBa_118021" target="new"><font color="#00DC00" size="+1">BBa_118021</font></a></html><br />
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<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The addition of the catechol to a solution of xylE will result in the production of 2-HMS.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00">BBa_118021</font></a></html> was transformed into <i>Escherichia coli</i> DH5α (E. coli) cells using our transformation protocol. We confirmed that the cells hosted the plasmid containing the BBa_118021 part and we begun experiments to characterize catechol degredation. <br />
<br><br><br />
In our first experiment we grew a 5 mL culture of our engineered <i>E. coli cells</i> in M9 minimal media overnight. The cells were spun down at 14000 rfc for 2 minutes. We raised the catechol concentration of this solution to 100 mM, the solution immediately turned bright yellow.<br />
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<p><br />
<font color="white">Figure 1. Left: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting the pUC19 plasmid. Right: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting part BBa_118021. The yellow colour suggests the production of 2-HMS.<br />
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In our second experiment we wanted to measure the absorbance of 2-hydroxymuconate semialdehyde over time. We grew a 5 mL culture of our engineered <i>E. coli</i> cells in M9 media overnight. 1 mL of the cell solution was removed for analysis. The cell solution was raised to 100 mM catachol. Immediately the formation of 2-HMS was tracked. The formation of 2-HMS can easily be tracked, as it absorbs light at 375 nm.<br />
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<font color="white">Figure 2. Production of 2-HMS over time.<br />
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xylE contains an iron molecule in its active site. Our hypothesis was that the iron molecule is oxidized after converting a single catechol molecule to 2-HMS; rendering the xylE inactive. We also want to test how xylE would behave in vitro rather than <i>in vivo</i>.<br />
<br><br><br />
To test this hypothesis we grew <i>E. coli</i> containing BBa_K118021 in 500 mL of Lysogeny broth (LB) media with ampicillin. <i>E.coli</i> containing the pUC19 plasmid were also grown in 500 mL of Lysogeny broth (LB) media with ampicillin to act as a negative control through out the course experiment. The cells were grown to a final optical density of 4.55 AU (measured at 600 nm). The cells were first spun down at 3800 rcf for 5 minutes. The supernatant was decanted and the cells were re-suspended in 40 mL M9 minimal media and incubated with 10 mg of lysozyme for 10 minutes. This cell suspension was spun down at 10000 rcf for 30 minutes. The supernatant was taken off and spun at 30000 rcf (S30) for 1 hour. The supernatant of the S30 samples was divided. Half the samples were flash frozen in liquid nitrogen and stored at -80<sup>o</sup>C. The second half was spun at 100000 rcf for 45 minutes. <br />
<br><br><br />
In order to measure the 2-HMS production we needed to determine the concentration of protein per volume of sedimentation material. To determine the concentration of the S30 and S100 xylE sedimentation a Bradford assay was conducted using a standard curve of BSA. Concentrations of the negative controls (pUC 19) for each of the S30 and S100 samples were also determined. Absorbance readings were taken at a 595 nm wavelength and concentration reported in µg/mL.<br />
<br><br><br />
<br />
===<font color="white">Results of the Bradford Assay===<br />
<hr><br />
We determined the concentrations of S30, S30 negative control, S100, and S100 negative control to be 779 µg/mL, 104.5 µg/mL, 519.3 µg/mL, and 100.1 µg/mL respectively.<br />
<br><br><br />
The different volumes of S30 and S100 extracts of xylE cells and pUC19 cells were incubated in a 0.05 mM catechol solution and the production of 2-HMS was observed over time. This observation was measured by recording absorbance readings at 375 nm over a ten minute period immediately after the introduction of catechol. To measure the absorbance of 2-HMS the S30 and S100 samples had to be diluted. The samples were diluted with 20 mM Tris pH 8.0 and also with d<sub>2</sub>H<sub>2</sub>O. <br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
We did not observe a difference in the catechol production in samples containing Tris vs. samples containing d<sub>2</sub>H<sub>2</sub>O. <font color="red" size="+2">Justin and Adam to fill in!</font><br />
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<font color="white">Figure 3. <font color="red" size="+2">Justin and Adam to fill in!</font><br />
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===<font color="white">Conclusion===<br />
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<font color="red" size="+2">Justin and Adam to fill in!</font><br />
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<br />
=<font color="white">Compartmentalization Parts=<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
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===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
<br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br />
<br><br />
<br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br />
[[image:UofLmms6growth.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/Parts/Characterized_Existing_PartsTeam:Lethbridge/Parts/Characterized Existing Parts2010-10-27T21:30:45Z<p>Liszabruder: /* Characterized Parts */</p>
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<font color="white">Check out the parts we have submitted and the parts we have characterized! You can also access our <a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results page here</font></a>!<br />
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<BLOCKQUOTE><br />
=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
==<font color="white">Characterized Parts</font>==<br />
<br />
You can view <html><a href="http://partsregistry.org/Part:BBa_118021" target="new"><font color="#00DC00">BBa_118021</font></a></html> on the registry and find the experimental data on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Placement_of_Oligoarginine_Tail_on_Proteins"><font color="#00DC00">Results</font></a></html> page.<br />
<br><br><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathways within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so the system can be optimized. These parts we have received directly from the Registry and were further characterized for future use in characterization of the microcompartment system.<br />
<br />
==<font color="white">N-terminal Arginine Fusion Vector==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Placement_of_Oligoarginine_Tail_on_Proteins"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
==<font color="white">C-terminal Arginine Fusion Vector==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00">BBa_K249005</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Placement_of_Oligoarginine_Tail_on_Proteins"><font color="#00DC00">Results</font></a></html> page.<br />
<br><br><br />
<br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br />
==<font color="white">Mms6 (Nanoparticles)==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Characterization_of_Mms6"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/Parts/Characterized_Existing_PartsTeam:Lethbridge/Parts/Characterized Existing Parts2010-10-27T21:30:14Z<p>Liszabruder: </p>
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<center><br />
<font color="white">Check out the parts we have submitted and the parts we have characterized! You can also access our <a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results page here</font></a>!<br />
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<br />
<BLOCKQUOTE><br />
=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
==<font color="white">Characterized Parts</font>==<br />
<br />
You can view <html><a href="http://partsregistry.org/Part:BBa_118021" target="new"><font color="#00DC00" size="+1">BBa_118021</font></a></html> on the registry and find the experimental data on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Placement_of_Oligoarginine_Tail_on_Proteins"><font color="#00DC00">Results</font></a></html> page.<br />
<br><br><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathways within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so the system can be optimized. These parts we have received directly from the Registry and were further characterized for future use in characterization of the microcompartment system.<br />
<br />
==<font color="white">N-terminal Arginine Fusion Vector==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Placement_of_Oligoarginine_Tail_on_Proteins"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
==<font color="white">C-terminal Arginine Fusion Vector==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00">BBa_K249005</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Placement_of_Oligoarginine_Tail_on_Proteins"><font color="#00DC00">Results</font></a></html> page.<br />
<br><br><br />
<br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br />
==<font color="white">Mms6 (Nanoparticles)==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Characterization_of_Mms6"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/Parts/Characterized_Existing_PartsTeam:Lethbridge/Parts/Characterized Existing Parts2010-10-27T21:29:03Z<p>Liszabruder: </p>
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</a><br />
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</a><br />
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</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Safety"><br />
<img src="https://static.igem.org/mediawiki/2010/0/00/UofLSafetybutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Art"><br />
<img src="https://static.igem.org/mediawiki/2010/0/0a/UofLArt.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/News"><br />
<img src="https://static.igem.org/mediawiki/2010/c/c3/UofLNewsButton.jpg" width="80"/><br />
</a><br />
</th><br />
</table><br />
</body><br />
</html><br />
<hr><br />
<html><br />
<center><br />
<font color="white">Check out the parts we have submitted and the parts we have characterized! You can also access our <a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results page here</font></a>!<br />
</center><br />
</html><br />
<br />
<html><br />
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<hr><br />
<br />
<BLOCKQUOTE><br />
=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
==<font color="white">Characterized Parts</font>==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00" size="+1">BBa_118021</font></a></html> on the registry and find the experimental data on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Placement_of_Oligoarginine_Tail_on_Proteins"><font color="#00DC00">Results</font></a></html> page.<br />
<br><br><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathways within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so the system can be optimized. These parts we have received directly from the Registry and were further characterized for future use in characterization of the microcompartment system.<br />
<br />
==<font color="white">N-terminal Arginine Fusion Vector==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Placement_of_Oligoarginine_Tail_on_Proteins"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
==<font color="white">C-terminal Arginine Fusion Vector==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00">BBa_K249005</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Placement_of_Oligoarginine_Tail_on_Proteins"><font color="#00DC00">Results</font></a></html> page.<br />
<br><br><br />
<br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br />
==<font color="white">Mms6 (Nanoparticles)==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Characterization_of_Mms6"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T21:25:28Z<p>Liszabruder: </p>
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<a href="https://2010.igem.org/Team:Lethbridge"><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Project"><br />
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<center><br />
Check out these important project links!<br />
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<hr><br />
<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
==<font color="white">Characterization of xylE==<br />
<br />
===<font color="white">Characterized Parts</font>===<br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00" size="+1">BBa_118021</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The addition of the catechol to a solution of xylE will result in the production of 2-HMS.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00">BBa_118021</font></a></html> was transformed into <i>Escherichia coli</i> DH5α (E. coli) cells using our transformation protocol. We confirmed that the cells hosted the plasmid containing the BBa_118021 part and we begun experiments to characterize catechol degredation. <br />
<br><br><br />
In our first experiment we grew a 5 mL culture of our engineered <i>E. coli cells</i> in M9 minimal media overnight. The cells were spun down at 14000 rfc for 2 minutes. We raised the catechol concentration of this solution to 100 mM, the solution immediately turned bright yellow.<br />
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<p><br />
<font color="white">Figure 1. Left: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting the pUC19 plasmid. Right: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting part BBa_118021. The yellow colour suggests the production of 2-HMS.<br />
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In our second experiment we wanted to measure the absorbance of 2-hydroxymuconate semialdehyde over time. We grew a 5 mL culture of our engineered <i>E. coli</i> cells in M9 media overnight. 1 mL of the cell solution was removed for analysis. The cell solution was raised to 100 mM catachol. Immediately the formation of 2-HMS was tracked. The formation of 2-HMS can easily be tracked, as it absorbs light at 375 nm.<br />
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<font color="white">Figure 2. Production of 2-HMS over time.<br />
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xylE contains an iron molecule in its active site. Our hypothesis was that the iron molecule is oxidized after converting a single catechol molecule to 2-HMS; rendering the xylE inactive. We also want to test how xylE would behave in vitro rather than <i>in vivo</i>.<br />
<br><br><br />
To test this hypothesis we grew <i>E. coli</i> containing BBa_K118021 in 500 mL of Lysogeny broth (LB) media with ampicillin. <i>E.coli</i> containing the pUC19 plasmid were also grown in 500 mL of Lysogeny broth (LB) media with ampicillin to act as a negative control through out the course experiment. The cells were grown to a final optical density of 4.55 AU (measured at 600 nm). The cells were first spun down at 3800 rcf for 5 minutes. The supernatant was decanted and the cells were re-suspended in 40 mL M9 minimal media and incubated with 10 mg of lysozyme for 10 minutes. This cell suspension was spun down at 10000 rcf for 30 minutes. The supernatant was taken off and spun at 30000 rcf (S30) for 1 hour. The supernatant of the S30 samples was divided. Half the samples were flash frozen in liquid nitrogen and stored at -80<sup>o</sup>C. The second half was spun at 100000 rcf for 45 minutes. <br />
<br><br><br />
In order to measure the 2-HMS production we needed to determine the concentration of protein per volume of sedimentation material. To determine the concentration of the S30 and S100 xylE sedimentation a Bradford assay was conducted using a standard curve of BSA. Concentrations of the negative controls (pUC 19) for each of the S30 and S100 samples were also determined. Absorbance readings were taken at a 595 nm wavelength and concentration reported in µg/mL.<br />
<br><br><br />
<br />
===<font color="white">Results of the Bradford Assay===<br />
<hr><br />
We determined the concentrations of S30, S30 negative control, S100, and S100 negative control to be 779 µg/mL, 104.5 µg/mL, 519.3 µg/mL, and 100.1 µg/mL respectively.<br />
<br><br><br />
The different volumes of S30 and S100 extracts of xylE cells and pUC19 cells were incubated in a 0.05 mM catechol solution and the production of 2-HMS was observed over time. This observation was measured by recording absorbance readings at 375 nm over a ten minute period immediately after the introduction of catechol. To measure the absorbance of 2-HMS the S30 and S100 samples had to be diluted. The samples were diluted with 20 mM Tris pH 8.0 and also with d<sub>2</sub>H<sub>2</sub>O. <br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
We did not observe a difference in the catechol production in samples containing Tris vs. samples containing d<sub>2</sub>H<sub>2</sub>O. <font color="red" size="+2">Justin and Adam to fill in!</font><br />
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<font color="white">Figure 3. <font color="red" size="+2">Justin and Adam to fill in!</font><br />
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<br />
===<font color="white">Conclusion===<br />
<hr><br />
<font color="red" size="+2">Justin and Adam to fill in!</font><br />
<br><br><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
<br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br />
<br><br />
<br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br />
[[image:UofLmms6growth.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/File:UofLcatecholfigure3.jpgFile:UofLcatecholfigure3.jpg2010-10-27T21:17:54Z<p>Liszabruder: </p>
<hr />
<div></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T21:15:12Z<p>Liszabruder: </p>
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<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
==<font color="white">Characterization of xylE==<br />
<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00" size="+1">BBa_118021</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The addition of the catechol to a solution of xylE will result in the production of 2-HMS.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00">BBa_118021</font></a></html> was transformed into <i>Escherichia coli</i> DH5α (E. coli) cells using our transformation protocol. We confirmed that the cells hosted the plasmid containing the BBa_118021 part and we begun experiments to characterize catechol degredation. <br />
<br><br><br />
In our first experiment we grew a 5 mL culture of our engineered <i>E. coli cells</i> in M9 minimal media overnight. The cells were spun down at 14000 rfc for 2 minutes. We raised the catechol concentration of this solution to 100 mM, the solution immediately turned bright yellow.<br />
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<img src="https://static.igem.org/mediawiki/2010/1/1a/UofLcatecholfigure1.jpg"/><br />
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<p><br />
<font color="white">Figure 1. Left: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting the pUC19 plasmid. Right: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting part BBa_118021. The yellow colour suggests the production of 2-HMS.<br />
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In our second experiment we wanted to measure the absorbance of 2-hydroxymuconate semialdehyde over time. We grew a 5 mL culture of our engineered <i>E. coli</i> cells in M9 media overnight. 1 mL of the cell solution was removed for analysis. The cell solution was raised to 100 mM catachol. Immediately the formation of 2-HMS was tracked. The formation of 2-HMS can easily be tracked, as it absorbs light at 375 nm.<br />
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<p><br />
<font color="white">Figure 2. Production of 2-HMS over time.<br />
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<br />
xylE contains an iron molecule in its active site. Our hypothesis was that the iron molecule is oxidized after converting a single catechol molecule to 2-HMS; rendering the xylE inactive. We also want to test how xylE would behave in vitro rather than <i>in vivo</i>.<br />
<br><br><br />
To test this hypothesis we grew <i>E. coli</i> containing BBa_K118021 in 500 mL of Lysogeny broth (LB) media with ampicillin. <i>E.coli</i> containing the pUC19 plasmid were also grown in 500 mL of Lysogeny broth (LB) media with ampicillin to act as a negative control through out the course experiment. The cells were grown to a final optical density of 4.55 AU (measured at 600 nm). The cells were first spun down at 3800 rcf for 5 minutes. The supernatant was decanted and the cells were re-suspended in 40 mL M9 minimal media and incubated with 10 mg of lysozyme for 10 minutes. This cell suspension was spun down at 10000 rcf for 30 minutes. The supernatant was taken off and spun at 30000 rcf (S30) for 1 hour. The supernatant of the S30 samples was divided. Half the samples were flash frozen in liquid nitrogen and stored at -80<sup>o</sup>C. The second half was spun at 100000 rcf for 45 minutes. <br />
<br><br><br />
In order to measure the 2-HMS production we needed to determine the concentration of protein per volume of sedimentation material. To determine the concentration of the S30 and S100 xylE sedimentation a Bradford assay was conducted using a standard curve of BSA. Concentrations of the negative controls (pUC 19) for each of the S30 and S100 samples were also determined. Absorbance readings were taken at a 595 nm wavelength and concentration reported in µg/mL.<br />
<br><br><br />
<br />
===<font color="white">Results of the Bradford Assay===<br />
[[image:UofLcatecholfigure3.jpg]]<br />
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<p><br />
<font color="white">Figure 3. Standard curve of BSA.<br />
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<br />
We determined the concentrations of S30, S30 negative control, S100, and S100 negative control to be 779 µg/mL, 104.5 µg/mL, 519.3 µg/mL, and 100.1 µg/mL respectively.<br />
<br><br><br />
The different volumes of S30 and S100 extracts of xylE cells and pUC19 cells were incubated in a 0.05 mM catechol solution and the production of 2-HMS was observed over time. This observation was measured by recording absorbance readings at 375 nm over a ten minute period immediately after the introduction of catechol. To measure the absorbance of 2-HMS the S30 and S100 samples had to be diluted. The samples were diluted with 20 mM Tris pH 8.0 and also with d<sub>2</sub>H<sub>2</sub>O. <br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<br />
We did not observe a difference in the catechol production in samples containing Tris vs. samples containing d<sub>2</sub>H<sub>2</sub>O.<br />
<br><br><br />
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<p><br />
<font color="white">Figure 4. <br />
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<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
<br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br />
<br><br />
<br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br />
[[image:UofLmms6growth.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T21:14:00Z<p>Liszabruder: </p>
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<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
==<font color="white">Characterization of xylE==<br />
<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00" size="+1">BBa_118021</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The addition of the catechol to a solution of xylE will result in the production of 2-HMS.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00">BBa_118021</font></a></html> was transformed into <i>Escherichia coli</i> DH5α (E. coli) cells using our transformation protocol. We confirmed that the cells hosted the plasmid containing the BBa_118021 part and we begun experiments to characterize catechol degredation. <br />
<br><br><br />
In our first experiment we grew a 5 mL culture of our engineered <i>E. coli cells</i> in M9 minimal media overnight. The cells were spun down at 14000 rfc for 2 minutes. We raised the catechol concentration of this solution to 100 mM, the solution immediately turned bright yellow.<br />
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<img src="https://static.igem.org/mediawiki/2010/1/1a/UofLcatecholfigure1.jpg"/><br />
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<br />
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<p><br />
<font color="white">Figure 1. Left: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting the pUC19 plasmid. Right: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting part BBa_118021. The yellow colour suggests the production of 2-HMS.<br />
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<br />
In our second experiment we wanted to measure the absorbance of 2-hydroxymuconate semialdehyde over time. We grew a 5 mL culture of our engineered <i>E. coli</i> cells in M9 media overnight. 1 mL of the cell solution was removed for analysis. The cell solution was raised to 100 mM catachol. Immediately the formation of 2-HMS was tracked. The formation of 2-HMS can easily be tracked, as it absorbs light at 375 nm.<br />
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<p><br />
<font color="white">Figure 2. Production of 2-HMS over time.<br />
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<br />
xylE contains an iron molecule in its active site. Our hypothesis was that the iron molecule is oxidized after converting a single catechol molecule to 2-HMS; rendering the xylE inactive. We also want to test how xylE would behave in vitro rather than <i>in vivo</i>.<br />
<br><br><br />
To test this hypothesis we grew <i>E. coli</i> containing BBa_K118021 in 500 mL of Lysogeny broth (LB) media with ampicillin. <i>E.coli</i> containing the pUC19 plasmid were also grown in 500 mL of Lysogeny broth (LB) media with ampicillin to act as a negative control through out the course experiment. The cells were grown to a final optical density of 4.55 AU (measured at 600 nm). The cells were first spun down at 3800 rcf for 5 minutes. The supernatant was decanted and the cells were re-suspended in 40 mL M9 minimal media and incubated with 10 mg of lysozyme for 10 minutes. This cell suspension was spun down at 10000 rcf for 30 minutes. The supernatant was taken off and spun at 30000 rcf (S30) for 1 hour. The supernatant of the S30 samples was divided. Half the samples were flash frozen in liquid nitrogen and stored at -80<sup>o</sup>C. The second half was spun at 100000 rcf for 45 minutes. <br />
<br><br><br />
In order to measure the 2-HMS production we needed to determine the concentration of protein per volume of sedimentation material. To determine the concentration of the S30 and S100 xylE sedimentation a Bradford assay was conducted using a standard curve of BSA. Concentrations of the negative controls (pUC 19) for each of the S30 and S100 samples were also determined. Absorbance readings were taken at a 595 nm wavelength and concentration reported in µg/mL.<br />
<br><br><br />
<br />
===<font color="white">Results of the Bradford Assay===<br />
[[image:UofLcatecholfigure3.jpg]]<br />
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<p><br />
<font color="white">Figure 3. Standard curve of BSA.<br />
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We determined the concentrations of S30, S30 negative control, S100, and S100 negative control to be 779 µg/mL, 104.5 µg/mL, 519.3 µg/mL, and 100.1 µg/mL respectively.<br />
<br><br><br />
The different volumes of S30 and S100 extracts of xylE cells and pUC19 cells were incubated in a 0.05 mM catechol solution and the production of 2-HMS was observed over time. This observation was measured by recording absorbance readings at 375 nm over a ten minute period immediately after the introduction of catechol. To measure the absorbance of 2-HMS the S30 and S100 samples had to be diluted. The samples were diluted with 20 mM Tris pH 8.0 and also with d<sub>2</sub>H<sub>2</sub>O. <br />
<br><br><br />
[[image:UofLcatecholfigure3.jpg]]<br />
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<font color="white">Figure 3. Standard curve of BSA.<br />
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===<font color="white">Results===<br />
<br />
We did not observe a difference in the catechol production in samples containing Tris vs. samples containing d<sub>2</sub>H<sub>2</sub>O.<br />
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<font color="white">Figure 4. <br />
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<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
<br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br />
<br><br />
<br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br />
[[image:UofLmms6growth.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/File:UofLcatecholfigure4.jpgFile:UofLcatecholfigure4.jpg2010-10-27T21:12:29Z<p>Liszabruder: </p>
<hr />
<div></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T21:11:37Z<p>Liszabruder: </p>
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<BLOCKQUOTE><br />
<br />
=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
==<font color="white">Characterization of xylE==<br />
<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00" size="+1">BBa_118021</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The addition of the catechol to a solution of xylE will result in the production of 2-HMS.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00">BBa_118021</font></a></html> was transformed into <i>Escherichia coli</i> DH5α (E. coli) cells using our transformation protocol. We confirmed that the cells hosted the plasmid containing the BBa_118021 part and we begun experiments to characterize catechol degredation. <br />
<br><br><br />
In our first experiment we grew a 5 mL culture of our engineered <i>E. coli cells</i> in M9 minimal media overnight. The cells were spun down at 14000 rfc for 2 minutes. We raised the catechol concentration of this solution to 100 mM, the solution immediately turned bright yellow.<br />
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<p><br />
<font color="white">Figure 1. Left: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting the pUC19 plasmid. Right: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting part BBa_118021. The yellow colour suggests the production of 2-HMS.<br />
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In our second experiment we wanted to measure the absorbance of 2-hydroxymuconate semialdehyde over time. We grew a 5 mL culture of our engineered <i>E. coli</i> cells in M9 media overnight. 1 mL of the cell solution was removed for analysis. The cell solution was raised to 100 mM catachol. Immediately the formation of 2-HMS was tracked. The formation of 2-HMS can easily be tracked, as it absorbs light at 375 nm.<br />
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<p><br />
<font color="white">Figure 2. Production of 2-HMS over time.<br />
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<br />
xylE contains an iron molecule in its active site. Our hypothesis was that the iron molecule is oxidized after converting a single catechol molecule to 2-HMS; rendering the xylE inactive. We also want to test how xylE would behave in vitro rather than <i>in vivo</i>.<br />
<br><br><br />
To test this hypothesis we grew <i>E. coli</i> containing BBa_K118021 in 500 mL of Lysogeny broth (LB) media with ampicillin. <i>E.coli<i> containing the pUC19 plasmid were also grown in 500 mL of Lysogeny broth (LB) media with ampicillin to act as a negative control through out the course experiment. The cells were grown to a final optical density of 4.55 AU (measured at 600 nm). The cells were first spun down at 3800 rcf for 5 minutes. The supernatant was decanted and the cells were re-suspended in 40 mL M9 minimal media and incubated with 10 mg of lysozyme for 10 minutes. This cell suspension was spun down at 10000 rcf for 30 minutes. The supernatant was taken off and spun at 30000 rcf (S30) for 1 hour. The supernatant of the S30 samples was divided. Half the samples were flash frozen in liquid nitrogen and stored at -80<sup>o</sup>C. The second half was spun at 100000 rcf for 45 minutes. <br />
<br><br><br />
In order to measure the 2-HMS production we needed to determine the concentration of protein per volume of sedimentation material. To determine the concentration of the S30 and S100 xylE sedimentation a Bradford assay was conducted using a standard curve of BSA. Concentrations of the negative controls (pUC 19) for each of the S30 and S100 samples were also determined. Absorbance readings were taken at a 595 nm wavelength and concentration reported in µg/mL.<br />
<br><br><br />
<br />
===<font color="white">Results of the Bradford Assay===<br />
[[image:UofLcatecholfigure3.jpg]]<br />
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<font color="white">Figure 3. Standard curve of BSA.<br />
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<br />
We determined the concentrations of S30, S30 negative control, S100, and S100 negative control to be 779 µg/mL, 104.5 µg/mL, 519.3 µg/mL, and 100.1 µg/mL respectively.<br />
<br><br><br />
The different volumes of S30 and S100 extracts of xylE cells and pUC19 cells were incubated in a 0.05 mM catechol solution and the production of 2-HMS was observed over time. This observation was measured by recording absorbance readings at 375 nm over a ten minute period immediately after the introduction of catechol. To measure the absorbance of 2-HMS the S30 and S100 samples had to be diluted. The samples were diluted with 20 mM Tris pH 8.0 and also with d<sub>2</sub>H<sub>2</sub>O. <br />
<br><br><br />
[[image:UofLcatecholfigure3.jpg]]<br />
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<font color="white">Figure 3. Standard curve of BSA.<br />
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===<font color="white">Results===<br />
<br />
We did not observe a difference in the catechol production in samples containing Tris vs. samples containing d<sub>2</sub>H<sub>2</sub>O.<br />
<br />
[[image:UofLcatecholfigure4.jpg]]<br />
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<font color="white">Figure 4. <br />
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=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
<br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br />
<br><br />
<br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br />
[[image:UofLmms6growth.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/File:UofLcatecholfigure2.jpgFile:UofLcatecholfigure2.jpg2010-10-27T21:10:13Z<p>Liszabruder: </p>
<hr />
<div></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T21:09:44Z<p>Liszabruder: </p>
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=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
==<font color="white">Characterization of xylE==<br />
<br />
===<font color="white">Characterized Parts</font>===<br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00" size="+1">BBa_118021</font></a></html><br />
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===<font color="white">Hypothesis</font>===<br />
<hr><br />
The addition of the catechol to a solution of xylE will result in the production of 2-HMS.<br />
<br><br><br />
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===<font color="white">Method</font>===<br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00">BBa_118021</font></a></html> was transformed into <i>Escherichia coli</i> DH5α (E. coli) cells using our transformation protocol. We confirmed that the cells hosted the plasmid containing the BBa_118021 part and we begun experiments to characterize catechol degredation. <br />
<br><br><br />
In our first experiment we grew a 5 mL culture of our engineered <i>E. coli cells</i> in M9 minimal media overnight. The cells were spun down at 14000 rfc for 2 minutes. We raised the catechol concentration of this solution to 100 mM, the solution immediately turned bright yellow.<br />
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<font color="white">Figure 1. Left: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting the pUC19 plasmid. Right: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting part BBa_118021. The yellow colour suggests the production of 2-HMS.<br />
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In our second experiment we wanted to measure the absorbance of 2-hydroxymuconate semialdehyde over time. We grew a 5 mL culture of our engineered <i>E. coli</i> cells in M9 media overnight. 1 mL of the cell solution was removed for analysis. The cell solution was raised to 100 mM catachol. Immediately the formation of 2-HMS was tracked. The formation of 2-HMS can easily be tracked, as it absorbs light at 375 nm.<br />
<br><br><br />
[[image:UofLcatecholfigure2.jpg]]<br />
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<font color="white">Figure 2. Production of 2-HMS over time.<br />
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xylE contains an iron molecule in its active site. Our hypothesis was that the iron molecule is oxidized after converting a single catechol molecule to 2-HMS; rendering the xylE inactive. We also want to test how xylE would behave in vitro rather than <i>in vivo</i>.<br />
<br><br><br />
To test this hypothesis we grew <i>E. coli</i> containing BBa_K118021 in 500 mL of Lysogeny broth (LB) media with ampicillin. <i>E.coli<i> containing the pUC19 plasmid were also grown in 500 mL of Lysogeny broth (LB) media with ampicillin to act as a negative control through out the course experiment. The cells were grown to a final optical density of 4.55 AU (measured at 600 nm). The cells were first spun down at 3800 rcf for 5 minutes. The supernatant was decanted and the cells were re-suspended in 40 mL M9 minimal media and incubated with 10 mg of lysozyme for 10 minutes. This cell suspension was spun down at 10000 rcf for 30 minutes. The supernatant was taken off and spun at 30000 rcf (S30) for 1 hour. The supernatant of the S30 samples was divided. Half the samples were flash frozen in liquid nitrogen and stored at -80<sup>o</sup>C. The second half was spun at 100000 rcf for 45 minutes. <br />
<br><br><br />
In order to measure the 2-HMS production we needed to determine the concentration of protein per volume of sedimentation material. To determine the concentration of the S30 and S100 xylE sedimentation a Bradford assay was conducted using a standard curve of BSA. Concentrations of the negative controls (pUC 19) for each of the S30 and S100 samples were also determined. Absorbance readings were taken at a 595 nm wavelength and concentration reported in µg/mL.<br />
<br><br><br />
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===<font color="white">Results of the Bradford Assay===<br />
[[image:UofLcatecholfigure3.jpg]]<br />
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<font color="white">Figure 3. Standard curve of BSA.<br />
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We determined the concentrations of S30, S30 negative control, S100, and S100 negative control to be 779 µg/mL, 104.5 µg/mL, 519.3 µg/mL, and 100.1 µg/mL respectively.<br />
<br><br><br />
The different volumes of S30 and S100 extracts of xylE cells and pUC19 cells were incubated in a 0.05 mM catechol solution and the production of 2-HMS was observed over time. This observation was measured by recording absorbance readings at 375 nm over a ten minute period immediately after the introduction of catechol. To measure the absorbance of 2-HMS the S30 and S100 samples had to be diluted. The samples were diluted with 20 mM Tris pH 8.0 and also with d<sub>2</sub>H<sub>2</sub>O. <br />
<br><br><br />
[[image:UofLcatecholfigure3.jpg]]<br />
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<font color="white">Figure 3. Standard curve of BSA.<br />
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===<font color="white">Results===<br />
<br />
We did not observe a difference in the catechol production in samples containing Tris vs. samples containing d<sub>2</sub>H<sub>2</sub>O.<br />
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[[image:UofLcatecholfigure4.jpg]]<br />
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<font color="white">Figure 4. <br />
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=<font color="white">Compartmentalization Parts=<br />
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One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
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===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
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===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
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===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
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===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
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<br><br />
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==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
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===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
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===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
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===<font color="white">Method</font>===<br />
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In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
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===<font color="white">Conclusion</font>===<br />
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The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
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===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
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<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br />
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=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
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===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
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===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
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===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
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[[image:UofLmms6growth.jpg|450px|center]]<br />
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===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/File:UofLcatecholfigure1.jpgFile:UofLcatecholfigure1.jpg2010-10-27T21:08:48Z<p>Liszabruder: </p>
<hr />
<div></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T21:05:53Z<p>Liszabruder: /* Catechol Degradation */</p>
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<BLOCKQUOTE><br />
<br />
=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
==<font color="white">Characterization of xylE==<br />
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===<font color="white">Characterized Parts</font>===<br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00" size="+1">BBa_118021</font></a></html><br />
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===<font color="white">Hypothesis</font>===<br />
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The addition of the catechol to a solution of xylE will result in the production of 2-HMS.<br />
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===<font color="white">Method</font>===<br />
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<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00">BBa_118021</font></a></html> was transformed into <i>Escherichia coli</i> DH5α (E. coli) cells using our transformation protocol. We confirmed that the cells hosted the plasmid containing the BBa_118021 part and we begun experiments to characterize catechol degredation. <br />
<br><br><br />
In our first experiment we grew a 5 mL culture of our engineered <i>E. coli cells</i> in M9 minimal media overnight. The cells were spun down at 14000 rfc for 2 minutes. We raised the catechol concentration of this solution to 100 mM, the solution immediately turned bright yellow.<br />
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[[image:UofLcatecholfigure1.jpg]]<br />
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<font color="white">Figure 1. Left: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting the pUC19 plasmid. Right: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting part BBa_118021. The yellow colour suggests the production of 2-HMS.<br />
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In our second experiment we wanted to measure the absorbance of 2-hydroxymuconate semialdehyde over time. We grew a 5 mL culture of our engineered <i>E. coli</i> cells in M9 media overnight. 1 mL of the cell solution was removed for analysis. The cell solution was raised to 100 mM catachol. Immediately the formation of 2-HMS was tracked. The formation of 2-HMS can easily be tracked, as it absorbs light at 375 nm.<br />
<br><br><br />
[[image:UofLcatecholfigure2.jpg]]<br />
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<font color="white">Figure 2. Production of 2-HMS over time.<br />
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xylE contains an iron molecule in its active site. Our hypothesis was that the iron molecule is oxidized after converting a single catechol molecule to 2-HMS; rendering the xylE inactive. We also want to test how xylE would behave in vitro rather than <i>in vivo</i>.<br />
<br><br><br />
To test this hypothesis we grew <i>E. coli</i> containing BBa_K118021 in 500 mL of Lysogeny broth (LB) media with ampicillin. <i>E.coli<i> containing the pUC19 plasmid were also grown in 500 mL of Lysogeny broth (LB) media with ampicillin to act as a negative control through out the course experiment. The cells were grown to a final optical density of 4.55 AU (measured at 600 nm). The cells were first spun down at 3800 rcf for 5 minutes. The supernatant was decanted and the cells were re-suspended in 40 mL M9 minimal media and incubated with 10 mg of lysozyme for 10 minutes. This cell suspension was spun down at 10000 rcf for 30 minutes. The supernatant was taken off and spun at 30000 rcf (S30) for 1 hour. The supernatant of the S30 samples was divided. Half the samples were flash frozen in liquid nitrogen and stored at -80<sup>o</sup>C. The second half was spun at 100000 rcf for 45 minutes. <br />
<br><br><br />
In order to measure the 2-HMS production we needed to determine the concentration of protein per volume of sedimentation material. To determine the concentration of the S30 and S100 xylE sedimentation a Bradford assay was conducted using a standard curve of BSA. Concentrations of the negative controls (pUC 19) for each of the S30 and S100 samples were also determined. Absorbance readings were taken at a 595 nm wavelength and concentration reported in µg/mL.<br />
<br><br><br />
<br />
===<font color="white">Results of the Bradford Assay===<br />
[[image:UofLcatecholfigure3.jpg]]<br />
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<font color="white">Figure 3. Standard curve of BSA.<br />
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We determined the concentrations of S30, S30 negative control, S100, and S100 negative control to be 779 µg/mL, 104.5 µg/mL, 519.3 µg/mL, and 100.1 µg/mL respectively.<br />
<br><br><br />
The different volumes of S30 and S100 extracts of xylE cells and pUC19 cells were incubated in a 0.05 mM catechol solution and the production of 2-HMS was observed over time. This observation was measured by recording absorbance readings at 375 nm over a ten minute period immediately after the introduction of catechol. To measure the absorbance of 2-HMS the S30 and S100 samples had to be diluted. The samples were diluted with 20 mM Tris pH 8.0 and also with d<sub>2</sub>H<sub>2</sub>O. <br />
<br><br><br />
[[image:UofLcatecholfigure3.jpg]]<br />
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<font color="white">Figure 3. Standard curve of BSA.<br />
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===<font color="white">Results===<br />
<br />
We did not observe a difference in the catechol production in samples containing Tris vs. samples containing d<sub>2</sub>H<sub>2</sub>O.<br />
<br />
[[image:UofLcatecholfigure4.jpg]]<br />
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<font color="white">Figure 4. <br />
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=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
<br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br />
<br><br />
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=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br />
[[image:UofLmms6growth.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T21:04:53Z<p>Liszabruder: </p>
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Check out these important project links!<br />
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<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Catechol Degradation=<br />
<br />
The focus of our <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">project</font></a></html> is to decrease the toxicity of tailing pond water through bioremediation. We are specifically interested in the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">degradation of the toxic molecule catechol</font></a></html> into 2-hydroxymuconate semialdyhyde (2-HMS); a bright yellow substrate that can be metabolized by the cell. This conversion is accomplished by catechol 2,3-dioxygenase (xylE).<br />
<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00" size="+1">BBa_118021</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The addition of the catechol to a solution of xylE will result in the production of 2-HMS.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_118021" target="new"><font color="#00DC00">BBa_118021</font></a></html> was transformed into <i>Escherichia coli</i> DH5α (E. coli) cells using our transformation protocol. We confirmed that the cells hosted the plasmid containing the BBa_118021 part and we begun experiments to characterize catechol degredation. <br />
<br><br><br />
In our first experiment we grew a 5 mL culture of our engineered <i>E. coli cells</i> in M9 minimal media overnight. The cells were spun down at 14000 rfc for 2 minutes. We raised the catechol concentration of this solution to 100 mM, the solution immediately turned bright yellow.<br />
<br><br><br />
[[image:UofLcatecholfigure1.jpg]]<br />
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<font color="white">Figure 1. Left: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting the pUC19 plasmid. Right: M9 media spiked with 100 mM catechol. The solution contains <i>E. coli</i> cells hosting part BBa_118021. The yellow colour suggests the production of 2-HMS.<br />
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<br />
In our second experiment we wanted to measure the absorbance of 2-hydroxymuconate semialdehyde over time. We grew a 5 mL culture of our engineered <i>E. coli</i> cells in M9 media overnight. 1 mL of the cell solution was removed for analysis. The cell solution was raised to 100 mM catachol. Immediately the formation of 2-HMS was tracked. The formation of 2-HMS can easily be tracked, as it absorbs light at 375 nm.<br />
<br><br><br />
[[image:UofLcatecholfigure2.jpg]]<br />
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<font color="white">Figure 2. Production of 2-HMS over time.<br />
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<br />
xylE contains an iron molecule in its active site. Our hypothesis was that the iron molecule is oxidized after converting a single catechol molecule to 2-HMS; rendering the xylE inactive. We also want to test how xylE would behave in vitro rather than <i>in vivo</i>.<br />
<br><br><br />
To test this hypothesis we grew <i>E. coli</i> containing BBa_K118021 in 500 mL of Lysogeny broth (LB) media with ampicillin. <i>E.coli<i> containing the pUC19 plasmid were also grown in 500 mL of Lysogeny broth (LB) media with ampicillin to act as a negative control through out the course experiment. The cells were grown to a final optical density of 4.55 AU (measured at 600 nm). The cells were first spun down at 3800 rcf for 5 minutes. The supernatant was decanted and the cells were re-suspended in 40 mL M9 minimal media and incubated with 10 mg of lysozyme for 10 minutes. This cell suspension was spun down at 10000 rcf for 30 minutes. The supernatant was taken off and spun at 30000 rcf (S30) for 1 hour. The supernatant of the S30 samples was divided. Half the samples were flash frozen in liquid nitrogen and stored at -80<sup>o</sup>C. The second half was spun at 100000 rcf for 45 minutes. <br />
<br><br><br />
In order to measure the 2-HMS production we needed to determine the concentration of protein per volume of sedimentation material. To determine the concentration of the S30 and S100 xylE sedimentation a Bradford assay was conducted using a standard curve of BSA. Concentrations of the negative controls (pUC 19) for each of the S30 and S100 samples were also determined. Absorbance readings were taken at a 595 nm wavelength and concentration reported in µg/mL.<br />
<br><br><br />
<br />
===<font color="white">Results of the Bradford Assay===<br />
[[image:UofLcatecholfigure3.jpg]]<br />
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<font color="white">Figure 3. Standard curve of BSA.<br />
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We determined the concentrations of S30, S30 negative control, S100, and S100 negative control to be 779 µg/mL, 104.5 µg/mL, 519.3 µg/mL, and 100.1 µg/mL respectively.<br />
<br><br><br />
The different volumes of S30 and S100 extracts of xylE cells and pUC19 cells were incubated in a 0.05 mM catechol solution and the production of 2-HMS was observed over time. This observation was measured by recording absorbance readings at 375 nm over a ten minute period immediately after the introduction of catechol. To measure the absorbance of 2-HMS the S30 and S100 samples had to be diluted. The samples were diluted with 20 mM Tris pH 8.0 and also with d<sub>2</sub>H<sub>2</sub>O. <br />
<br><br><br />
[[image:UofLcatecholfigure3.jpg]]<br />
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<font color="white">Figure 3. Standard curve of BSA.<br />
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===<font color="white">Results===<br />
<br />
We did not observe a difference in the catechol production in samples containing Tris vs. samples containing d<sub>2</sub>H<sub>2</sub>O.<br />
<br />
[[image:UofLcatecholfigure4.jpg]]<br />
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<font color="white">Figure 4. <br />
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=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
<br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br />
<br><br />
<br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br />
[[image:UofLmms6growth.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/JudgingTeam:Lethbridge/Judging2010-10-27T19:51:51Z<p>Liszabruder: </p>
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Check out these important project links!<br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><br />
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<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Judging=<br />
<br />
This page is to help everyone find the information that they need quickly and easily. Happy browsing!<br />
<br />
=<font color="FFD700">Gold Criteria=<br />
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We took a closer look at the previously submitted xylE construct. You can take a look at what we did on our <a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Existing_Parts"><font color="#00DC00"> Characterized Existing Parts</font></a> page, our <a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a> page and on the registry for part <a href="http://partsregistry.org/Part:BBa_K118021" target="new"><font color="#00DC00"> BBa_K118021</font></a>.<br />
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Take a look at our <a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><font color="#00DC00"> collaborations</font></a>!<br />
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=<font color="C0C0C0">Silver Criteria=<br />
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They worked!! Take a look at some of our new BioBrick parts that we made and characterized! You can find them on our <a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Parts"><font color="#00DC00"> Characterized Parts</font></a> page and our <a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a> page. These pages also have the link to the parts on the Registry.<br />
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=<font color="8C7853">Bronze Criteria=<br />
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We had a SPECTACULAR summer!! Check out our <a href="https://2010.igem.org/Team:Lethbridge/Art"><font color="#00DC00"> Artsmith</font></a> and <a href="https://2010.igem.org/Team:Lethbridge/News"><font color="#00DC00"> Publicity</font></a> pages!<br />
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We are no doubt having fun at the Jamboree! <br />
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Come and check out our poster and presentation! We love talking about our project and hearing alternate ways to approach it.<br />
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Definitely registered for the Jamboree.<br />
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Submitted the iGEM 2010 Judging form.<br />
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Have a look at our team's <a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00"> project description</font></a>. We have four subsections to our project; <a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00"> catechol degradation</font></a>, <a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> compartmentalization</font></a>, <a href="https://2010.igem.org/Team:Lethbridge/Project/DNA_Degradation"><font color="#00DC00"> DNA degradation</font></a> and <a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00"> magnetic nanoparticles</font></a>.<br />
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Take a look at the <a href="https://2010.igem.org/Team:Lethbridge/Parts"><font color="#00DC00"> list of parts </font></a>we have designed and the 9 that we submitted.<br />
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We have submitted 9 new BioBricks to the registry along with entering their information.<br />
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<br />
=<font color="white">Attribution and Contributions=<br />
The <html><a href="https://2010.igem.org/Team:Lethbridge/Team"><font color="#00DC00"> Lethbridge iGEM team</font></a></html> declare there is no scientific overlap between our project and the projects of the <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Graduate_Students"><font color="#00DC00"> advisors</font></a></html> or <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Supervisors"><font color="#00DC00"> instructors</font></a></html>. The <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Undergraduate_Students"><font color="#00DC00"> undergraduates</font></a></html>, in order to apply for the <html><a href="https://2010.igem.org/Oil_Sands" target="new"><font color="#00DC00"> Oil Sands Initiative Grant</font></a></html>, independently determined the benefits of using the catechol-2,3-dioxygenase for bioremediation of the tailings ponds. We were ecstatic to receive the grant in May 2010. The characterization and development of the <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00"> project</font></a></html> in the laboratory was done by the undergraduates with the supervision of the advisors and instructors as necessary. All the information found within our wiki was done by the team's undergraduates with the exception of the <html><a href="https://2010.igem.org/Team:Lethbridge/Modeling"><font color="#00DC00"> Modelling</font></a></html> which was carried out by the advisors and the <html><a href="https://2010.igem.org/Team:Lethbridge/Art"><font color="#00DC00"> Artsmith</font></a></html> which was done primarily by or in collaboration with <html><a href="https://2010.igem.org/Team:Lethbridge/Team#A._William_Smith"><font color="#00DC00"> Anonymous Smith</font></a></html> from our new media department at the University of Lethbridge.<br />
<br />
=<font color="white">Other Awesome Stuff!=<br />
<br />
==<font color="white">Important Team Parts==<br />
<br />
Take a look at part <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00">BBa_K331030</font></a></html>, <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00">BBa_K331031</font></a></html>, and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>. They are a few of the parts that we have characterize that you can find on our <html><a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Parts"><font color="#00DC00"> Characterized Parts</font></a></html> and <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> pages.<br />
<br><br />
<br><br />
<br />
==<font color="white">Human Practice==<br />
===<font color="white">“Study the past, if you would divine the future” – Confucius===<br />
<br />
Scientific study is generally perceived as the development of new ideas and novel data, but underlying this is the fact that scientific advancement is made by building formerly known information on top of new innovation. Indeed, without the discovery of the cell, synthetic biology would never exist.<br />
<br><br><br />
In the same way that scientific advancements can be made by looking at prior invention, the Lethbridge iGEM Team believes that synthetic biology ethical advancements can also be made by looking at ethical concerns of the past. Due to the fact that synthetic biology is such a new science, we are in the position to dictate ethical rules that should be implemented as new discoveries are made. <br />
<br><br><br />
Lethbridge iGEM Team has chosen to look at significant scientific discoveries of the past and analyze them from ethical, environmental, economic, legal and social standpoints. Learning how ethics has been dealt with (or should have been dealt with!) in the past can significantly shape the direction of ethical development in the field of synthetic biology. <br />
<br><br><br />
Through our analysis of cloning, antibiotics, the steam engine, internet and nuclear power, the Lethbridge iGEM Team will “divine the future” of ethics and its relationship with the newly developing field of synthetic biology.<br />
<br><br />
<br><br />
Click <html><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><font color="#00DC00"> here</font></a></html> for more ethics information!<br />
<br><br />
<br><br />
==<font color="white">Safety==<br />
<br />
What has our team looked at to ensure the lab, the public and the environment are safe? Take a look at our <html><a href="https://2010.igem.org/Team:Lethbridge/Safety"><font color="#00DC00"> Safety</font></a></html> page!<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/JudgingTeam:Lethbridge/Judging2010-10-27T19:42:43Z<p>Liszabruder: /* Gold Criteria */</p>
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Check out these important project links!<br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><br />
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<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Judging=<br />
<br />
This page is to help everyone find the information that they need quickly and easily. Happy browsing!<br />
<br />
=<font color="FFD700">Gold Criteria=<br />
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We took a closer look at the previously submitted xylE construct. You can take a look at what we did on our <a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Existing_Parts"><font color="#00DC00"> Characterized Existing Parts</font></a></html> page, our <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> page and on the registry for part <a href="http://partsregistry.org/Part:BBa_K118021" target="new"><font color="#00DC00"> BBa_K118021</font></a>.<br />
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Take a look at our <a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><font color="#00DC00"> collaborations</font></a>!<br />
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=<font color="C0C0C0">Silver Criteria=<br />
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It worked!! Take a look at some of our new BioBrick parts that we made and characterized! You can find it on our <a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Parts"><font color="#00DC00"> Characterized Parts</font></a> page and on the Registry for part <a href="http://partsregistry.org/Part:BBa_K331027" target="new"><font color="#00DC00"> BBa_K331027</font></a>.<br />
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=<font color="8C7853">Bronze Criteria=<br />
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We had a SPECTACULAR summer!! Check out our <a href="https://2010.igem.org/Team:Lethbridge/Art"><font color="#00DC00"> Artsmith</font></a> and <a href="https://2010.igem.org/Team:Lethbridge/News"><font color="#00DC00"> Publicity</font></a> pages!<br />
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We are no doubt having fun at the Jamboree! <br />
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Come and check out our poster and presentation! We love talking about our project and hearing alternate ways to approach it.<br />
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Definitely registered for the Jamboree.<br />
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Submitted the iGEM 2010 Judging form.<br />
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Have a look at our team's <a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00"> project description</font></a>. We have four subsections to our project; <a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00"> catechol degradation</font></a>, <a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> compartmentalization</font></a>, <a href="https://2010.igem.org/Team:Lethbridge/Project/DNA_Degradation"><font color="#00DC00"> DNA degradation</font></a> and <a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00"> magnetic nanoparticles</font></a>.<br />
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Take a look at the <a href="https://2010.igem.org/Team:Lethbridge/Parts"><font color="#00DC00"> list of parts </font></a>we have designed and the 9 that we submitted.<br />
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We have submitted 9 new BioBricks to the registry along with entering their information.<br />
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<br />
=<font color="white">Attribution and Contributions=<br />
The <html><a href="https://2010.igem.org/Team:Lethbridge/Team"><font color="#00DC00"> Lethbridge iGEM team</font></a></html> declare there is no scientific overlap between our project and the projects of the <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Graduate_Students"><font color="#00DC00"> advisors</font></a></html> or <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Supervisors"><font color="#00DC00"> instructors</font></a></html>. The <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Undergraduate_Students"><font color="#00DC00"> undergraduates</font></a></html>, in order to apply for the <html><a href="https://2010.igem.org/Oil_Sands" target="new"><font color="#00DC00"> Oil Sands Initiative Grant</font></a></html>, independently determined the benefits of using the catechol-2,3-dioxygenase for bioremediation of the tailings ponds. We were ecstatic to receive the grant in May 2010. The characterization and development of the <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00"> project</font></a></html> in the laboratory was done by the undergraduates with the supervision of the advisors and instructors as necessary. All the information found within our wiki was done by the team's undergraduates with the exception of the <html><a href="https://2010.igem.org/Team:Lethbridge/Modeling"><font color="#00DC00"> Modelling</font></a></html> which was carried out by the advisors and the <html><a href="https://2010.igem.org/Team:Lethbridge/Art"><font color="#00DC00"> Artsmith</font></a></html> which was done primarily by or in collaboration with <html><a href="https://2010.igem.org/Team:Lethbridge/Team#A._William_Smith"><font color="#00DC00"> Anonymous Smith</font></a></html> from our new media department at the University of Lethbridge.<br />
<br />
=<font color="white">Other Awesome Stuff!=<br />
<br />
==<font color="white">Important Team Parts==<br />
<br />
Take a look at part <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00">BBa_K331030</font></a></html>, <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00">BBa_K331031</font></a></html>, and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>. They are a few of the parts that we have characterize that you can find on our <html><a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Parts"><font color="#00DC00"> Characterized Parts</font></a></html> and <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> pages.<br />
<br><br />
<br><br />
<br />
==<font color="white">Human Practice==<br />
===<font color="white">“Study the past, if you would divine the future” – Confucius===<br />
<br />
Scientific study is generally perceived as the development of new ideas and novel data, but underlying this is the fact that scientific advancement is made by building formerly known information on top of new innovation. Indeed, without the discovery of the cell, synthetic biology would never exist.<br />
<br><br><br />
In the same way that scientific advancements can be made by looking at prior invention, the Lethbridge iGEM Team believes that synthetic biology ethical advancements can also be made by looking at ethical concerns of the past. Due to the fact that synthetic biology is such a new science, we are in the position to dictate ethical rules that should be implemented as new discoveries are made. <br />
<br><br><br />
Lethbridge iGEM Team has chosen to look at significant scientific discoveries of the past and analyze them from ethical, environmental, economic, legal and social standpoints. Learning how ethics has been dealt with (or should have been dealt with!) in the past can significantly shape the direction of ethical development in the field of synthetic biology. <br />
<br><br><br />
Through our analysis of cloning, antibiotics, the steam engine, internet and nuclear power, the Lethbridge iGEM Team will “divine the future” of ethics and its relationship with the newly developing field of synthetic biology.<br />
<br><br />
<br><br />
Click <html><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><font color="#00DC00"> here</font></a></html> for more ethics information!<br />
<br><br />
<br><br />
==<font color="white">Safety==<br />
<br />
What has our team looked at to ensure the lab, the public and the environment are safe? Take a look at our <html><a href="https://2010.igem.org/Team:Lethbridge/Safety"><font color="#00DC00"> Safety</font></a></html> page!<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/JudgingTeam:Lethbridge/Judging2010-10-27T19:40:42Z<p>Liszabruder: </p>
<hr />
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</a><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><br />
<img src="https://static.igem.org/mediawiki/2010/2/26/UofLEthicsbutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Safety"><br />
<img src="https://static.igem.org/mediawiki/2010/0/00/UofLSafetybutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Art"><br />
<img src="https://static.igem.org/mediawiki/2010/0/0a/UofLArt.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/News"><br />
<img src="https://static.igem.org/mediawiki/2010/c/c3/UofLNewsButton.jpg" width="80"/><br />
</a><br />
</th><br />
</table><br />
</body><br />
</html><br />
<hr><br />
<center><br />
Check out these important project links!<br />
</center><br />
<html><br />
<body><br />
<center><br />
<table border="0" width="20%" style="background-color:#000000"><br />
<tr><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Judging"><br />
<img src="https://static.igem.org/mediawiki/2010/f/fd/UofLjudgingbutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Results"><br />
<img src="https://static.igem.org/mediawiki/2010/3/3f/UofLresultsbutton.jpg" width="60"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><br />
<img src="https://static.igem.org/mediawiki/2010/b/b2/UofLcollaborationbutton.png" width="60"/><br />
</a><br />
</th><br />
<br />
<tr><br />
</table><br />
</center><br />
</body><br />
</html><br />
<hr><br />
<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Judging=<br />
<br />
This page is to help everyone find the information that they need quickly and easily. Happy browsing!<br />
<br />
=<font color="FFD700">Gold Criteria=<br />
<br />
<html><br />
<body><br />
<table border="0" cellpadding="8" width="90%" style="background-color:#000000"><br />
<br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f1/UofLgoldcheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="FFD700"><br />
We took a closer look at the previously submitted xylE construct. You can take a look at what we did on our <a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Existing_Parts"><font color="#00DC00"> Characterized Existing Parts</font></a> page, our <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> page and on the registry for part <a href="http://partsregistry.org/Part:BBa_K118021" target="new"><font color="#00DC00"> BBa_K118021</font></a>.<br />
<br />
</th><br />
<br />
</tr><br />
<br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f1/UofLgoldcheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="FFD700"><br />
Take a look at our <a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><font color="#00DC00"> collaborations</font></a>!<br />
</th><br />
<br />
</tr><br />
<br />
</table><br />
</body><br />
</html><br />
<br />
=<font color="C0C0C0">Silver Criteria=<br />
<br />
<html><br />
<body><br />
<table border="0" cellpadding="8" width="90%" style="background-color:#000000"><br />
<br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/b/bd/UofLsilvercheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="C0C0C0"><br />
It worked!! Take a look at some of our new BioBrick parts that we made and characterized! You can find it on our <a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Parts"><font color="#00DC00"> Characterized Parts</font></a> page and on the Registry for part <a href="http://partsregistry.org/Part:BBa_K331027" target="new"><font color="#00DC00"> BBa_K331027</font></a>.<br />
<br />
</th><br />
<br />
</tr><br />
<br />
</table><br />
</body><br />
</html><br />
<br />
=<font color="8C7853">Bronze Criteria=<br />
<br />
<html><br />
<body><br />
<table border="0" cellpadding="8" width="90%" style="background-color:#000000"><br />
<br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
We had a SPECTACULAR summer!! Check out our <a href="https://2010.igem.org/Team:Lethbridge/Art"><font color="#00DC00"> Artsmith</font></a> and <a href="https://2010.igem.org/Team:Lethbridge/News"><font color="#00DC00"> Publicity</font></a> pages!<br />
<br />
</th><br />
<br />
</tr><br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
We are no doubt having fun at the Jamboree! <br />
<br />
</th><br />
<br />
</tr><br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
Come and check out our poster and presentation! We love talking about our project and hearing alternate ways to approach it.<br />
<br />
</th><br />
<br />
</tr><br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
Definitely registered for the Jamboree.<br />
<br />
</th><br />
<br />
</tr><br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
Submitted the iGEM 2010 Judging form.<br />
<br />
</th><br />
<br />
</tr><br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
Have a look at our team's <a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00"> project description</font></a>. We have four subsections to our project; <a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00"> catechol degradation</font></a>, <a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> compartmentalization</font></a>, <a href="https://2010.igem.org/Team:Lethbridge/Project/DNA_Degradation"><font color="#00DC00"> DNA degradation</font></a> and <a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00"> magnetic nanoparticles</font></a>.<br />
<br />
</th><br />
<br />
</tr><br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
Take a look at the <a href="https://2010.igem.org/Team:Lethbridge/Parts"><font color="#00DC00"> list of parts </font></a>we have designed and the 9 that we submitted.<br />
<br />
</th><br />
<br />
</tr><br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
We have submitted 9 new BioBricks to the registry along with entering their information.<br />
<br />
</th><br />
<br />
</tr><br />
<br />
<br />
</table><br />
</body><br />
</html><br />
<br />
=<font color="white">Attribution and Contributions=<br />
The <html><a href="https://2010.igem.org/Team:Lethbridge/Team"><font color="#00DC00"> Lethbridge iGEM team</font></a></html> declare there is no scientific overlap between our project and the projects of the <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Graduate_Students"><font color="#00DC00"> advisors</font></a></html> or <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Supervisors"><font color="#00DC00"> instructors</font></a></html>. The <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Undergraduate_Students"><font color="#00DC00"> undergraduates</font></a></html>, in order to apply for the <html><a href="https://2010.igem.org/Oil_Sands" target="new"><font color="#00DC00"> Oil Sands Initiative Grant</font></a></html>, independently determined the benefits of using the catechol-2,3-dioxygenase for bioremediation of the tailings ponds. We were ecstatic to receive the grant in May 2010. The characterization and development of the <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00"> project</font></a></html> in the laboratory was done by the undergraduates with the supervision of the advisors and instructors as necessary. All the information found within our wiki was done by the team's undergraduates with the exception of the <html><a href="https://2010.igem.org/Team:Lethbridge/Modeling"><font color="#00DC00"> Modelling</font></a></html> which was carried out by the advisors and the <html><a href="https://2010.igem.org/Team:Lethbridge/Art"><font color="#00DC00"> Artsmith</font></a></html> which was done primarily by or in collaboration with <html><a href="https://2010.igem.org/Team:Lethbridge/Team#A._William_Smith"><font color="#00DC00"> Anonymous Smith</font></a></html> from our new media department at the University of Lethbridge.<br />
<br />
=<font color="white">Other Awesome Stuff!=<br />
<br />
==<font color="white">Important Team Parts==<br />
<br />
Take a look at part <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00">BBa_K331030</font></a></html>, <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00">BBa_K331031</font></a></html>, and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>. They are a few of the parts that we have characterize that you can find on our <html><a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Parts"><font color="#00DC00"> Characterized Parts</font></a></html> and <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> pages.<br />
<br><br />
<br><br />
<br />
==<font color="white">Human Practice==<br />
===<font color="white">“Study the past, if you would divine the future” – Confucius===<br />
<br />
Scientific study is generally perceived as the development of new ideas and novel data, but underlying this is the fact that scientific advancement is made by building formerly known information on top of new innovation. Indeed, without the discovery of the cell, synthetic biology would never exist.<br />
<br><br><br />
In the same way that scientific advancements can be made by looking at prior invention, the Lethbridge iGEM Team believes that synthetic biology ethical advancements can also be made by looking at ethical concerns of the past. Due to the fact that synthetic biology is such a new science, we are in the position to dictate ethical rules that should be implemented as new discoveries are made. <br />
<br><br><br />
Lethbridge iGEM Team has chosen to look at significant scientific discoveries of the past and analyze them from ethical, environmental, economic, legal and social standpoints. Learning how ethics has been dealt with (or should have been dealt with!) in the past can significantly shape the direction of ethical development in the field of synthetic biology. <br />
<br><br><br />
Through our analysis of cloning, antibiotics, the steam engine, internet and nuclear power, the Lethbridge iGEM Team will “divine the future” of ethics and its relationship with the newly developing field of synthetic biology.<br />
<br><br />
<br><br />
Click <html><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><font color="#00DC00"> here</font></a></html> for more ethics information!<br />
<br><br />
<br><br />
==<font color="white">Safety==<br />
<br />
What has our team looked at to ensure the lab, the public and the environment are safe? Take a look at our <html><a href="https://2010.igem.org/Team:Lethbridge/Safety"><font color="#00DC00"> Safety</font></a></html> page!<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/JudgingTeam:Lethbridge/Judging2010-10-27T19:36:08Z<p>Liszabruder: </p>
<hr />
<div><div style="background-color:#000000; color:white"><br />
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<br><br />
<br />
<table border="0" width="100%" style="background-color:#000000"><br />
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<tr><br />
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<th><br />
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<image src="https://static.igem.org/mediawiki/2010/2/29/UofLteamlogo.jpg" width="200px"/><br />
<br />
</th><br />
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<th><br />
<br />
<image src="https://static.igem.org/mediawiki/2010/4/4e/UofLwewantyou.jpg" height="300px"/><br />
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</th><br />
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<th><br />
<br />
<image src="https://static.igem.org/mediawiki/2010/2/29/UofLteamlogo.jpg" width="200px"/><br />
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</tr><br />
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</table><br />
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<br />
<align="centre"><br />
<table border="0" width="100%" style="background-color:#000000"><br />
<br />
<tr><br />
<br />
<th><br />
<br />
<a href="https://2010.igem.org/Team:Lethbridge"><br />
<img src="https://static.igem.org/mediawiki/2010/2/22/UofLHome.jpg" width="80"/><br />
</a><br />
<br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Team"><br />
<img src="https://static.igem.org/mediawiki/2010/0/0d/UofLTeam.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Project"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8d/UofLProjectbutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work"><br />
<img src="https://static.igem.org/mediawiki/2010/7/73/UofLNotebookbutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Parts"><br />
<img src="https://static.igem.org/mediawiki/2010/8/84/UofLPartsSubmittedToTheRegistrybutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Modeling"><br />
<img src="https://static.igem.org/mediawiki/2010/e/e1/UofLModelingbutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><br />
<img src="https://static.igem.org/mediawiki/2010/2/26/UofLEthicsbutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Safety"><br />
<img src="https://static.igem.org/mediawiki/2010/0/00/UofLSafetybutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Art"><br />
<img src="https://static.igem.org/mediawiki/2010/0/0a/UofLArt.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/News"><br />
<img src="https://static.igem.org/mediawiki/2010/c/c3/UofLNewsButton.jpg" width="80"/><br />
</a><br />
</th><br />
</table><br />
</body><br />
</html><br />
<hr><br />
<center><br />
Check out these important project links!<br />
</center><br />
<html><br />
<body><br />
<center><br />
<table border="0" width="20%" style="background-color:#000000"><br />
<tr><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Judging"><br />
<img src="https://static.igem.org/mediawiki/2010/f/fd/UofLjudgingbutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Results"><br />
<img src="https://static.igem.org/mediawiki/2010/3/3f/UofLresultsbutton.jpg" width="60"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><br />
<img src="https://static.igem.org/mediawiki/2010/b/b2/UofLcollaborationbutton.png" width="60"/><br />
</a><br />
</th><br />
<br />
<tr><br />
</table><br />
</center><br />
</body><br />
</html><br />
<hr><br />
<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Judging=<br />
<br />
This page is to help everyone find the information that they need quickly and easily. Happy browsing!<br />
<br />
=<font color="FFD700">Gold Criteria=<br />
<br />
<html><br />
<body><br />
<table border="0" cellpadding="8" width="90%" style="background-color:#000000"><br />
<br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f1/UofLgoldcheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="FFD700"><br />
We took a closer look at the previously submitted xylE construct. You can take a look at what we did on our <a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Existing_Parts"><font color="#00DC00"> Characterized Existing Parts</font></a> page and on the registry for part <a href="http://partsregistry.org/Part:BBa_K118021" target="new"><font color="#00DC00"> BBa_K118021</font></a>.<br />
<br />
</th><br />
<br />
</tr><br />
<br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f1/UofLgoldcheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="FFD700"><br />
Take a look at our <a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><font color="#00DC00"> collaborations</font></a>!<br />
</th><br />
<br />
</tr><br />
<br />
</table><br />
</body><br />
</html><br />
<br />
=<font color="C0C0C0">Silver Criteria=<br />
<br />
<html><br />
<body><br />
<table border="0" cellpadding="8" width="90%" style="background-color:#000000"><br />
<br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/b/bd/UofLsilvercheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="C0C0C0"><br />
It worked!! Take a look at some of our new BioBrick parts that we made and characterized! You can find it on our <a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Parts"><font color="#00DC00"> Characterized Parts</font></a> page and on the Registry for part <a href="http://partsregistry.org/Part:BBa_K331027" target="new"><font color="#00DC00"> BBa_K331027</font></a>.<br />
<br />
</th><br />
<br />
</tr><br />
<br />
</table><br />
</body><br />
</html><br />
<br />
=<font color="8C7853">Bronze Criteria=<br />
<br />
<html><br />
<body><br />
<table border="0" cellpadding="8" width="90%" style="background-color:#000000"><br />
<br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
We had a SPECTACULAR summer!! Check out our <a href="https://2010.igem.org/Team:Lethbridge/Art"><font color="#00DC00"> Artsmith</font></a> and <a href="https://2010.igem.org/Team:Lethbridge/News"><font color="#00DC00"> Publicity</font></a> pages!<br />
<br />
</th><br />
<br />
</tr><br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
We are no doubt having fun at the Jamboree! <br />
<br />
</th><br />
<br />
</tr><br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
Come and check out our poster and presentation! We love talking about our project and hearing alternate ways to approach it.<br />
<br />
</th><br />
<br />
</tr><br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
Definitely registered for the Jamboree.<br />
<br />
</th><br />
<br />
</tr><br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
Submitted the iGEM 2010 Judging form.<br />
<br />
</th><br />
<br />
</tr><br />
<tr><br />
<br />
<th align="left"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
</th><br />
<br />
<th align="left"><font color="8C7853"><br />
Have a look at our team's <a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00"> project description</font></a>. We have four subsections to our project; <a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00"> catechol degradation</font></a>, <a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> compartmentalization</font></a>, <a href="https://2010.igem.org/Team:Lethbridge/Project/DNA_Degradation"><font color="#00DC00"> DNA degradation</font></a> and <a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00"> magnetic nanoparticles</font></a>.<br />
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<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
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Take a look at the <a href="https://2010.igem.org/Team:Lethbridge/Parts"><font color="#00DC00"> list of parts </font></a>we have designed and the 9 that we submitted.<br />
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We have submitted 9 new BioBricks to the registry along with entering their information.<br />
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</table><br />
</body><br />
</html><br />
<br />
=<font color="white">Attribution and Contributions=<br />
The <html><a href="https://2010.igem.org/Team:Lethbridge/Team"><font color="#00DC00"> Lethbridge iGEM team</font></a></html> declare there is no scientific overlap between our project and the projects of the <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Graduate_Students"><font color="#00DC00"> advisors</font></a></html> or <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Supervisors"><font color="#00DC00"> instructors</font></a></html>. The <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Undergraduate_Students"><font color="#00DC00"> undergraduates</font></a></html>, in order to apply for the <html><a href="https://2010.igem.org/Oil_Sands" target="new"><font color="#00DC00"> Oil Sands Initiative Grant</font></a></html>, independently determined the benefits of using the catechol-2,3-dioxygenase for bioremediation of the tailings ponds. We were ecstatic to receive the grant in May 2010. The characterization and development of the <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00"> project</font></a></html> in the laboratory was done by the undergraduates with the supervision of the advisors and instructors as necessary. All the information found within our wiki was done by the team's undergraduates with the exception of the <html><a href="https://2010.igem.org/Team:Lethbridge/Modeling"><font color="#00DC00"> Modelling</font></a></html> which was carried out by the advisors and the <html><a href="https://2010.igem.org/Team:Lethbridge/Art"><font color="#00DC00"> Artsmith</font></a></html> which was done primarily by or in collaboration with <html><a href="https://2010.igem.org/Team:Lethbridge/Team#A._William_Smith"><font color="#00DC00"> Anonymous Smith</font></a></html> from our new media department at the University of Lethbridge.<br />
<br />
=<font color="white">Other Awesome Stuff!=<br />
<br />
==<font color="white">Important Team Parts==<br />
<br />
Take a look at part <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00">BBa_K331030</font></a></html>, <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00">BBa_K331031</font></a></html>, and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>. They are a few of the parts that we have characterize that you can find on our <html><a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Parts"><font color="#00DC00"> Characterized Parts</font></a></html> and <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> pages.<br />
<br><br />
<br><br />
<br />
==<font color="white">Human Practice==<br />
===<font color="white">“Study the past, if you would divine the future” – Confucius===<br />
<br />
Scientific study is generally perceived as the development of new ideas and novel data, but underlying this is the fact that scientific advancement is made by building formerly known information on top of new innovation. Indeed, without the discovery of the cell, synthetic biology would never exist.<br />
<br><br><br />
In the same way that scientific advancements can be made by looking at prior invention, the Lethbridge iGEM Team believes that synthetic biology ethical advancements can also be made by looking at ethical concerns of the past. Due to the fact that synthetic biology is such a new science, we are in the position to dictate ethical rules that should be implemented as new discoveries are made. <br />
<br><br><br />
Lethbridge iGEM Team has chosen to look at significant scientific discoveries of the past and analyze them from ethical, environmental, economic, legal and social standpoints. Learning how ethics has been dealt with (or should have been dealt with!) in the past can significantly shape the direction of ethical development in the field of synthetic biology. <br />
<br><br><br />
Through our analysis of cloning, antibiotics, the steam engine, internet and nuclear power, the Lethbridge iGEM Team will “divine the future” of ethics and its relationship with the newly developing field of synthetic biology.<br />
<br><br />
<br><br />
Click <html><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><font color="#00DC00"> here</font></a></html> for more ethics information!<br />
<br><br />
<br><br />
==<font color="white">Safety==<br />
<br />
What has our team looked at to ensure the lab, the public and the environment are safe? Take a look at our <html><a href="https://2010.igem.org/Team:Lethbridge/Safety"><font color="#00DC00"> Safety</font></a></html> page!<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/JudgingTeam:Lethbridge/Judging2010-10-27T19:32:46Z<p>Liszabruder: /* Important Team Parts */</p>
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<a href="https://2010.igem.org/Team:Lethbridge"><br />
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</a><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Team"><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work"><br />
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</a><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Art"><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/News"><br />
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<center><br />
Check out these important project links!<br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Results"><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><br />
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<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Judging=<br />
<br />
This page is to help everyone find the information that they need quickly and easily. Happy browsing!<br />
<br />
=<font color="FFD700">Gold Criteria=<br />
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We took a closer look at the previously submitted xylE construct. You can take a look at what we did on our <a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Existing_Parts"><font color="#00DC00"> Characterized Existing Parts</font></a> page and on the registry for part <a href="http://partsregistry.org/Part:BBa_K118021" target="new"><font color="#00DC00"> BBa_K118021</font></a>.<br />
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Take a look at our <a href="https://2010.igem.org/Team:Lethbridge/Collaboration"><font color="#00DC00"> collaborations</font></a>!<br />
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<br />
=<font color="C0C0C0">Silver Criteria=<br />
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It worked!! Take a look at some of our new BioBrick parts that we made and characterized! You can find it on our <a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Parts"><font color="#00DC00"> Characterized Parts</font></a> page and on the Registry for part <a href="http://partsregistry.org/Part:BBa_K331027" target="new"><font color="#00DC00"> BBa_K331027</font></a>.<br />
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=<font color="8C7853">Bronze Criteria=<br />
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We had a SPECTACULAR summer!! Check out our <a href="https://2010.igem.org/Team:Lethbridge/Art"><font color="#00DC00"> Artsmith</font></a> and <a href="https://2010.igem.org/Team:Lethbridge/News"><font color="#00DC00"> Publicity</font></a> pages!<br />
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We are no doubt having fun at the Jamboree! <br />
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Come and check out our poster and presentation! We love talking about our project and hearing alternate ways to approach it.<br />
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Definitely registered for the Jamboree.<br />
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<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
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Submitted the iGEM 2010 Judging form.<br />
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<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
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<br />
<th align="left"><font color="8C7853"><br />
Have a look at our team's <a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00"> project description</font></a>. We have four subsections to our project; <a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00"> catechol degradation</font></a>, <a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> compartmentalization</font></a>, <a href="https://2010.igem.org/Team:Lethbridge/Project/DNA_Degradation"><font color="#00DC00"> DNA degradation</font></a> and <a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00"> magnetic nanoparticles</font></a>.<br />
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Take a look at the <a href="https://2010.igem.org/Team:Lethbridge/Parts"><font color="#00DC00"> list of parts </font></a>we have submitted.<br />
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<img src="https://static.igem.org/mediawiki/2010/f/f2/UofLbronzecheck.jpg"/><br />
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We have submitted ___ new BioBricks to the registry along with entering their information.<br />
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</tr><br />
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</body><br />
</html><br />
<br />
=<font color="white">Attribution and Contributions=<br />
The <html><a href="https://2010.igem.org/Team:Lethbridge/Team"><font color="#00DC00"> Lethbridge iGEM team</font></a></html> declare there is no scientific overlap between our project and the projects of the <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Graduate_Students"><font color="#00DC00"> advisors</font></a></html> or <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Supervisors"><font color="#00DC00"> instructors</font></a></html>. The <html><a href="https://2010.igem.org/Team:Lethbridge/Team#Undergraduate_Students"><font color="#00DC00"> undergraduates</font></a></html>, in order to apply for the <html><a href="https://2010.igem.org/Oil_Sands" target="new"><font color="#00DC00"> Oil Sands Initiative Grant</font></a></html>, independently determined the benefits of using the catechol-2,3-dioxygenase for bioremediation of the tailings ponds. We were ecstatic to receive the grant in May 2010. The characterization and development of the <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00"> project</font></a></html> in the laboratory was done by the undergraduates with the supervision of the advisors and instructors as necessary. All the information found within our wiki was done by the team's undergraduates with the exception of the <html><a href="https://2010.igem.org/Team:Lethbridge/Modeling"><font color="#00DC00"> Modelling</font></a></html> which was carried out by the advisors and the <html><a href="https://2010.igem.org/Team:Lethbridge/Art"><font color="#00DC00"> Artsmith</font></a></html> which was done primarily by or in collaboration with <html><a href="https://2010.igem.org/Team:Lethbridge/Team#A._William_Smith"><font color="#00DC00"> Anonymous Smith</font></a></html> from our new media department at the University of Lethbridge.<br />
<br />
=<font color="white">Other Awesome Stuff!=<br />
<br />
==<font color="white">Important Team Parts==<br />
<br />
Take a look at part <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00">BBa_K331030</font></a></html>, <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00">BBa_K331031</font></a></html>, and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>. They are a few of the parts that we have characterize that you can find on our <html><a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized_Parts"><font color="#00DC00"> Characterized Parts</font></a></html> and <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> pages.<br />
<br><br />
<br><br />
<br />
==<font color="white">Human Practice==<br />
===<font color="white">“Study the past, if you would divine the future” – Confucius===<br />
<br />
Scientific study is generally perceived as the development of new ideas and novel data, but underlying this is the fact that scientific advancement is made by building formerly known information on top of new innovation. Indeed, without the discovery of the cell, synthetic biology would never exist.<br />
<br><br><br />
In the same way that scientific advancements can be made by looking at prior invention, the Lethbridge iGEM Team believes that synthetic biology ethical advancements can also be made by looking at ethical concerns of the past. Due to the fact that synthetic biology is such a new science, we are in the position to dictate ethical rules that should be implemented as new discoveries are made. <br />
<br><br><br />
Lethbridge iGEM Team has chosen to look at significant scientific discoveries of the past and analyze them from ethical, environmental, economic, legal and social standpoints. Learning how ethics has been dealt with (or should have been dealt with!) in the past can significantly shape the direction of ethical development in the field of synthetic biology. <br />
<br><br><br />
Through our analysis of cloning, antibiotics, the steam engine, internet and nuclear power, the Lethbridge iGEM Team will “divine the future” of ethics and its relationship with the newly developing field of synthetic biology.<br />
<br><br />
<br><br />
Click <html><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><font color="#00DC00"> here</font></a></html> for more ethics information!<br />
<br><br />
<br><br />
==<font color="white">Safety==<br />
<br />
What has our team looked at to ensure the lab, the public and the environment are safe? Take a look at our <html><a href="https://2010.igem.org/Team:Lethbridge/Safety"><font color="#00DC00"> Safety</font></a></html> page!<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/OctoberTeam:Lethbridge/Notebook/Lab Work/October2010-10-27T19:27:43Z<p>Liszabruder: /* Js,Jv */</p>
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<a href="https://2010.igem.org/Team:Lethbridge"><br />
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</a><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Team"><br />
<img src="https://static.igem.org/mediawiki/2010/0/0d/UofLTeam.jpg" width="80"/><br />
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<BLOCKQUOTE><br />
<br />
=<font color="white">October=<br />
==<font color="white">October 1, 2010==<br />
===<font color="white">KG===<br />
<b>Objective:</b> PCR of xylE for conformation of transformation.<br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x24)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>13.8<td>331.2<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>48<br />
<tr><td>dNTPs<td>1<td>24<br />
<tr><td>Forward VF2 Primer<td>1<td>24<br />
<tr><td>Reverse VR Primer<td>1<td>24<br />
<tr><td>Template DNA<td>1<td><br />
<tr><td>Pfu polymerase<td>0.2<td>4.8<br />
</table><br><br />
<br />
Ran with PFV program on thermocycler with extension time of 3 minutes.<br />
<br />
==<font color="white">October 2, 2010==<br />
===<font color="white">ADS===<br />
<b>Objective:</b> Purify plasmid DNA from transformed Mr. GENE synthesized DNA<br />
*<partinfo>K331022</partinfo><br />
*<partinfo>K331023</partinfo><br />
*<partinfo>K331024</partinfo><br />
*<partinfo>K331025</partinfo><br />
<b>Method:</b> Use Qiagen miniprep method with BioBasic EZ-10 Spin columns.<br><br />
------------------------------------------------------------------------------------------------------------------------<br />
<b>Objective:</b> Create 20 new biobricks using recently arrived synthesized DNA and terminator <partinfo>B0015</partinfo>, pLac <partinfo>R0010</partinfo>, and pTet <partinfo>R0040</partinfo> to create the following new biobricks:<br />
*1)R0010 + K331022 to obtain <partinfo>K331026</partinfo><br />
*2)R0040 + K331022 to obtain <partinfo>K331030</partinfo><br />
*3)K331022 + B0015 to obtain <partinfo>K331034</partinfo><br />
*4)K331022 + K331024 to obtain (<font color="red">NAME BIOBRICK!!!!</font>)<br />
*5)R0010 + K331023 to obtain <partinfo>K331027</partinfo><br />
*6)R0040 + K331023 to obtain <partinfo>K331031</partinfo><br />
*7)K331023 + B0015 to obtain <partinfo>K331035</partinfo><br />
*8)K331023 + K331025 to obtain (<font color="red">NAME BIOBRICK!!!!</font>)<br />
*9)R0010 + K331024 to obtain <partinfo>K331028</partinfo><br />
*10)R0040 + K331024 to obtain <partinfo>K331032</partinfo><br />
*11)K331024 + B0015 to obtain <partinfo>K331036</partinfo><br />
*12)K331024 + K331022 to obtain (<font color="red">NAME BIOBRICK!!!!</font>)<br />
*13)R0010 + K331025 to obtain <partinfo>K331029</partinfo><br />
*14)R0040 + K331025 to obtain <partinfo>K331033</partinfo><br />
*15)K331025 + B0015 to obtain <partinfo>K331037</partinfo><br />
*16)K331025 + K331023 to obtain (<font color="red">NAME BIOBRICK!!!!</font>)<br />
*17)K331022 to obtain <partinfo>K331022</partinfo><br />
*18)K331023 to obtain <partinfo>K331023</partinfo><br />
*19)K331024 to obtain <partinfo>K331024</partinfo><br />
*20)K331025 to obtain <partinfo>K331025</partinfo><br />
All parts will be inserted into pSB1C3.<br><br />
<b>Method:</b> Use BioBrick Assembly Method <br><br />
<b>Results:</b> Obtained <font color="red">RESULTS!!!</font> positive colonies.<br />
----<br />
<br />
<br />
===<font color="white">HB===<br />
<br />
<b>Objective:</b> PCR mms6 from Mr. Gene to attach fusion standard to the N-term.<br><br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x6.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>12.8<td>83.2<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>13<br />
<tr><td>dNTPs<td>1<td>6.5<br />
<tr><td>mms6 Prefix Fusion Forward Primer<td>1<td>6.5<br />
<tr><td>Standard Suffix Reverse Primer<td>1<td>6.5<br />
<tr><td>Template DNA<td>2<td>13<br />
<tr><td>Pfu polymerase<td>0.2<td><br />
</table><br><br />
<br />
Prefix fusion sense primer has melting temperature of 56.1<sup>o</sup>C. Standard suffix primer has melting temperature of 61.3<sup>o</sup>C. <br />
<br />
PCR- Conditions:<br />
1. 95<sup>o</sup>C for 180 sec<br />
2. 95<sup>o</sup>C for 30 sec<br />
3. 56.3<sup>o</sup>C for 30 sec<br />
4. 72<sup>o</sup>C for 150 sec<br />
5. 72<sup>o</sup>C for 600 sec<br />
(35 cycles)<br />
<br />
Gradient - <br />
1. 54.3<sup>o</sup>C 2. 55.4<sup>o</sup>C 3. 56.0<sup>o</sup>C 4. 56.6<sup>o</sup>C 5. 57.8<sup>o</sup>C 6. 58.7<sup>o</sup>C<br />
<br />
----<br />
<br />
<b>Objective:</b> PCR mms6 from Mr. Gene to attach fusion standard to the C-term.<br><br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x6.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>12.8<td>83.2<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>13<br />
<tr><td>dNTPs<td>1<td>6.5<br />
<tr><td>Prefix Forward Primer<td>1<td>6.5<br />
<tr><td>mms6 Fusion Suffix Reverse Primer<td>1<td>6.5<br />
<tr><td>Template DNA<td>2<td>13<br />
<tr><td>Pfu polymerase<td>0.2<td><br />
</table><br><br />
<br />
Prefix fusion sense primer has melting temperature of 58.8<sup>o</sup>C. Standard suffix primer has melting temperature of 75<sup>o</sup>C. <br />
<br />
PCR- Conditions:<br />
1. 95<sup>o</sup>C for 180 sec<br />
2. 95<sup>o</sup>C for 30 sec<br />
3. 53.8<sup>o</sup>C for 30 sec<br />
4. 72<sup>o</sup>C for 150 sec<br />
5. 72<sup>o</sup>C for 600 sec<br />
(35 cycles)<br />
<br />
Gradient - <br />
1. 51.4<sup>o</sup>C 2. 52.3<sup>o</sup>C 3. 53.5<sup>o</sup>C 4. 54.1<sup>o</sup>C 5. 55.3<sup>o</sup>C 6. 56.2<sup>o</sup>C<br />
<br />
----<br />
===<font color="white">JV===<br />
<b>Objective:</b> Analyzed KG and HB's PCRs.<br><br />
<b>Objective:</b> Run samples on 2% agarose gel at 100V for 70 minutes.<br><br />
<br />
GEL PICTURE!!!<br />
<br />
==<font color="white">October 5, 2010==<br />
===<font color="white">ADS===<br />
<b>Objective:</b> Purify plasmid DNA from successfully assembled biobricks.<br><br />
<b>Method:</b> Use Qiagen miniprep method with BioBasic EZ-10 columns.<br><br />
Will send these minipreps for sequencing at Macrogen.<br />
<br />
<br />
<br />
<br />
<br><br />
<br />
==<font color="white">October 16, 2010==<br />
===<font color="white">JV===<br />
<br />
<b>Objective:</b> Separate different fractions of the cytosol of cells containing catechol 2,3-dioxygenase <br><br />
<b>Method:</b><br />
*Grow DH5&alpha; cells in 500mL of LB media with Amp<sup>+</sup>.<br />
*Optical density of cell suspension (measured at 600nm): 4.55<br />
*Spun cells down: 10 minutes, 4<sup>o</sup>C, at 5000RPM<br />
*Cell pellet weight is 3.03g<br />
*Flash froze cells and stored at -80<sup>o</sup>C <br><br />
<br />
Plasmid DNA was also isolated using QIAGEN spin column protocol. DNA was eluted to 60&micro;L.<br><br />
<br />
==<font color="white">October 19, 2010==<br />
===<font color="white">Js===<br />
<br />
<b>Objective</b> Ligate biobrick parts<br><br />
<b>Method</b> restrict and ligate into RFP plasmid using the biobrick standard method then transform cells<br />
*pLacI+mRBS<br />
*mRBS+Lum<br />
*Lum+dT<br />
*K118021+dT<br />
*pBad+mRBS<br />
*mRBS+TetR<br />
*TetR+dT <br />
<br />
** forgot to add RFP plasmid at ligation step<br />
*** repeated all protocol<br />
*** ligated the RFP plasmid into the ligated linear DNA as well<br />
<br />
==<font color="white">October 21, 2010==<br />
===<font color="white">Js,Jv===<br />
<br />
<b>Objective</b> Colony PCR for conformation of ligation<br />
<b>Method</b> pick white colonies and perform colony pcr<br />
*pick white colonies from plates<br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x24)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>6.8<td>261.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>77<br />
<tr><td>dNTPs<td>2<td>77<br />
<tr><td>Forward VF2 Primer<td>2<td>77<br />
<tr><td>Reverse VR Primer<td>2<td>77<br />
<tr><td>Template DNA<td>5<td><br />
<tr><td>Pfu polymerase<td>0.2<td>77<br />
</table><br><br />
<br />
PCR- Conditions:<br />
1. 95<sup>o</sup>C for 180 sec<br />
2. 95<sup>o</sup>C for 30 sec<br />
3. 53.8<sup>o</sup>C for 30 sec<br />
4. 72<sup>o</sup>C for 150 sec<br />
5. 72<sup>o</sup>C for 600 sec<br />
(35 cycles)<br />
<br><br />
<br><br />
ran a 2% agarose gel for conformation<br />
*no XYLE(K118021)-dT bands were observed, grew cells overnight.<br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/OctoberTeam:Lethbridge/Notebook/Lab Work/October2010-10-27T19:27:15Z<p>Liszabruder: </p>
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<BLOCKQUOTE><br />
<br />
=<font color="white">October=<br />
==<font color="white">October 1, 2010==<br />
===<font color="white">KG===<br />
<b>Objective:</b> PCR of xylE for conformation of transformation.<br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x24)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>13.8<td>331.2<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>48<br />
<tr><td>dNTPs<td>1<td>24<br />
<tr><td>Forward VF2 Primer<td>1<td>24<br />
<tr><td>Reverse VR Primer<td>1<td>24<br />
<tr><td>Template DNA<td>1<td><br />
<tr><td>Pfu polymerase<td>0.2<td>4.8<br />
</table><br><br />
<br />
Ran with PFV program on thermocycler with extension time of 3 minutes.<br />
<br />
==<font color="white">October 2, 2010==<br />
===<font color="white">ADS===<br />
<b>Objective:</b> Purify plasmid DNA from transformed Mr. GENE synthesized DNA<br />
*<partinfo>K331022</partinfo><br />
*<partinfo>K331023</partinfo><br />
*<partinfo>K331024</partinfo><br />
*<partinfo>K331025</partinfo><br />
<b>Method:</b> Use Qiagen miniprep method with BioBasic EZ-10 Spin columns.<br><br />
------------------------------------------------------------------------------------------------------------------------<br />
<b>Objective:</b> Create 20 new biobricks using recently arrived synthesized DNA and terminator <partinfo>B0015</partinfo>, pLac <partinfo>R0010</partinfo>, and pTet <partinfo>R0040</partinfo> to create the following new biobricks:<br />
*1)R0010 + K331022 to obtain <partinfo>K331026</partinfo><br />
*2)R0040 + K331022 to obtain <partinfo>K331030</partinfo><br />
*3)K331022 + B0015 to obtain <partinfo>K331034</partinfo><br />
*4)K331022 + K331024 to obtain (<font color="red">NAME BIOBRICK!!!!</font>)<br />
*5)R0010 + K331023 to obtain <partinfo>K331027</partinfo><br />
*6)R0040 + K331023 to obtain <partinfo>K331031</partinfo><br />
*7)K331023 + B0015 to obtain <partinfo>K331035</partinfo><br />
*8)K331023 + K331025 to obtain (<font color="red">NAME BIOBRICK!!!!</font>)<br />
*9)R0010 + K331024 to obtain <partinfo>K331028</partinfo><br />
*10)R0040 + K331024 to obtain <partinfo>K331032</partinfo><br />
*11)K331024 + B0015 to obtain <partinfo>K331036</partinfo><br />
*12)K331024 + K331022 to obtain (<font color="red">NAME BIOBRICK!!!!</font>)<br />
*13)R0010 + K331025 to obtain <partinfo>K331029</partinfo><br />
*14)R0040 + K331025 to obtain <partinfo>K331033</partinfo><br />
*15)K331025 + B0015 to obtain <partinfo>K331037</partinfo><br />
*16)K331025 + K331023 to obtain (<font color="red">NAME BIOBRICK!!!!</font>)<br />
*17)K331022 to obtain <partinfo>K331022</partinfo><br />
*18)K331023 to obtain <partinfo>K331023</partinfo><br />
*19)K331024 to obtain <partinfo>K331024</partinfo><br />
*20)K331025 to obtain <partinfo>K331025</partinfo><br />
All parts will be inserted into pSB1C3.<br><br />
<b>Method:</b> Use BioBrick Assembly Method <br><br />
<b>Results:</b> Obtained <font color="red">RESULTS!!!</font> positive colonies.<br />
----<br />
<br />
<br />
===<font color="white">HB===<br />
<br />
<b>Objective:</b> PCR mms6 from Mr. Gene to attach fusion standard to the N-term.<br><br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x6.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>12.8<td>83.2<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>13<br />
<tr><td>dNTPs<td>1<td>6.5<br />
<tr><td>mms6 Prefix Fusion Forward Primer<td>1<td>6.5<br />
<tr><td>Standard Suffix Reverse Primer<td>1<td>6.5<br />
<tr><td>Template DNA<td>2<td>13<br />
<tr><td>Pfu polymerase<td>0.2<td><br />
</table><br><br />
<br />
Prefix fusion sense primer has melting temperature of 56.1<sup>o</sup>C. Standard suffix primer has melting temperature of 61.3<sup>o</sup>C. <br />
<br />
PCR- Conditions:<br />
1. 95<sup>o</sup>C for 180 sec<br />
2. 95<sup>o</sup>C for 30 sec<br />
3. 56.3<sup>o</sup>C for 30 sec<br />
4. 72<sup>o</sup>C for 150 sec<br />
5. 72<sup>o</sup>C for 600 sec<br />
(35 cycles)<br />
<br />
Gradient - <br />
1. 54.3<sup>o</sup>C 2. 55.4<sup>o</sup>C 3. 56.0<sup>o</sup>C 4. 56.6<sup>o</sup>C 5. 57.8<sup>o</sup>C 6. 58.7<sup>o</sup>C<br />
<br />
----<br />
<br />
<b>Objective:</b> PCR mms6 from Mr. Gene to attach fusion standard to the C-term.<br><br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x6.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>12.8<td>83.2<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>13<br />
<tr><td>dNTPs<td>1<td>6.5<br />
<tr><td>Prefix Forward Primer<td>1<td>6.5<br />
<tr><td>mms6 Fusion Suffix Reverse Primer<td>1<td>6.5<br />
<tr><td>Template DNA<td>2<td>13<br />
<tr><td>Pfu polymerase<td>0.2<td><br />
</table><br><br />
<br />
Prefix fusion sense primer has melting temperature of 58.8<sup>o</sup>C. Standard suffix primer has melting temperature of 75<sup>o</sup>C. <br />
<br />
PCR- Conditions:<br />
1. 95<sup>o</sup>C for 180 sec<br />
2. 95<sup>o</sup>C for 30 sec<br />
3. 53.8<sup>o</sup>C for 30 sec<br />
4. 72<sup>o</sup>C for 150 sec<br />
5. 72<sup>o</sup>C for 600 sec<br />
(35 cycles)<br />
<br />
Gradient - <br />
1. 51.4<sup>o</sup>C 2. 52.3<sup>o</sup>C 3. 53.5<sup>o</sup>C 4. 54.1<sup>o</sup>C 5. 55.3<sup>o</sup>C 6. 56.2<sup>o</sup>C<br />
<br />
----<br />
===<font color="white">JV===<br />
<b>Objective:</b> Analyzed KG and HB's PCRs.<br><br />
<b>Objective:</b> Run samples on 2% agarose gel at 100V for 70 minutes.<br><br />
<br />
GEL PICTURE!!!<br />
<br />
==<font color="white">October 5, 2010==<br />
===<font color="white">ADS===<br />
<b>Objective:</b> Purify plasmid DNA from successfully assembled biobricks.<br><br />
<b>Method:</b> Use Qiagen miniprep method with BioBasic EZ-10 columns.<br><br />
Will send these minipreps for sequencing at Macrogen.<br />
<br />
<br />
<br />
<br />
<br><br />
<br />
==<font color="white">October 16, 2010==<br />
===<font color="white">JV===<br />
<br />
<b>Objective:</b> Separate different fractions of the cytosol of cells containing catechol 2,3-dioxygenase <br><br />
<b>Method:</b><br />
*Grow DH5&alpha; cells in 500mL of LB media with Amp<sup>+</sup>.<br />
*Optical density of cell suspension (measured at 600nm): 4.55<br />
*Spun cells down: 10 minutes, 4<sup>o</sup>C, at 5000RPM<br />
*Cell pellet weight is 3.03g<br />
*Flash froze cells and stored at -80<sup>o</sup>C <br><br />
<br />
Plasmid DNA was also isolated using QIAGEN spin column protocol. DNA was eluted to 60&micro;L.<br><br />
<br />
==<font color="white">October 19, 2010==<br />
===<font color="white">Js===<br />
<br />
<b>Objective</b> Ligate biobrick parts<br><br />
<b>Method</b> restrict and ligate into RFP plasmid using the biobrick standard method then transform cells<br />
*pLacI+mRBS<br />
*mRBS+Lum<br />
*Lum+dT<br />
*K118021+dT<br />
*pBad+mRBS<br />
*mRBS+TetR<br />
*TetR+dT <br />
<br />
** forgot to add RFP plasmid at ligation step<br />
*** repeated all protocol<br />
*** ligated the RFP plasmid into the ligated linear DNA as well<br />
<br />
==<font color="white">October 21, 2010==<br />
===<font color="white">Js,Jv===<br />
<br />
<b>Objective</b> Colony PCR for conformation of ligation<br />
<b>Method</b> pick white colonies and perform colony pcr<br />
*pick white colonies from plates<br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x24)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>6.8<td>261.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>77<br />
<tr><td>dNTPs<td>2<td>77<br />
<tr><td>Forward VF2 Primer<td>2<td>77<br />
<tr><td>Reverse VR Primer<td>2<td>77<br />
<tr><td>Template DNA<td>5<td><br />
<tr><td>Pfu polymerase<td>0.2<td>77<br />
</table><br><br />
<br />
PCR- Conditions:<br />
1. 95<sup>o</sup>C for 180 sec<br />
2. 95<sup>o</sup>C for 30 sec<br />
3. 53.8<sup>o</sup>C for 30 sec<br />
4. 72<sup>o</sup>C for 150 sec<br />
5. 72<sup>o</sup>C for 600 sec<br />
(35 cycles)<br />
<br><br />
<br />
ran a 2% agarose gel for conformation<br />
*no XYLE(K118021)-dT bands were observed, grew cells overnight.<br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/SeptemberTeam:Lethbridge/Notebook/Lab Work/September2010-10-27T19:19:00Z<p>Liszabruder: /* AV */</p>
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<BLOCKQUOTE><br />
=<font color="white">September=<br />
==<font color="white">September 2, 2010==<br />
===<font color="white">ADS===<br />
<b>Objective:</b> Assemble lumazine-dT into pSB1C3 using NEB BioBrick Assembly Kit.<br><br />
<b>Method:</b> Have the following parts: dT [28 ng/(&micro;L)]; lumazine [78 ng/(&micro;L)]; psB1C3 [14 ng/(&micro;L)].<br><br />
*Require 500 ng DNA each part in a 50(&micro;L) rxn therefore 17.9(&micro;L) dT and 6.4(&micro;L) Lumazine. Need 250 ng pSB1C3 or 17.9(&micro;L)<br />
<br />
Restriction Reactions - <br />
<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b></td><td>Volume(&micro;L)</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>36.1</td></tr><br />
<tr><td>NEBuffer 2 (10x)</td><td>5</td></tr><br />
<tr><td>Upstream Part (Lumazine)</td><td>26.4</td></tr><br />
<tr><td>EcoRI-HF</td><td>1</td></tr><br />
<tr><td>SpeI</td><td>1</td></tr><br />
<tr><td>100X BSA</td><td>0.5</td></tr><br />
</table><br />
<br />
<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b></td><td>Volume(&micro;L)</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>24.6</td></tr><br />
<tr><td>NEBuffer 2 (10x)</td><td>5</td></tr><br />
<tr><td>Downstream Part (dT)</td><td>17.9</td></tr><br />
<tr><td>XbaI</td><td>1</td></tr><br />
<tr><td>PstI</td><td>1</td></tr><br />
<tr><td>100X BSA</td><td>0.5</td></tr><br />
</table><br />
<br />
<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b></td><td>Volume(&micro;L)</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>3.35</td></tr><br />
<tr><td>NEBuffer 2 (10x)</td><td>2.5</td></tr><br />
<tr><td>Destination Plasmid (pSB1C3)</td><td>17.9</td></tr><br />
<tr><td>EcoRI-HF</td><td>.5</td></tr><br />
<tr><td>PstI</td><td>.5</td></tr><br />
<tr><td>100X BSA</td><td>0.25</td></tr><br />
</table><br />
<br />
<br />
* 2(&micro;L) was removed prior to adding enzymes for analysis on gel. <br />
** Split digestion reactions in half: dT - 2 @ 24(&micro;L); Lum - 2 @ 24(&micro;L); psB1C3 - 2 @ 11.5(&micro;L)<br />
*** Ran 10 minute and 60 minute reactions at 37<sup>o</sup>C. Followed by 20 minute heat shock at 80<sup>o</sup>C. <br />
**** 2(&micro;L) was removed from the heat killed reactions for analysis on gel.<br />
<br />
---- <br />
<br />
* Performed 4 combinations of ligation: 10 min Restriction + 10 min Ligation; 10 min Restriction + Overnight Ligation; 60 min Restriction + 10 min Ligation; 60 min Restriction + Overnight Ligation<br />
<br />
<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>2X Master Mix(x2.5)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>11<td>77.5<br />
<tr><td>T4 Ligase Buffer (10x)</td><td>2<td>5<br />
<tr><td>Upstream Part</td><td>2<td>5<br />
<tr><td>Downstream Part </td><td>2<td>5<br />
<tr><td>T4 DNA Ligase</td><td>1<td>2.5<br />
<tr><td>Plasmid Part</td><td>2<td>5<br />
</table><br />
<br />
Incubated 10 minute and overnight ligations at room temperature ( 25<sup>o</sup>C). Heat killed ligase at 80<sup>o</sup>C for 20 min. <br />
* 2(&micro;L) of each reaction was taken for analysis on gel. <br />
<br />
----<br />
<br />
* Confirmed ligation with PCR analysis. Analyzed restriction, ligation and PCR on a 1.5% TAE agarose gel. <br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x5.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>36.5<td>202.4<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>27.5<br />
<tr><td>dNTPs<td>2<td>11<br />
<tr><td>Forward VF2 Primer<td>2<td>11<br />
<tr><td>Reverse VR Primer<td>2<td>11<br />
<tr><td>Template DNA<td>2<td><br />
<tr><td>Pfu polymerase<td>0.2<td>1.1<br />
</table><br><br />
<br />
Added 48(&micro;L) to each reaction. <br><br />
[[image:Lethbridge_100903AssemblyADS.jpg|200px]]<br />
<br />
----<br />
<b>Objective:</b> Characterize xylE in LB Media vs. M9 Minimal Media.<br><br />
<b>Method:</b> Three experiments with spectra. <br><br />
<br />
* Experiment 1 - Measure absorption spectra of 1:10 dilution of: 1. Cells suspended in m9 media +/- catechol. 2. Cells suspended in LB media +/- catechol. 3. m9 media from spun down cells +/- catechol. 4. LB media from spun down cells +/- catechol. 5. Sterile LB media +/- catechol. 6. Water +/- catechol. <br />
<br />
* Experiment 2 - Catechol Breakdown. Follow absorbance of 2-hydroxymucanate semialdehyde at 375 nm. Measured all the above samples for 10 min at that wavelength.<br />
<br />
==<font color="white">September 14, 2010==<br />
===<font color="white">JF===<br />
<b>Objective:</b> Miniprep of RFP expression construct (J04450) via Qiaquick/Qiaprep spin column. Protocol followed as per kit instructions.<br><br />
<br />
----<br />
===<font color="white">JV===<br />
<br />
<b>Objective:</b> PCR the pSB1C3 plasmid from part J04450 miniprep.<br><br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>12.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<br />
<tr><td>dNTPs<td>1<br />
<tr><td>SB-prep-26 Primer<td>1<br />
<tr><td>SB-prep-3P Primer<td>1<br />
<tr><td>Template DNA<td>2<br />
<tr><td>Pfu polymerase<td>0.2<br />
</table><br><br />
<br />
Used iGEM cycle PSB1C3 in thermocycler.<br />
Ran PCR product on 1% TAE agarose gel. <br />
<br />
----<br />
===<font color="white">KG===<br />
<br />
<b>Objective:</b> PCR of xylE (mRBS-xylE K118021) with fusion standard so that an arginine tail can be attached.<br><br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x6)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>12.8<td>76.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>12<br />
<tr><td>dNTPs<td>1<td>6<br />
<tr><td>xylE Fusion Prefix Forward Primer<td>1<td>6<br />
<tr><td>xylE Fusion Suffix Reverse Primer<td>1<td>6<br />
<tr><td>Template DNA<td>2<td>12<br />
<tr><td>Pfu polymerase<td>0.2<td>1.2<br />
</table><br><br />
<br />
PCR- Conditions:<br />
1. 95<sup>o</sup>C for 180 sec<br />
2. 95<sup>o</sup>C for 30 sec<br />
3. 56.6 +/- 5<sup>o</sup>C for 30 sec<br />
4. 72<sup>o</sup>C for 150 sec<br />
5. 72<sup>o</sup>C for 600 sec<br />
(35 cycles)<br />
<br />
Analyzed PCR on 2% agarose gel which ran at 100 V for 90 minutes.<br><br />
[[image:100915PCR AV KG JV.jpg|200px]]<br />
<br />
===<font color="white">AS===<br />
<b>Objective:</b> Assemble xylE-dT using 3AB assembly and 3AB assembly.<br><br />
<br />
Restriction Reactions - <br />
<br />
3AB Upstream Part (xylE)<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b></td><td>Volume(&micro;L)</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>30.6</td></tr><br />
<tr><td>NEBuffer 2 (10x)</td><td>5</td></tr><br />
<tr><td>Plasmid DNA </td><td>11.9</td></tr><br />
<tr><td>EcoRI-HF</td><td>1</td></tr><br />
<tr><td>SpeI</td><td>1</td></tr><br />
<tr><td>100X BSA</td><td>0.5</td></tr><br />
</table><br />
<br />
3AB Downstream Part (dT)<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b></td><td>Volume(&micro;L)</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>24.6</td></tr><br />
<tr><td>NEBuffer 2 (10x)</td><td>5</td></tr><br />
<tr><td>Plasmid DNA</td><td>17.9</td></tr><br />
<tr><td>XbaI</td><td>1</td></tr><br />
<tr><td>PstI</td><td>1</td></tr><br />
<tr><td>100X BSA</td><td>0.5</td></tr><br />
</table><br />
<br />
3AB Plasmid (pSB1C3)<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b></td><td>Volume(&micro;L)</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td></td></tr><br />
<tr><td>NEBuffer 2 (10x)</td><td>5</td></tr><br />
<tr><td>Plasmid DNA</td><td>42.5</td></tr><br />
<tr><td>EcoRI-HF</td><td>1</td></tr><br />
<tr><td>PstI</td><td>1</td></tr><br />
<tr><td>100X BSA</td><td>0.5</td></tr><br />
</table><br />
<br />
*Ran 10 minute and 60 minute reactions at 37<sup>o</sup>C. Followed by 20 minute heat shock at 80<sup>o</sup>C. <br />
<br />
Ligation Reaction -<br />
<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>11<br />
<tr><td>T4 Ligase Buffer (10x)</td><td>2<br />
<tr><td>Upstream Part</td><td>2<br />
<tr><td>Downstream Part </td><td>2<br />
<tr><td>T4 DNA Ligase</td><td>1<br />
<tr><td>Plasmid Part</td><td>2<br />
</table><br />
<br />
Incubated 10 minute and overnight ligations at room temperature ( 25<sup>o</sup>C). Heat killed ligase at 80<sup>o</sup>C for 20 min. <br />
<br />
Transformation - <br />
<br />
A) Thawed 200(&micro;L) Library Efficiency DH5alpha Competent Cells on ice.<br />
B) Gently mixed cells and then aliquoted 100(&micro;L) into chilled polypropylene tubes.<br />
C) Added 1(&micro;L) of ligation mix to cells. Added 5(&micro;L) of pUC19 DNA to 100(&micro;L) cells to determine efficiency. <br />
D) Incubated cells on ice for 30 minutes.<br />
E) Heat shocked cells for 45 seconds in a 42<sup>o</sup>C water bath.<br />
F) Placed on ice for 2 minutes.<br />
G) Added 0.9 mL of room temperature SOC medium.<br />
H) Shook at 225 rpm for 1 hour.<br />
I) Diluted control cells 1:100 with SOC medium.<br />
J) Spread 100(&micro;L) of this dilution on LB-Amp agar plates<br />
K) Spread 50 and 250(&micro;L) of experimental cells on LB-Cam agar plates. <br />
L) Incubated overnight at 37<sup>o</sup>C<br />
<br />
==<font color="white">September 15, 2010==<br />
===<font color="white"> JV, ADS===<br />
<b>Objective:</b> Determine if dT ligated onto p-rbs-xylE using PCR analysis.<br><br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x4.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>11.8<td>53.1<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>9<br />
<tr><td>dNTPs<td>1<td>4.5<br />
<tr><td>Forward VF2 Primer<td>1<td>4.5<br />
<tr><td>Reverse VR Primer<td>1<td>4.5<br />
<tr><td>Template DNA<td>1<td><br />
<tr><td>Pfu polymerase<td>0.2<td><br />
</table><br><br />
<br />
Used iGEM program 7 in thermocycler.<br />
<br />
----<br />
<b>Objective:</b> Determine if minipreps from Sept 15, 2010 worked. Analyze assembly intermediates. <br><br />
<b>Method:</b> Run 1% TAE agarose gels at 100 V for 90 minutes. <br><br />
<br />
[[image:100914ADSAssemblyPrep.jpg|100px]]<br />
<br />
==<font color="white">September 16, 2010==<br />
===<font color="white"> JV===<br />
<b>Objective:</b> Isolate RFP in pSB1C3 from DH5alpha cells.<br><br />
<b>Method:</b> Used Qiagen minipep protocol and spin column.<br><br />
<b>Results:</b> Gel of miniprepped RFP against previously prepared RFP showed a band of the right size.<br><br />
<br />
[[image:100916 JV RFP BW.jpg|50px]]<br />
<br />
==<font color="white">September 17, 2010==<br />
===<font color="white"> JV===<br />
<b>Objective:</b> Isolate plasmid DNA from DH5alpha cells: K249024; B0015; K118021/B0015<br><br />
<b>Method:</b> Used Qiagen minipep protocol and spin column. Ran a 1% agarose gel to view DNA<br><br />
<br />
GEL PICTURE!<br />
----<br />
===<font color="white">AV===<br />
<br />
<b>Objective:</b> PCR out RFP from the complete construct (J04450) with a N-term fusion standard.<br><br />
<br />
Composition of each PCR tube:<br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td>Volume(&micro;L)</td></tr><br />
<tr><td>10x Pfu Buffer w/ MgSO<sub>4</sub></td><td>2</td></tr><br />
<tr><td>dNTP (10mM)</td><td>1</td></tr><br />
<tr><td>N-term Fus Prefix</td><td>1</td></tr><br />
<tr><td>Biobrick Suffix</td><td>1</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>O</td><td>12.8</td></tr><br />
<tr><td>Template DNA (J04450)</td><td>2</td></tr><br />
<tr><td>Pfu DNA Polymerase</td><td>0.2</td></tr><br />
</table><br />
<br />
PCR conditions:<br />
<table><table border ="3"><br />
<tr><td><b>Step</b></td><td><b>Temperature (<sup>o</sup>C)</b></td><td><b>Time (mins)</b></td><td><b>Number of cycles</b></td></tr><br />
<tr><td>Initial Denaturation</td><td>95</td><td>2</td><td>1</td></tr><br />
<tr><td>Denaturation</td><td>95</td><td>0.5</td><td>25</td></tr><br />
<tr><td>Annealing</td><td>48.2, 52.4, 56.0 (gradient)</td><td>0.5</td><td>25</td></tr><br />
<tr><td>Extension</td><td>72</td><td>2</td><td>25</td></tr><br />
<tr><td>Final Extension</td><td>72</td><td>10</td><td>1</td></tr><br />
</table><br />
<br />
[[image:100915PCR AV KG JV.jpg|200px]]<br />
<br />
Results: Amplification showing in gel, but fragment amplified was ~1000bp. RFP and dT should be ~800bp. Primers were checked and discovered the YFP/CFP primers were not compatible with the RFP. Concluded the amplification resulted from sloppy annealing of N-term fus prefix to the bio prefix resulting in the full construct being amplified (~1000bp)<br />
----<br />
<br />
===<font color="white">KG===<br />
<br />
<b>Objective:</b> PCR xylE to attach fusion standard to the N-term and standard suffix to C-term.<br><br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x6)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>12.8<td>76.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>12<br />
<tr><td>dNTPs<td>1<td>6<br />
<tr><td>Fusion Forward Primer<td>1<td>6<br />
<tr><td>Standard Reverse Primer<td>1<td>6<br />
<tr><td>Template DNA<td>2<td>12<br />
<tr><td>Pfu polymerase<td>0.2<td>1.2<br />
</table><br><br />
<br />
Fusion standard prefix has melting temperature of 66.5<sup>o</sup>C. Standard suffix has melting temperature of 70.5<sup>o</sup>C. <br />
Annealing temperature of 61.5<sup>o</sup>C with 5 <sup>o</sup>C gradient. <br />
PCR- Conditions:<br />
1. 95<sup>o</sup>C for 180 sec<br />
2. 95<sup>o</sup>C for 30 sec<br />
3. 61.5 +/- 5<sup>o</sup>C for 30 sec<br />
4. 72<sup>o</sup>C for 150 sec<br />
5. 72<sup>o</sup>C for 600 sec<br />
(35 cycles)<br />
<br />
----<br />
<b>Objective:</b> PCR xylE to attach fusion standard to the C-term and standard suffix to N-term.<br><br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x6)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>12.8<td>76.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>12<br />
<tr><td>dNTPs<td>1<td>6<br />
<tr><td>Standard Forward Primer<td>1<td>6<br />
<tr><td>Fusion Reverse Primer<td>1<td>6<br />
<tr><td>Template DNA<td>2<td>12<br />
<tr><td>Pfu polymerase<td>0.2<td>1.2<br />
</table><br><br />
<br />
Standard prefix has melting temperature of 68.7<sup>o</sup>C. Standard suffix has melting temperature of 61.6<sup>o</sup>C. <br />
Annealing temperature of 56.6<sup>o</sup>C with 5 <sup>o</sup>C gradient. <br />
<br />
PCR- Conditions:<br />
1. 95<sup>o</sup>C for 180 sec<br />
2. 95<sup>o</sup>C for 30 sec<br />
3. 56.6 +/- 5<sup>o</sup>C for 30 sec<br />
4. 72<sup>o</sup>C for 150 sec<br />
5. 72<sup>o</sup>C for 600 sec<br />
(35 cycles)<br />
<br />
==<font color="white">September 18, 2010==<br />
===<font color="white">JS, DM===<br />
<b>Objective:</b> Overexpress mms6.<br><br />
Method: Followed overexpression protocol. One flask was induced with 250(&micro;L) IPTG while another was induced with 50(&micro;L).<br />
<br />
<table><table border ="3"><br />
<tr><td><b>Time (minutes)</b></td><td><b>OD #1 (600&lambda;)</b></td><td><b>OD #2 (600&lambda;)</b></td></tr><br />
<tr><td>30</td><td>0.139</td><td>0.133</td></tr><br />
<tr><td>60</td><td>0.200</td><td>0.188</td></tr><br />
<tr><td>90</td><td>0.372</td><td>0.360</td></tr><br />
<tr><td>120</td><td>0.702</td><td>0.704</td></tr><br />
<tr><td>150(T<sub>0</sub>)</td><td>0.871</td><td>0.810</td></tr><br />
<tr><td>210(T<sub>1</sub>)</td><td>2.600</td><td>2.500</td></tr><br />
<tr><td>270(T<sub>2</sub>)</td><td>3.317</td><td>3.300</td></tr><br />
<tr><td>330(T<sub>3</sub>)</td><td>4.408</td><td>3.946</td></tr><br />
</table><br><br />
<br />
* Plated cells in presence of IPTG, Fe or IPTG and Fe on AMP(+) plates. Incubated at 37 <sup>o</sup>C. <br />
** Preparation of these samples was achieved by taking a 60(&micro;L) cell sample and inoculating 5 mL LB media. These solutions were incubated at 37 <sup>o</sup>C for 30 min. To 1 mL of culture, the appropriate amount of Fe2+ [0.938(&micro;L)], Fe3+[1.88(&micro;L)] or IPTG [1(&micro;L)] was added.<br />
----<br />
===<font color="white">TF===<br />
<br />
<b>Objective:</b> Obtain preparations of B0015 (dT) and J04450 (RFP).<br />
<br />
<b>Method:</b> Used [[Team:Lethbridge/Notebook/Protocols|Plasmid DNA Purification by Alkaline Lysis (Large Scale AKA Maxiprep]] protocol.<br />
<br />
<b>Results:</b> Chloroform, DNA and isopropanol were mixed and therefore no products could be recovered.<br />
----<br />
<br />
<br />
<br />
==<font color="white">September 20, 2010==<br />
===<font color="white">ADS===<br />
<b>Objective:</b> Analyze PCR of mms6 (HB) and fluorescent proteins (AV).<br><br />
<b>Method:</b> Analyzed on 2% TAE agarose gel.<br><br />
Load order:<br><br />
<table><table border ="3"><br />
<tr><td><b>Lane</b></td><td><b>Sample</b></td><td><b>Volume<br>Sample (&micro;L)</b></td><td><b>Volume Loading<br>Dye (&micro;L)</b></td></tr><br />
<tr><td>1</td><td>HB1</td><td>10</td><td>2</td></tr><br />
<tr><td>2</td><td>HB2</td><td>10</td><td>2</td></tr><br />
<tr><td>3</td><td>HB3</td><td>10</td><td>2</td></tr><br />
<tr><td>4</td><td>HB4</td><td>10</td><td>2</td></tr><br />
<tr><td>5</td><td>HB5</td><td>10</td><td>2</td></tr><br />
<tr><td>6</td><td>100bp Ladder (Fermentas)</td><td>0.5</td><td>2(+10 H<sub>2</sub>0)</td></tr><br />
<tr><td>7</td><td>RFP1</td><td>10</td><td>2</td></tr><br />
<tr><td>8</td><td>RFP2</td><td>10</td><td>2</td></tr><br />
<tr><td>9</td><td>RFP3</td><td>10</td><td>2</td></tr></table><br><br />
<b>Results:</b> <br><br />
[[image:100920.jpg|100px]]<br />
*mms6 was not amplified<br />
*RFP was amplified<br />
**Expected size is ~800bp<br />
**Actual size is >1000bp<br />
***We believe that the suffix segment of the fusion primer annealed rather than the FP segment. This would cause the promoter (R0040) of J04450 to be amplified, adding ~200bp, giving a final size of >1000bp.<br />
----------------------------------------------------------------------------------------------------------------------<br />
<b>Objective:</b> Confirm assembly (3 antibiotic) of K118021 and B0015 from Sept. 14, 2010 via PCR.<br><br />
<b>Method:</b> Set up 50uL reaction mixture with 2uL of ligated DNA. Used VF2 and VR primers.<br><br />
Used PFU setting on Thermocycler<br><br />
<b>Results:</b> <br><br />
[[image:100915Assembly.jpg|100px]]<br />
<b>Conclusion:</b> Assembly did not work as intended.<br />
----<br />
<br />
===<font color="white">HB===<br />
<br />
<b>Objective:</b> PCR mms6 to attach fusion standard to the N-term and standard suffix to C-term.<br><br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x6)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>12.8<td>76.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>12<br />
<tr><td>dNTPs<td>1<td>6<br />
<tr><td>Fusion Forward Primer<td>1<td>6<br />
<tr><td>Standard Reverse Primer<td>1<td>6<br />
<tr><td>Template DNA<td>2<td>12<br />
<tr><td>Pfu polymerase<td>0.2<td>1.2<br />
</table><br><br />
<br />
Prefix fusion sense primer has melting temperature of 56.1<sup>o</sup>C. Standard suffix primer has melting temperature of 61.3<sup>o</sup>C. <br />
<br />
PCR- Conditions:<br />
1. 95<sup>o</sup>C for 180 sec<br />
2. 95<sup>o</sup>C for 30 sec<br />
3. 51.1<sup>o</sup>C for 30 sec<br />
4. 72<sup>o</sup>C for 150 sec<br />
5. 72<sup>o</sup>C for 600 sec<br />
(35 cycles)<br />
<br />
Program HBPREFU:<br />
1. 49.1<sup>o</sup>C 2. 50.2<sup>o</sup>C 3. 51.4<sup>o</sup>C 4. 52.6<sup>o</sup>C 5. 53.5<sup>o</sup>C<br />
<br />
==<font color="white">September 21, 2010==<br />
===<font color="white">ADS===<br />
<b>Objective:</b> Insert xylE (with N and C terminal fusion standards, obtained by PCR of K118021 by KG) into pSB1C3 plasmid for submission to registry.<br><br />
<br />
<b>Method:</b><br />
*Restriction of xylE PCR product and pSB1C3 (containing J04450 biobrick) via BioBrick Method using EcoRI-HF and PstI (Enzymes from NEB)<br />
*Ligation of xylE PCR product and pSB1C3 via BioBrick Method using T4 DNA ligase (Enzyme from NEB)<br />
*Transform into Library Efficiency Compentent DH5&alpha; Cells (Invitrogen)<br />
<br />
<b>Results:</b><br />
*Obtained too numerous to count (TNTC) colonies. <br />
**Obtained ~100 white colonies (indicating removal of RFP and insertion of new BioBrick)<br />
<br />
<b>Note:</b><br><br />
Parent plasmid (from PCR) not digested, possibly K118021 moved into pSB1C3 backbone.<br />
<br />
<b>Follow-up:</b><br><br />
Screen white colonies by addition of catechol to solution containing white cells.<br />
----<br />
===<font color="white">TF, MC===<br />
<br />
<b>Objective:</b> Obtain a preparation of B0015 (dT).<br />
<br />
<b>Method:</b> Used [[Team:Lethbridge/Notebook/Protocols|Plasmid DNA Purification by Alkaline Lysis (Large Scale AKA Maxiprep]] protocol.<br />
----<br />
===<font color="white">AV===<br />
<b>Objective:</b> PCR amplify the signal sequences synthesized by Mr. Gene (K331007, K331008, K331009, K331012).<br><br />
<br />
Composition of each PCR tube:<br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume(&micro;L)</b></td></tr><br />
<tr><td>10x Pfu Buffer w/ MgSO<sub>4</sub></td><td>2</td></tr><br />
<tr><td>dNTP (10mM)</td><td>1</td></tr><br />
<tr><td>VF2</td><td>1</td></tr><br />
<tr><td>VR</td><td>1</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>O</td><td>12.8</td></tr><br />
<tr><td>Template DNA</td><td>2</td></tr><br />
<tr><td>Pfu DNA Polymerase</td><td>0.2</td></tr><br />
</table><br><br />
Used iGEM thermocycler setting: PFU.<br><br />
<br />
==<font color="white">September 21, 2010==<br />
===<font color="white">ADS===<br />
<b>Objective:</b> Analyze PCR of K118021-B0015 and subsequent PCR (ADS) and PCR of <partinfo>K118021</partinfo> to add either N or C terminal fusion standard (KG).<br><br />
<b>Method:</b> Analyze on 2% TAE agarose gel.<br><br />
Load Order:<br><br />
<table><table border ="3"><br />
<tr><td><b>Lane</b></td><td><b>Sample</b></td><td><b>Volume<br>Sample (&micro;L)</b></td><td><b>Volume Loading<br>Dye (&micro;L)</b></td></tr><br />
<tr><td>1</td><td>xylE F-S 1</td><td>5</td><td>1</td></tr><br />
<tr><td>2</td><td>xylE F-S 2</td><td>5</td><td>1</td></tr><br />
<tr><td>3</td><td>xylE F-S 3</td><td>5</td><td>1</td></tr><br />
<tr><td>4</td><td>xylE F-S 4</td><td>5</td><td>1</td></tr><br />
<tr><td>5</td><td>xylE F-S 5</td><td>5</td><td>1</td></tr><br />
<tr><td>6</td><td>xylE F-S 6</td><td>5</td><td>1</td></tr><br />
<tr><td>7</td><td>100bp Ladder (NEB)</td><td>0.5</td><td>1(+5 H<sub>2</sub>0)</td></tr><br />
<tr><td>8</td><td>xylE S-F 1</td><td>5</td><td>1</td></tr><br />
<tr><td>9</td><td>xylE S-F 2</td><td>5</td><td>1</td></tr><br />
<tr><td>10</td><td>xylE S-F 3</td><td>5</td><td>1</td></tr><br />
<tr><td>11</td><td>xylE S-F 4</td><td>5</td><td>1</td></tr><br />
<tr><td>12</td><td>xylE S-F 5</td><td>5</td><td>1</td></tr><br />
<tr><td>13</td><td>xylE S-F 6</td><td>5</td><td>1</td></tr><br />
<tr><td>14</td><td>Empty</td><td></td><td></td></tr><br />
<tr><td>15</td><td>PCR of K118021-B0015 (ADS)</td><td>1</td><td>1</td></tr><br />
<tr><td>16</td><td>Empty</td><td></td><td></td></tr><br />
<tr><td>17</td><td>Empty</td><td></td><td></td></tr></table><br><br />
<b>Results:</b> <br><br />
[[image:100921.jpg|200px]]<br />
*Both KG PCR (Standard-Fusion and Fusion-Standard) amplified<br />
*ADS PCR Amplified, but no insert present.<br />
<br />
==<font color="white">September 23, 2010==<br />
===<font color="white">JV===<br />
<b>Objective:</b> Characterized catechol degradation by xylE enzyme<br><br />
<br />
<b>Method:</b> Measured absorbance of catechol (275nm) and 2-hydroxymuconate semialdehyde (380nm).<br><br />
<br />
*<b>Protocol:</b><br />
*1) Grow cells in M9 minimal medium<br />
*2) Take 1/10 dilution of cells<br />
*3) Introduce 1&micro;L of 0.05M catechol solution into the cell dilution. (Final concentration of 50&microM;).<br />
*4) Quench the reaction with 5A% w/v trichloroacetate at certain time points. (0,15sec, 30sec, 45sec, 60sec, 2min, 3min, 4min, 5min, 10min).<br />
*5) Spin down cells.<br />
*6) Measure absorbance of supernatant.<br><br />
<br />
<b>Results: Cuvette used interfered with Spectra. </b><br />
<br />
===<font color="white">ADS===<br />
<b><font size="+1">NOTE:</font></b> In all transformations, heat shock step was missed. HOWEVER, all transformations showed significant number of colony forming units.<br><br />
<b>Objective:</b> Move xylE (two biobrick; one with Fusion prefix, one with fusion suffix) into pSB1C3.<br><br />
<b>Method:</b><br />
*Restriction of xylE PCR product and pSB1C3 (containing J04450 biobrick) via BioBrick Method using EcoRI-HF and PstI (Enzymes from NEB)<br />
*Ligation of xylE PCR product and pSB1C3 via BioBrick Method using T4 DNA ligase (Enzyme from NEB) <br />
**Incubated 30 min at RT<br />
*Transform into Subcloning Efficiency Compentent DH5&alpha; Cells (Invitrogen)<br />
<b>Results:</b> TBD <br><br />
<b>Follow-up:</b> TBD<br><br />
----------------------------------------------------------------------------------------------------------------------<br />
<b>Objective:</b> Create glycerol stocks of <partinfo>J04450</partinfo> in pSB1A3 and pSB1T3 for use in RFP-BioBrick Assembly.<br><br />
<b>Method:</b> Transform into Subcloning Efficiency Competent DH5&alpha; Cells (Invitrogen)<br><br />
Obtained all plasmid DNA from 2010 Kit Plate 1<br />
*J04450 in pSB1A3 - Well 1C<br />
*J04450 in pSB1T3 - Well 7A<br />
<b>Results:</b> Obtained TNTC colonies<br><br />
<b>Follow-up:</b> <br />
*Grow overnight cultures<br />
*Generate Glycerol Stocks<br />
*Generate Plasmid DNA via Maxiprep<br />
----------------------------------------------------------------------------------------------------------------------<br />
<b>Objective:</b> Create glycerol stocks of received synthesized (Mr. Gene) signal peptides.<br><br />
<b>Method:</b> Transform into Subcloning Efficiency Competent DH5&alpha; Cells (Invitrogen) plasmid DNA containing the following BioBricks:<br />
*1) <partinfo>K331007</partinfo> - &beta;-lactamase Bla Signal Sequence<br />
*2) <partinfo>K331008</partinfo> - Outer Membrane Protein ompA<br />
*3) <partinfo>K331009</partinfo> - Heat Stable Toxin I<br />
*4) <partinfo>K331012</partinfo> - Penicillin Binding Protein DacA<br />
*All inserts in pMA-T vector (Standard Mr. Gene vector)<br />
<b>Results:</b> Obtained TNTC Cells <br><br />
<b>Follow-up:</b> <br />
*1) Grow overnight cultures<br />
*2) Purify pDNA<br />
*3) Move into pSB1C3 plasmid<br />
*4) Verify sequence <br />
*5) Submit to registry for sequencing<br />
----<br />
===<font color="white">TF, MC===<br />
<br />
<b>Objective:</b> Obtain a preparation of J04450 (RFP).<br><br />
<br />
<b>ethod:</b> Used [[Team:Lethbridge/Notebook/Protocols|Plasmid DNA Purification by Alkaline Lysis (Large Scale AKA Maxiprep]] protocol.<br />
<br />
==<font color="white">September 24, 2010==<br />
===<font color="white">ADS===<br />
<b>Objective:</b> Generate plasmid DNA of <partinfo>E1010</partinfo> for downstream PCR<br><br />
<b>Method:</b> Transform plasmid DNA into Subcloning Efficiency Competent DH5&alpha; Cells (Invitrogen)<br><br />
DNA obtained from 2010 Kit Plate 1 Well 18F (E1010 in pSB2K3)<br><br />
<b>Results:</b> Obtained <font color="red"> TBD</font> colonies<br><br />
<b>Follow-up:</b><br />
*Grow overnight cultures (Generate glycerol stocks)<br />
*Purify plasmid DNA (Generate pDNA stocks)<br />
*PCR to add terminal fusion standards<br />
----------------------------------------------------------------------------------------------------------------------<br />
<b>Objective:</b> Create glycerol stocks of J04450 in pSB1K3 for use in RFP-BioBrick Assembly.<br><br />
<b>Method:</b> Transform into Subcloning Efficiency Competent DH5&alpha; Cells (Invitrogen)<br><br />
Obtained plasmid DNA from 2010 Kit Plate 1 well 5A (J04450 in pSB1K3)<br><br />
<b>Results:</b> Obtained TNTC colonies<br><br />
<b>Follow-up:</b> <br />
*Grow overnight cultures<br />
*Generate Glycerol Stocks<br />
*Generate Plasmid DNA via Maxiprep<br />
<br />
==<font color="white">September 25, 2010==<br />
===<font color="white">JV===<br />
<b>Objective:</b> Extract Plasmid DNA from DH5&alpha; cells.<br><br />
<br />
<b>Method:</b>Qiagen spin column protocol.<br><br />
<br />
*<partinfo>K331007</partinfo> (in pMA-T vector)<br />
*<partinfo>K331008</partinfo> (in pMA-T vector)<br />
*<partinfo>K331009</partinfo> (in pMA-T vector)<br />
*<partinfo>K331012</partinfo> (in pMA-T vector)<br><br />
<br />
Cells containing plasmids were put into glycerol stocks and put into HJ's -80<sup>o</sup>C.<br><br />
<br />
===<font color="white">ADS===<br />
<b>Objective:</b> Move plasmid DNA made by JV (above, Sept 25, 2010) into pSB1C3 for submission to the Standard Registry of Parts.<br><br />
<b>Method:</b><br />
*1) Digest at 37<sup>o</sup> for 10 min with PstI and EcoRI:<br />
**pSB1C3 containing <partinfo>J04450</partinfo><br />
**pMA-T with <partinfo>K331007</partinfo>, <partinfo>K331008</partinfo>, <partinfo>K331009</partinfo>, and <partinfo>K331012</partinfo>.<br />
*2) Heat Kill at 80<sup>o</sup>C for 20 min<br />
*3) Mix 2&micro;L of pSB1C3 with each biobrick, ligate with T4 DNA ligase for 10 min<br />
*4) Transform into subcloning efficiency DH5&alpha; cells, plate on ampicillin plates.<br><br />
<b>Results:</b> Obtained <font color="red">RESULT!!!</font color> positive colonies<br><br />
----------------------------------------------------------------------------------------------------------------------<br />
<b>Objective:</b> Assemble KG's C-terminal fusion xylE (<partinfo>K331021</partinfo>) (<font color="red">UNCONFIRMED</font>) and C-terminal Arginine + dT (<partinfo>S04261</partinfo>) to create <partinfo>K331011</partinfo>. <br><br />
<b>Method:</b><br />
*1) Digest at 37<sup>o</sup> for 10 min:<br />
**pSB1C3 containing <partinfo>J04450</partinfo> with PstI and EcoRI<br />
**<partinfo>K331021</partinfo> with EcoRI, SpeI<br />
**<partinfo>S04261</partinfo> with XbaI and PstI<br />
*2) Heat Kill at 80<sup>o</sup>C for 20 min<br />
*3) Mix 2&micro;L of pSB1C3 with each biobrick, ligate with T4 DNA ligase for 10 min<br />
*4) Transform into subcloning efficiency DH5&alpha; cells, plate on ampicillin plates.<br><br />
<b>Results:</b> Obtained <font color="red">RESULT!!!</font color> positive colonies<br><br />
<br />
==<font color="white">September 26, 2010==<br />
===<font color="white">ADS===<br />
<b>Objective:</b> Purify plasmid DNA from 5mL cultures grown from transformations of KG's ligated PCRs containing (<font color="red">UNCONFIRMED</font>) <partinfo>K331020</partinfo> and <partinfo>K331021</partinfo>.<br><br />
<b>Method:</b> Used Qiagen spin column method with BioBasic EZ-10 spin columns.<br><br />
------------------------------------------------------------------------------------------------------------------------<br />
<b>Objective:</b> Assemble the following:<br><br />
*<partinfo>K331020</partinfo> + <partinfo>B0015</partinfo> to obtain <font color="red"> To be named</font> biobrick<br />
*<partinfo>K331021</partinfo> + <partinfo>S04261</partinfo> to obtain <font color="red"> To be named</font> biobrick<br />
<b>Method:</b><br> <br />
*Digest <partinfo>K331020</partinfo> and <partinfo>K331021</partinfo> with EcoRI and SpeI<br />
*Digest <partinfo>B0015</partinfo> and <partinfo>S04261</partinfo> with XbaI and PstI<br />
*Digest <partinfo>J04450</partinfo> in pSB1C3 with EcoRI and PstI<br />
Incubate each at 37<sup>o</sup>C for 10 min<br><br />
Heat kill at 80<sup>o</sup>C for 20 min <br><br />
Ligate with T4 Ligase for 10 min at room temperature<br><br />
Transform into DH5&alpha subcloning efficiency cells<br><br />
<b>Results:</b> Obtained <font color="red">RESULTS!!!!</font color> positive colonies.<br><br />
Also analyzed minipreps on 1.5% TAE Agarose Gel<br><br />
[[image:100926Minipreps.jpg|200px]]<br />
<br />
==<font color="white">September 29, 2010==<br />
===<font color="white">ADS===<br />
Received synthesized parts from Mr. GENE (all in pMA vectors)<br><br />
*<partinfo>K331022</partinfo><br />
*<partinfo>K331023</partinfo><br />
*<partinfo>K331024</partinfo><br />
*<partinfo>K331025</partinfo><br />
Each tube contained 5&micro;g pDNA; reconstitute in 50&micro;L of TE buffer to get 100ng/&micro;L concentration<br><br />
Transform 1&micro;L into DH5&alpha; subcloning efficiency cells<br><br />
Obtained TNTC colonies<br><br />
Started overnight 5mL cultures for miniprep and insertion into pSB1C3 vector.<br />
<br />
----<br />
===<font color="white">AV===<br />
<br />
<b>Objective:</b> PCR the N-term fusion tag onto RFP (E1010) using sloppy annealing of the YFP/CFP fusion primers. <br><br><br />
<br />
Composition of each PCR tube:<br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td>Volume(&micro;L)</td></tr><br />
<tr><td>10x Pfu Buffer w/ MgSO<sub>4</sub></td><td>2</td></tr><br />
<tr><td>dNTP (10mM)</td><td>1</td></tr><br />
<tr><td>N-term Fus Prefix</td><td>1</td></tr><br />
<tr><td>Biobrick Suffix</td><td>1</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>O</td><td>12.8</td></tr><br />
<tr><td>Template DNA (J04450)</td><td>2</td></tr><br />
<tr><td>Pfu DNA Polymerase</td><td>0.2</td></tr><br />
</table><br><br />
<br />
PCR conditions:<br />
<table><table border ="3"><br />
<tr><td><b>Step</b></td><td><b>Temperature (<sup>o</sup>C)</b></td><td><b>Time (mins)</b></td><td><b>Number of cycles</b></td></tr><br />
<tr><td>Initial Denaturation</td><td>95</td><td>2</td><td>1</td></tr><br />
<tr><td>Denaturation</td><td>95</td><td>0.5</td><td>25</td></tr><br />
<tr><td>Annealing</td><td>47.6, 50.5, 53.8 (gradient)</td><td>0.5</td><td>25</td></tr><br />
<tr><td>Extension</td><td>72</td><td>2</td><td>25</td></tr><br />
<tr><td>Final Extension</td><td>72</td><td>10</td><td>1</td></tr><br />
</table><br><br />
<br />
Results: Gel has not been run. Have decided to order RFP specific primers for next year.<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/AugustTeam:Lethbridge/Notebook/Lab Work/August2010-10-27T19:17:38Z<p>Liszabruder: </p>
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<font color="white">Here you can check out the work we have done in the lab, click on a month to take a look!<br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/April"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8a/UofLapril.jpg" width="60"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/May"><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UofLmaybutton.jpg" width="60"/><br />
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<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/June"><br />
<img src="https://static.igem.org/mediawiki/2010/8/80/UofLjunebutton.jpg" width="60"/><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/July"><br />
<img src="https://static.igem.org/mediawiki/2010/5/53/UofLjulybutton.jpg" width="60"/><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/August"><br />
<img src="https://static.igem.org/mediawiki/2010/1/15/UofLaugustbutton.jpg" width="80"/><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/September"><br />
<img src="https://static.igem.org/mediawiki/2010/4/4d/UofLseptemberbutton.jpg" width="60"/><br />
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<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/October"><br />
<img src="https://static.igem.org/mediawiki/2010/4/4e/UofLoctoberbutton.jpg" width="60"/><br />
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<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">August=<br />
<br />
==<font color="white">August 3, 2010==<br />
(in Lab: HB, AV, JV)<br><br />
<br />
<b>Objective:</b> To restrict pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet<br><br />
<br />
<b>Method:</b> Used [[Team:Lethbridge/Notebook/Protocols|Restriction of Plasmid DNA]] protocol.<br />
<br />
* A front vector was made made in sRBS ,mRBS, dT plasmids using EcoRI and Xbal enzymes<br />
* pDNA that was cut out of plasmid for a front vector was pBAD, TetR, dT and pLacI using EcoRI and SpeI enzymes<br />
* A back vector was made in sRBS and mRBS plasmids with PstI and SpeI<br />
* pDNA that was cut out of plasmid for a back vector was TeTR and it was restricted with XbaI and PstI <br><br />
<br />
<table><table border ="3"><br />
<tr><td><b>Construct</b></td><td><b>pDNA</b></td><td><b>buffer</b></td><td><b>Enzymes</b></td></tr><br />
<tr><td>pBAD-sRBS/mRBS</td><td>pBAD</td><td>Red</td><td>EcoRI and SpeI</td></tr><br />
<tr><td>pBAD-sRBS/mRBS</td><td>sRBS</td><td>Orange</td><td>XbaI and EcoRI</td></tr><br />
<tr><td>pBAD-sRBS/mRBS</td><td>mRBS</td><td>Orange</td><td>XbaI and EcoRII</td></tr><br />
<tr><td>sRBS/mRBS-TetR</td><td>sRBS</td><td>Red</td><td>PstI and SpeI</td></tr><br />
<tr><td>sRBS/mRBS-TetR</td><td>mRBS</td><td>Red<td>PstI and SpeI</td></tr><br />
<tr><td>sRBS/mRBS-TetR</td><td>TetR</td><td>Tango</td><td>XbaI and PstI</td></tr><br />
<tr><td>TetR-dT</td><td>TetR</td><td>Red</td><td>EcoRI and SpeI</td></tr><br />
<tr><td>TetR-dT</td><td>dT</td><td>Orange</td><td>XbaI and EcoRI</td></tr><br />
<tr><td>dT-pTet</td><td>dT</td><td>Red</td><td>EcoRI and SpeI</td></tr><br />
<tr><td>dT-pTet</td><td>pTet</td><td>Orange</td><td>XbaI and EcoRI</td></tr><br />
<tr><td>pLAcI-sRBS/mRBS</td><td>pLacI</td><td>Red</td><td>EcoRI and SpeI</td></tr><br />
<tr><td>pLAcI-sRBS/mRBS</td><td>sRBS</td><td>Orange</td><td>XbaI and EcoRI</td></tr><br />
<tr><td>pLAcI-sRBS/mRBS</td><td>mRBS</td><td>Orange</td><td>XbaI and EcoRI</td></tr><br />
<tr><td>Mms6-PET28(a)</td><td>PET28(a)</td><td>Orange</td><td>NotI</td></tr><br />
</table><br />
* For all reactions <br />
** 158 (&micro;L) Milli-Q H<sub>2</sub>O<br />
** 10 (&micro;L) Buffer<br />
** 0.5(&micro;L) of each enzyme<br />
** 10 (&micro;L) pDNA<br />
<br />
Restriction was incubated for 1 hour at 37<sup>o</sup>C<br><br><br><br />
<b>Objective:</b> Run PCR of pBAD, TetR, dT, pLacI, and mms6.<br><br />
<b>Method:</b> Used PCR thermocycler iGEM program 7<br><br />
<table><table border="3"><br />
<tr><td><b>Component</b></td><td><b>Volume per tube (&micro;L)</b></td><td><b>Master Mix (x6)</b></td></tr><br />
<tr><td>MilliQ H<sub>2</sub>O</td><td>41.8</td><td>250.8</td></tr><br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub></td><td>5</td><td>30</td></tr><br />
<tr><td>dNTPs</td><td>1</td><td>6</td></tr><br />
<tr><td>Forward Primer</td><td>0.5</td><td>3</td></tr><br />
<tr><td>Reverse Primer</td><td>0.5</td><td>3</td></tr><br />
<tr><td>Template DNA</td><td>1</td><td>-</td></tr><br />
<tr><td>Pfu DNA polymerase</td><td>0.2</td><td>-</td></tr><br />
<tr><td>Total</td><td>50</td><td>292.8</td></tr><br />
</table><br />
*Added 48.8 &micro;L of Master Mix to each PCR reaction<br><br><br><br />
<br />
<br />
<b>Objective:</b> Complete maxipreps of Lumazine (K249002), EYFP (E0030), and ECFP (E0020) and run on 1% agarose gel.<br><br />
<b>Method:</b><br><br />
<table><table border="3"><br />
<tr><td><b>Lane</b></td><td><b>Sample</b></td><td><b>Components (&micro;L)</b></td></tr><br />
<tr><td>1</td><td>1 kb ladder</td><td>2 loading dye (5x) + 0.5 ladder + 3.5 MilliQ H<sub>2</sub>O</td></tr><br />
<tr><td>2</td><td>ECFP</td><td>2 DNA + 2 loading dye (5x) + 2 MilliQ H<sub>2</sub>O</td></tr><br />
<tr><td>3</td><td>EYFP</td><td>2 DNA + 2 loading dye (5x) + 2 MilliQ H<sub>2</sub>O</td></tr><br />
<tr><td>4</td><td>Lumazine</td><td>2 DNA + 2 loading dye (5x) + 2 MilliQ H<sub>2</sub>O</td></tr><br />
</table><br />
*Ran at 100V for 42 minutes. Stained in EtBr for 15 minutes.<br> <br />
<b>Results:</b> [[image:Lethbridge_100803JV Maxiprep.JPG|50px]]<br><br><br><br />
<br />
<br />
<b>Objective:</b> Ligate dT into pSB1C3.<br><br />
<b>Method:</b><br><br />
*PCR amplify and purify both pSB1C3 and dT<br />
*Restrict both with EcoRI and PstI<br />
*Restrict pSB1C3 with DpnI<br />
*Ligate pSB1C3 and dT<br><br />
<u>Restriction:</u><br />
<table><table border="3"><br />
<tr><td><b>Restriction</b></td><td><b>MilliQ H<sub>2</sub>O (&micro;L)</b></td><td><b>Buffer Orange (&micro;L)</b></td><td><b>pDNA (&micro;L)</b></td><td><b>Enzyme (&micro;L)</b></td></tr><br />
<tr><td>pSB1C3</td><td>79.25</td><td>10</td><td>10</td><td>0.25 EcoRI + 0.25 PstI + 0.25 DpnI</td></tr><br />
<tr><td>pSB1C3 control</td><td>80</td><td>10</td><td>10</td><td>-</td></tr><br />
<tr><td>dT</td><td>79.50</td><td>10</td><td>10</td><td>0.25 EcoRI + 0.25 PstI</td></tr><br />
<tr><td>dT control</td><td>80</td><td>10</td><td>10</td><td>-</td></tr><br />
</table><br />
*Restriction were incubated at 37<sup>o</sup>C for 90 minutes.<br />
*Enzymes were heat killed for 20 minutes at 80<sup>o</sup>C.<br />
<br />
==<font color="white">August 3, 2010 Evening==<br />
(in lab: KG, JS)<br><br />
<br />
<b>Objective:</b> To run 1.5% agarose of restrictions: pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet<br><br />
<br />
<b>Method:</b> Used a 1.5% agarose gel with 2 (&micro;L) of loading dye and 10 (&micro;L) of pDNA.<br><br />
<br />
<table border ="3"><br />
<tr><td><b>Lane</b><td><b>Contents</b><td><b>Result</b><br />
<tr><td>1<td>1kb ladder<td><br />
<tr><td>2<td>pTet (XbaI, EcoRI)<td>good<br />
<tr><td>3<td>pTet (orange buffer)<td>---<br />
<tr><td>4<td>dT (XbaI, EcoR)<td>no good<br />
<tr><td>5<td>dT (orange buffer)<td>---<br />
<tr><td>6<td>mRBS (SpeI, PstI)<td> good<br />
<tr><td>7<td>mRBS (red buffer)<td>---<br />
<tr><td>8<td>sRBS (SpeI, PstI)<td> good<br />
<tr><td>9<td>sRBS (red buffer)<td>---<br />
<tr><td>10<td>mRBS (XbaI, EcoRI)<td> good<br />
<tr><td>11<td>mRBS (orange buffer)<td>---<br />
<tr><td>12<td>sRBS (XbaI, EcoRl)<td> good<br />
<tr><td>13<td>sRBS (orange buffer)<td>---<br />
<tr><td>14<td>pet28(a)<td> good<br />
<tr><td>15<td>100 bp ladder<td><br />
<tr><td>16<td>PSB1C3<td> not good<br />
<tr><td>17<td>PSB1C3 restriction digest<td>---<br />
</table><br><br />
<br />
<table border ="3"><br />
<tr><td><b>Lane</b><td><b>Contents</b><td><b>Result</b><br />
<tr><td>1<td>TetR (EcorI, SpeI)<td> good<br />
<tr><td>2<td>TetR (red buffer)<td>---<br />
<tr><td>3<td>pLacI (EcoRI, SpeI)<td> good<br />
<tr><td>4<td>pLacI (red buffer)<td>---<br />
<tr><td>5<td>Mms6<td>can not tell<br />
<tr><td>6<td>Mms6 control<td>---<br />
<tr><td>7<td>TetR (Xbal, PstI)<td> good<br />
<tr><td>8<td>TetR (tango buffer)<td>---<br />
<tr><td>9<td>pBAD (EcoRI, SpeI)<td>good<br />
<tr><td>10<td>pBAD (red buffer)<td>---<br />
<tr><td>11<td>dT (EcoRI, SpeI)<td>good<br />
<tr><td>12<td>dT (red buffer)<td>---<br />
<tr><td>13<td>pLacI (2)<td>?<br />
<tr><td>14<td>dT control<td> not good<br />
<tr><td>15<td>dT restriction<td><br />
<tr><td>16<td>100 bp ladder<td><br />
<tr><td>17<td>dT PCR product<td> good<br />
<tr><td>18<td>Mms6 PCR product<td> good<br />
<tr><td>19<td>pBAD PCR product<td> good<br />
<tr><td>20<td>pLacI PCR product<td> good<br />
</table><br><br />
<br />
[[image:Lethbridge_100803AS.JPG|200px]]<br />
<br />
----<br />
<b>Objective:</b> To ligate: pBAD-sRBS/mRBS, sRBS/mRBS-TetR, TetR-dT, dT-pTet, pLacI-sRBS/mRBS, Mms6-ptet28(a), dT-PSB1C3<br><br />
<br />
<b>Method:</b> [[Team:Lethbridge/Notebook/Protocols|Ligation of Plasmid DNA]]<br><br />
15&micro;L pDNA in plasmid, and 15 &micro;L of pDNA biobrick<br />
<br />
==<font color="white">August 4, 2010==<br />
(in lab: JV)<br><br />
<br />
<b> Objective:</B> PCR analysis of ligation product of August 3, 2010<br><br />
* <u>Ligations</u><br />
** pBAD-mRBS<br />
** pBAD-SRBS<br />
** SRBS-tetR <br />
** mRBS-TetR<br />
** dt-pTet<br />
** mms6-pET-28a<br />
** dt-pSBIC3<br />
** pLacI-SRBS<br />
* <u>Controls</u><br />
** pBAD<br />
** TetR<br />
** TetR<br />
** pLacI<br />
** mms6<br />
<br />
<b>Method:</b><br><br />
<u>PCR:</u> Thermocycler set to iGEM program 7<br><br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x16)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>41.8<td>668.6<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>80<br />
<tr><td>dNTPs<td>1<td>16<br />
<tr><td>Forward Primer<td>0.5<td>8<br />
<tr><td>Reverse Primers<td>0.5<td>8<br />
<tr><td>Template DNA<td>1<td>16<br />
<tr><td>Pfu polymerase<td>0.2<td>3.2<br />
</table><br><br />
<br />
<u>2.5% agarose gel(1x TAE)</u><br><br />
<table border ="3"><br />
<tr><td>lane<td><b>contents</b><td><b>Successful Ligation ?</b><br />
<tr><td>1<td>50bp ladder<td>---<br />
<tr><td>2<td>dt pSBIC3<td>---<br />
<tr><td>3<td>dt pTet<td>x<br />
<tr><td>4<td>dt control<td>---<br />
<tr><td>5<td>sRBS-TetR<td>x<br />
<tr><td>6<td>mRBS-TetR<td>?<br />
<tr><td>7<td>TetR control<td>---<br />
<tr><td>8<td>pLacI-mRBS<td>x<br />
<tr><td>9<td>pLacI-sRBS<td>?<br />
<tr><td>10<td>pLacI control<td>---<br />
<tr><td>11<td>Mms6 pET-28a<td>no band<br />
<tr><td>12<td>Mms6 control<td>---<br />
<tr><td>13<td>pBad-SRBS<td>x<br />
<tr><td>14<td>pBad-mRBS<td>x<br />
<tr><td>15<td>pBad control<td>---<br />
</table><br />
*Ran at 100V for 70 minutes.<br />
<br />
<b>Results:</b>[[image:Lethbridge_100804 JV Ligation PCR AS.JPG|200px]] <br><br><br><br />
<br />
<br />
<br />
<b>Objective:</b> Transform the successful ligations<br><br />
<br />
<b>Method:</b> used [[Team:Lethbridge/Notebook/Protocols|Competent Cell Transformation]] protocol<br />
* changes:<br />
**used 50&micro;L aliquottes of DH5&alpha<br />
**did not pipette up and down once, the cells were just swirled 3 times<br />
**added 400&micro;L SOC media, shoock at 37<sup>0</sup>C for 90 min<br />
**platted 250&micro;L and 150&micro;L<br><br />
<br />
Incubated from 12:00AM to 4;00 PM<br><br />
<br />
<table border ="3"><br />
<td><b>results</b><br />
<tr><td>contents<td><b>&250&micro;L</b><td><b>150&micro;L</b><br />
<tr><td>dt-pTet<td>good<td>x<br />
<tr><td>- control<td>x<td>x<br />
<tr><td>mms6-pET-28a<td>good<td>good<br />
<tr><td>dt-pSBIC3<td>x<td>x<br />
<tr><td>mRBS-TetR<td>good<td>good<br />
<tr><td>pLacI-mRBS<td>good<td>good<br />
<tr><td>SRBS-TetR<td>x<td>x<br />
<tr><td>pBAD-SRBS<td>good<td>good<br />
<tr><td>+ contol<td>good<td>good<br />
</table><br><br />
<br />
==<font color="white">August 6, 2010==<br />
<br />
In Lab: JV<br />
<br />
<b>Objective:</b> Put lumazine synthase gene and mms6 into pET-28(A) for future overexpression. <br><br />
<br />
<b>Method:</b> Restrict mms6 from Mr. Gene with NotI. Large quantities of DNA can be used so a gel extraction of the part can be done. PCR lumazine synthase out of its backbone. Restrict off the extra DNA fragments from the PCR with NotI. Restrict pET-28A with notI. Ligate.<br><br />
<br />
<u>Restriction:</u><br />
<table><table border="3"><br />
<tr><td><b>Restriction</b></td><td><b>MilliQ H<sub>2</sub>O (&micro;L)</b></td><td><b>Buffer Orange (&micro;L)</b></td><td><b>pDNA (&micro;L)</b></td><td><b>Enzyme (&micro;L)</b></td></tr><br />
<tr><td>mms6</td><td>799.5</td><td>100</td><td>100</td><td>1 NotI</td></tr><br />
</table><br />
<br />
<u>PCR:</u><br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>41.85<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<br />
<tr><td>dNTPs<td>1<br />
<tr><td>Forward Primer (VF2)<td>0.5<br />
<tr><td>Reverse Primers (VR)<td>0.5<br />
<tr><td>Template DNA (Lumazine Synthase)<td>1<br />
<tr><td>Pfu polymerase<td>0.2<br />
</table><br><br />
<br />
<u>2% Agarose Gel for Gel Extraction of mms6:</u><br />
<table border ="3"><br />
<tr><td><b>lane</b><td><b>sample</b><td><b>loaded</b><br />
<tr><td>1<td>mms6 restricted with NotI<td>1 mL sample<br />
<tr><td>2<td>mms6 unrestricted control<td>10(&micro;L) sample, 2(&micro;L)dye<br />
<tr><td>3<td>50bp ladder<td>2(&micro;L) ladder, 2(&micro;L) dye, 8(&micro;L) H<sub>2</sub>0<br />
<tr><td>3<td>1 kb ladder<td>2(&micro;L) ladder, 2(&micro;L) dye, 8(&micro;L) H<sub>2</sub>0<br />
</table><br><br />
<br />
Gel ran for 60 minutes at 100 V. Small & faint band was slightly visible.<br />
Gel extraction was carried out using QIAGEN method. Eluted to 12 (&micro;L).<br><br />
[[image:Lethbridge_100809 JV Mms6 Gel Extract BW.jpg|200px]]<br />
<br />
==<font color="white">August 6, 2010 Evening==<br />
<br />
<b>Objective:</b> Attempt colony pcr for rapid screening<br><br />
<br />
<b>Method:</b> Followed two protocols from openwet<br><br />
<br />
*Knight Protocol<br><br />
**place 20(&micro;L) sterile H<sub>2</sub>O in 0.6mL sterile tube<br />
**with P10 pipette set to 3(&micro;L) dip tip into colony<br />
**place pipette tip into water and pipette up and down 20 times(this can be stored at 4<sup>0</sup>C for inoculation of overnight 5mL cultures)<br><br />
<br />
*Endy Protocol<br />
**place 50(&micro;L) sterile H<sub>2</sub>O in 0.6mL sterile tube <br />
**with PLO pipette (set 3(&micro;L)) dip sterile tip into colony <br />
**place pipette tip into water and pipette up and down 20 times<br />
<br />
<table border ="3"><br />
<tr><td><b>Knight cont'd</b><td><b>Endy cont'd</b><br />
<tr><td><b>setup 20(&micro;L) reaction</b><td><b>setup 20(&micro;L) reaction</b><br />
<tr><td>1(&micro;L) colony suspension<td>2(&micro;L) colony suspension<br />
<tr><td>2(&micro;L) 10x p.fu (+Mg SO<sub>4</sub><td>2(&micro;L) 10x p.fu (+Mg SO<sub>4</sub><br />
<tr><td>2(&micro;L) dNTP<td>2(&micro;L) dNTP<br />
<tr><td>1.25(&micro;L)VF2 Primer (10(&micro;M)<td>0.75(&micro;L)VF2 Primer (10(&micro;M)<br />
<tr><td>1.25(&micro;L)VF Primer (10(&micro;M)<td>0.75(&micro;L)VF Primer (10(&micro;M)<br />
<tr><td>0.2(&micro;L) Pfu polymerase<td>0.2(&micro;L) Pfu polymerase<br />
<tr><td>11.8 Milli-Q H<sub>2</sub>O<td>12.6 Milli-Q H<sub>2</sub>O<br />
</table><br><br />
<br />
*as control for each rxn used equal volume of mRBS maxiprep<br />
<br />
<table border ="3"><br />
<tr><td><b>Knight cont'd</b><td><b>Endy cont'd</b><br />
<tr><td><b>cycling conditions</b><td><b>cycling conditions</b><br />
<tr><td>95<sup>0</sup>C for 15 minutes<td>95<sup>0</sup>C for 6 minutes<br />
<tr><td>*94<sup>0</sup>C for 30 seconds<td>**95<sup>0</sup>C for 30 seconds<br />
<tr><td>*56<sup>0</sup>C for 30 seconds<td>**56<sup>0</sup>C for 30 seconds<br />
<tr><td>*68<sup>0</sup>C for 1 minutes<td>**70<sup>0</sup>C for 1 minutes<br />
<tr><td>68<sup>0</sup>C for 20 minutes<td>70<sup>0</sup>C for 10 minutes<br />
</table><br><br />
<br />
(*) were run 39 times<br><br />
(**) were run 35 times<br><br />
<br />
Made the following program (called COLONYY)<br />
Lid preheat 98<sup>0</sup>C<br />
*98<sup>0</sup>C for 15 minutes <br />
*<b>98<sup>0</sup>C for 30 seconds</b><br />
*<b>56<sup>0</sup>C for 30 seconds</b><br />
*<b>68-70<sup>0</sup>C gradient for 1 minute</b><br />
*68-70<sup>0</sup>C gradient for 20 minute<br />
*4<sup>0</sup>C indefinte<br />
<br />
<b>bold</b> selections were cycled 39 times<br><br />
<br />
<b> Objective:</b> Analyzed PCR products on 2.5% TAE Agarose gel.<br />
<br />
<table border ="3"><br />
<tr><td><b>lane</b><td><b>sample</b><td><b>loaded</b><br />
<tr><td>1<td>50bp ladder<td>1(&micro;L) ladder, 1(&micro;L) dye, 4(&micro;L) H<sub>2</sub>0<br />
<tr><td>2<td>Knight control<td>5(&micro;L) sample, 1(&micro;L)dye<br />
<tr><td>3<td>Knight colony<td>5(&micro;L) sample, 1(&micro;L)dye<br />
<tr><td>4<td>Endy control<td>5(&micro;L) sample, 1(&micro;L)dye<br />
<tr><td>5<td>Endy colony<td>5(&micro;L) sample, 1(&micro;L)dye<br />
<tr><td>6<td>50bp ladder<td>1(&micro;L) ladder, 1(&micro;L) dye, 4(&micro;L) H<sub>2</sub>0<br />
<tr><td>7<td>lumazine(justin's)<td>5(&micro;L) sample, 1(&micro;L)dye<br />
</table><br><br />
[[image:Lethbridge_100806ADSPCR.JPG|200px]]<br />
<br />
Repeat gel with template controls<br />
<br />
<table border ="3"><br />
<tr><td><b>lane</b><td><b>sample</b><td><b>loaded</b><br />
<tr><td>1<td>50bp ladder<td>0.5(&micro;L) ladder, 2(&micro;L) dye, 9.5(&micro;L) H<sub>2</sub>0<br />
<tr><td>2<td>Endy Template<td>5(&micro;L) colony suspension, 1(&micro;L)dye, 4(&micro;L) H<sub>2</sub>0 <br />
<tr><td>3<td>Endy mRBS Control (PLR)<td>5(&micro;L) sample, 1(&micro;L)dye<br />
<tr><td>4<td>Endy mRBS-TetR colony(PCR)<td>5(&micro;L) sample, 1(&micro;L)dye<br />
<tr><td>4<td>mRBS template<td>0.5(&micro;L) sample, 1(&micro;L)dye, 4(&micro;L) H<sub>2</sub>0<br />
<tr><td>5<td>Knight Template<td>0.25(&micro;L) colony suspension, 1(&micro;L)dye, 4(&micro;L) H<sub>2</sub>0<br />
<tr><td>6<td>Knight mRBS control<td>5(&micro;L) ladder, 1(&micro;L) dye<br />
<tr><td>7<td>Knight-mRBS-TetR colony<td>5(&micro;L) sample, 1(&micro;L)dye <br />
<tr><td>1<td>1Kb ladder<td>0.5(&micro;L) ladder, 2(&micro;L) dye, 9.5(&micro;L) H<sub>2</sub>0<br />
</table><br><br />
[[image:Lethbridge_100809AS-PCR.jpg|200px]]<br />
<br />
*ran at 100V for 75 minutes<br />
<br />
==<font color="white">Aug 9, 2010==<br />
(In Lab: HB)<br />
<br />
<b>Objective:</b> Run PCR of mRBS-xylE I1 and I2 and lumazine.<br><br />
<br />
<b>Method:</b><br><br />
<u>PCR:</u> Thermocycler set to iGEM program 7<br><br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x4)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>41.8<td>167.2<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>20<br />
<tr><td>dNTPs<td>1<td>4<br />
<tr><td>Forward Primer (VF2)<td>0.5<td>2<br />
<tr><td>Reverse Primers (VR)<td>0.5<td>2<br />
<tr><td>Template DNA<td>1<td><br />
<tr><td>Pfu polymerase<td>0.2<td><br />
</table><br><br />
<br />
Added 48.8&micro;L Master Mix to each reaction tube.<br />
<br />
<b>Objective:</b> Run 2% agarose gel to confirm PCR of mRBS-xylE I1 and I2 and lumazine worked.<br><br />
<br />
<table border ="3"><br />
<tr><td><b>lane</b><td><b>sample</b><td><b>loaded</b><br />
<tr><td>1<td>50bp ladder<td>0.5(&micro;L) ladder, 1(&micro;L) dye, 6.5(&micro;L) H<sub>2</sub>0<br />
<tr><td>2<td>mRBS-xylE I1<td>5(&micro;L) sample, 2(&micro;L)dye<br />
<tr><td>3<td>mRBS-xylE I2<td>5(&micro;L) sample, 2(&micro;L)dye<br />
<tr><td>4<td>Lumazine<td>5(&micro;L) sample, 2(&micro;L)dye<br />
</table><br><br />
<br />
Ran at 100 V for 45 minutes. mRBS-xylE did not amplify while lumazine did amplify. <br><br />
<br />
<b>Results:</b> Ligation of mRBS-xylE NOT confirmed<br><br />
[[image:Lethbridge_100809 HB xylE Lum PCR BW.jpg|50px]]<br />
<br />
<br />
----<br />
(In Lab: AV)<br><br />
<br />
<b> Objective:</b> Prepared glycerol stocks & Miniprepped the following using the Qiagen Protocol.<br><br />
<br />
<table border ="3"><br />
<tr><td>pLacI-mRBS Colony 1<br />
<tr><td>pLacI-mRBS Colony 2<br />
<tr><td>pLacI-sRBS Colony 2<br />
<tr><td>pLacI-sRBS Colony 3<br />
<tr><td>pBAD-mRBS Colony 1<br />
<tr><td>pBAD-mRBS Colony 2<br />
<tr><td>pBAD-sRBS Colony 1<br />
<tr><td>pBAD-sRBS Colony 2<br />
<tr><td>dT-pTet Colony 1<br />
<tr><td>dT-pTet Colony 3<br />
<tr><td>mRBS-TetR Colony 1<br />
<tr><td>mRBS-TetR Colony 3<br />
</table><br><br />
<br />
----<br />
<b>Objective:</b> To determine which of the previous ligations worked.<br><br />
<br />
<b>Method:</b> Restricted with single cutter and double cutter. <br><br />
<br />
<br />
Restriction Reaction (SINGLE)<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b></td><td>Volume(&micro;L)</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>15.75</td></tr><br />
<tr><td>Orange Buffer (10x)</td><td>2</td></tr><br />
<tr><td>pDNA</td><td>2</td></tr><br />
<tr><td>EcoRI</td><td>0.25</td></tr><br />
</table><br />
<br />
<br />
Restriction Reaction (DOUBLE)<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b></td><td>Volume(&micro;L)</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>15.50</td></tr><br />
<tr><td>Orange Buffer (10x)</td><td>2</td></tr><br />
<tr><td>pDNA</td><td>2</td></tr><br />
<tr><td>EcoRI</td><td>0.25</td></tr><br />
<tr><td>PstI</td><td>0.25</td></tr><br />
</table><br />
<br />
<br />
Unrestricted Control<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b></td><td>Volume(&micro;L)</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>16</td></tr><br />
<tr><td>Orange Buffer (10x)</td><td>2</td></tr><br />
<tr><td>pDNA </td><td>2</td></tr><br />
</table> <br><br />
<br />
DNA was restricted for 1 hour at 37<sup>o</sup>C.<br><br />
<br />
Analyzed results on a 2% agarose gel with 2 (&micro;L) of loading dye and 10 (&micro;L) of pDNA. Load order as follows:<br><br />
<br />
<table border ="3"><br />
<tr><td><b>Lane</b><td><b>Contents</b><br />
<tr><td>1<td>1kb ladder<br />
<tr><td>2<td>50 bp ladder<br />
<tr><td>3<td>dT-pTet 1 DRD<br />
<tr><td>4<td>dT-pTet 1 SRD<br />
<tr><td>5<td>dT-pTet 1 URD<br />
<tr><td>6<td>dT-pTet 3 DRD<br />
<tr><td>7<td>dT-pTet 3 SRD<br />
<tr><td>8<td>dT-pTet 3 URD<br />
<tr><td>9<td>pLacI-mRBS 1 DRD<br />
<tr><td>10<td>pLacI-mRBS 1 SRD<br />
<tr><td>11<td>pLacI-mRBS 1 URD<br />
<tr><td>12<td>pLacI-mRBS 2 DRD<br />
<tr><td>13<td>pLacI-mRBS 2 SRD<br />
<tr><td>14<td>pLacI-mRBS 2 URD <br />
<tr><td>15<td>pLacI-sRBS 1 DRD<br />
<tr><td>16<td>pLacI-sRBS 1 SRD<br />
<tr><td>17<td>pLacI-sRBS 1 URD<br />
<tr><td>18<td>pLacI-sRBS 3 DRD<br />
<tr><td>19<td>pLacI-sRBS 3 SRD<br />
<tr><td>20<td>pLacI-sRBS 3 URD<br />
</table><br><br />
[[image:Lethbridge_100809AVKG Top.jpg|200px]]<br />
<br />
<table border ="3"><br />
<tr><td><b>Lane</b><td><b>Contents</b><br />
<tr><td>1<td>1 kb ladder<br />
<tr><td>2<td>50 bp ladder<br />
<tr><td>3<td>pBAD-mRBS 1 DRD <br />
<tr><td>4<td>pBAD-mRBS 1 SRD<br />
<tr><td>5<td>pBAD-mRBS 1 URD<br />
<tr><td>6<td>pBAD-mRBS 2 DRD<br />
<tr><td>7<td>pBAD-mRBS 2 SRD<br />
<tr><td>8<td>pBAD-mRBS 2 URD<br />
<tr><td>9<td>pBAD-sRBS 1 DRD<br />
<tr><td>10<td>pBAD-sRBS 1 SRD<br />
<tr><td>11<td>pBAD-sRBS 1 URD<br />
<tr><td>12<td>pBAD-sRBS 2DRD<br />
<tr><td>13<td>pBAD-sRBS 2 SRD<br />
<tr><td>14<td>pBAD-sRBS 2 URD<br />
<tr><td>15<td>mRBS-TetR 1 DRD<br />
<tr><td>16<td>mRBS-TetR 1 SRD<br />
<tr><td>17<td>mRBS-TetR 1 URD<br />
<tr><td>18<td>mRBS-TetR 3 DRD <br />
<tr><td>19<td>mRBS-TetR 3 SRD<br />
<tr><td>20<td>mRBS-TetR 3 URD <br />
</table><br><br />
<br />
[[image:Lethbridge_100809AVKG Bottom.jpg|200px]]<br />
----<br />
<br />
==<font color="white">Aug 9,2010 Evening==<br />
(In Lab: JV, AS)<br><br />
<br />
<b>Objective:</b> To ligate: lumazine into vector upstream of dT. Lumazine and mms6 into pET28a. <br><br />
<br />
<b>Method:</b><br><br />
<br />
<u>Restrictions</u><br><br />
*Restrict Lumazine wit EcoRI and SpeI (Red Buffer)<br><br />
*Restrict the dT with XbaI and EcoRI (Orange Buffer)<br><br />
*Restrict Lumazine Synthase with NotI (Red Buffer)<br><br />
<br />
Set up reactions as follows:<br><br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td></tr><br />
<tr><td>MilliQ H<sub>2</sub>O</td><td>15.6 or 15.8</td></tr><br />
<tr><td>Buffer</td><td>2</td></tr><br />
<tr><td>pDNA</td><td>2</td></tr><br />
<tr><td>Enzyme</td><td>0.20 + 0.20</td></tr></table><br />
<br />
Incubated reactions for 60 minutes at 37<sup>o</sup>C<br><br />
<br />
<u>Ligation</u><br><br />
Reaction set up as follows:<br />
*T4 DNA ligase - 0.25&micro;L<br><br />
*DNA 1 - 8&micro;L<br><br />
*DNA 2 - 8&micro;L<br><br />
*10x Ligation Buffer - 2&micro;L<br><br />
*MilliQ H<sub>2</sub>O - 1.75&micro;L<br><br />
Incubated reactions overnight at room temperature.<br />
<br />
==<font color="white">Aug 10, 2010==<br />
<br />
(In Lab: JV)<br><br />
<br />
<b> Objective:</b> Reran large gel from Aug 9/2010. <br><br />
<br />
Load order was as follows: <br />
<br />
<table border ="3"><br />
<tr><td><b>Lane</b><td><b>Contents</b><br />
<tr><td>1<td>50 bp ladder<br />
<tr><td>2<td>pBAD-mRBS 2 URD<br />
<tr><td>3<td>pBAD-mRBS 2 SRD<br />
<tr><td>4<td>pBAD-mRBS 2 DRD<br />
<tr><td>5<td>pLacI-sRBS 3 URD<br />
<tr><td>6<td>pLacI-sRBS 3 SRD<br />
<tr><td>7<td>pLacI-sRBS 3 DRD<br />
<tr><td>8<td>pLacI-mRBS 2 URD<br />
<tr><td>9<td>pLacI-mRBS 2 SRD<br />
<tr><td>10<td>pLacI-mRBS 1 DRD<br />
<tr><td>11<td>dT-pTet 3 URD<br />
<tr><td>12<td>dT-pTet 3 SRD<br />
<tr><td>13<td>dT-pTet 3 DRD<br />
<tr><td>14<td>pBAD-mRBS 1 URD <br />
<tr><td>15<td>pBAD-mRBS 1 SRD<br />
<tr><td>16<td>pBAD-mRBS 1 DRD<br />
<tr><td>17<td>pLacI-sRBS 2 URD<br />
<tr><td>18<td>pLacI-sRBS 2 SRD<br />
<tr><td>19<td>pLacI-sRBS 2 DRD<br />
<tr><td>20<td>1 kb ladder<br />
</table><br><br />
<br />
<table border ="3"><br />
<tr><td><b>Lane</b><td><b>Contents</b><br />
<tr><td>1<td>50 bp ladder<br />
<tr><td>2<td>pLacI-mRBS 1 URD<br />
<tr><td>3<td>pLacI-mRBS 1 SRD <br />
<tr><td>4<td>pLacI-mRBS 1 DRD<br />
<tr><td>5<td>dT-pTet 1 URD<br />
<tr><td>6<td>dT-pTet 1 SRD<br />
<tr><td>7<td>dT-pTet 1 DRD<br />
<tr><td>8<td>mRBS-TetR 3 URD<br />
<tr><td>9<td>mRBS-TetR 3 SRD <br />
<tr><td>10<td>mRBS-TetR 3 DRD<br />
<tr><td>11<td>mRBS-TetR 1 URD<br />
<tr><td>12<td>mRBS-TetR 1 SRD<br />
<tr><td>13<td>mRBS-TetR 1 DRD<br />
<tr><td>14<td>pBAD-sRBS 2 URD<br />
<tr><td>15<td>pBAD-sRBS 2 SRD<br />
<tr><td>16<td>pBAD-sRBS 2 DRD<br />
<tr><td>17<td>pBAD-sRBS 1 URD<br />
<tr><td>18<td>pBAD-sRBS 1 SRD <br />
<tr><td>19<td>pBAD-sRBS 1 DRD<br />
<tr><td>20<td>1 kb ladder <br />
</table><br><br />
<br />
[[image:Lethbridge_100810ReRunRestrictionDigers.JPG|200px]]<br />
<br />
----<br />
(In Lab: JV)<br><br />
<br />
<b> Objective:</b> Determine which ligations/transformations worked from 08/04/10. <br><br />
<br />
<b> Method:</b> Colony PCR. <br><br />
<br />
A: dT-pTet (1-10)<br />
B: pBAD-sRBS (1-10)<br />
C: pLacI-sRBS (1-10)<br />
D: pBAD-mRBS (1-10)<br />
E: mRBS-TetR (1-10)<br />
F: pLacI-mRBS (1-10)<br />
<br />
Pick colony with pipette set at 3(&micro;L)<br />
Pipette colony up and dow in 20(&micro;L) sterile Milli-Q water.<br />
Will use 96 well plate.<br />
<br />
PCR- Conditions:<br />
1. 95<sup>o</sup>C for 15 min<br />
2. 98<sup>o</sup>C for 20 sec<br />
3. 55<sup>o</sup>C for 40 sec<br />
4. 72<sup>o</sup>C for 2 min<br />
5. 72<sup>o</sup>C for 20 min<br />
6. 4<sup>o</sup>C infinitely<br />
<br />
Reaction Mixture - <br />
<br />
<b>Method:</b><br><br />
<u>PCR:</u> Thermocycler set to iGEM program 7<br><br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x65)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>10.9<td>708.5<br />
<tr><td>5X Phusion HF Buffer<td>4<td>260 <br />
<tr><td>dNTPs<td>1<td>65<br />
<tr><td>Forward Primer (VF2)<td>1<td>65<br />
<tr><td>Reverse Primers (VR)<td>1<td>65<br />
<tr><td>Colony Template<td>2<td><br />
<tr><td>Phusion polymerase<td>0.17<td>11.05<br />
</table><br><br />
<br />
Controls: sRBS, pTET & mRBS - will PCR these to compare size using same conditions as above. <br> <br />
[[image:Lethbridge_100810MassiveColonyPCR JV AS.JPG|200px]]<br><br />
[[image:Lethbridge_100810MassiveColonyPCR Large Gel JV AS.JPG|200px]]<br />
<br />
==<font color="white">Aug 10, 2010 Evening==<br />
<br />
(In Lab: ADS)<br><br />
<br />
<b> Objective:</b> PCR amplify xylE from mRBS-xylE for creation of xylE BioBrick <br><br />
<br />
<b> Method:</b> 20&micro;L reactions <br><br />
<br />
PCR- Conditions:<br />
1. Initial Denaturation 98<sup>o</sup>C for 30 sec<br />
2. Denaturation 98<sup>o</sup>C for 10 sec<br />
3. Anneal (51<sup>o</sup>C, 55<sup>o</sup>C,59.8<sup>o</sup>C, 64.6<sup>o</sup>C, 69.1<sup>o</sup>C, 71<sup>o</sup>C) for 30 sec<br />
4. Extend 72<sup>o</sup>C for 30 sec<br />
5. Final Extend 72<sup>o</sup>C for 10 min<br />
6. Held 4<sup>o</sup>C for 30 hours<br />
<br />
6 tubes in gradient PCR. <br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x6.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>8.8<td>57.2<br />
<tr><td>5X Phusion HF Buffer<td>4<td>26 <br />
<tr><td>dNTPs<td>2<td>13<br />
<tr><td>Forward Primer <td>2<td>13<br />
<tr><td>Reverse Primer<td>2<td>13<br />
<tr><td>Template DNA<td>1<td>6.5<br />
<tr><td>Polymerase<td>0.2<td>1.3<br />
</table><br><br />
<br />
Ran samples on 1.5% agarose gel (1X TAE) for 60 minutes at 100V. <br><br />
<br />
<b>Results:</b><br><br />
[[image:Lethbridge_100810xylE PCR AS.JPG|100px]]<br />
<br />
==<font color="white">Aug 11, 2010==<br />
<br />
(In Lab: AS, JV, TF)<br><br />
<br />
<b>Objective:</b> Determine what transformations have the correct insert from Aug. 4, 2010. <br><br />
<b>Method:</b> Colony PCR. Changes - Used pipette tip instead of toothpick. <br><br />
<u>PCR:</u> Thermocycler set to iGEM PFUTEST<br><br />
<br />
1. pLacI-mRBS: 4 (A - E) 50(&micro;L)<br />
2. pBAD-mRBS: 5 (A - E) 20(&micro;L)<br />
3. mRBS-TetR: 6 (A -E) 20(&micro;L)<br />
Controls - mRBS<br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x7.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>9.8<td>73.5<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>15<br />
<tr><td>dNTPs<td>2<td>15<br />
<tr><td>Forward Primer (VF2)<td>2<td>15<br />
<tr><td>Reverse Primer (VR)<td>2<td>15<br />
<tr><td>Template DNA<td>2<td><br />
<tr><td>Pfu polymerase<td>0.2<td>1.5<br />
</table><br><br />
<br />
Added 18&micro;L Master Mix to each reaction tube.<br />
<br />
----<br />
(In Lab: ADS)<br><br />
<br />
<b>Objective:</b> Transform mRBS-xylE BioBrick into DH5alpha. <br><br />
<br />
Transformation - <br />
<br />
A) Thawed 50(&micro;L) Sub-Cloning Efficiency DH5alpha Competent Cells on ice.<br />
B) Gently mixed cells and then aliquoted 100(&micro;L) into chilled polypropylene tubes.<br />
C) Added 2(&micro;L) of BioBrick to cells. Added 5(&micro;L) of pUC19 DNA to 100(&micro;L) cells to determine efficiency. <br />
D) Incubated cells on ice for 30 minutes.<br />
E) Heat shocked cells for 45 seconds in a 42<sup>o</sup>C water bath.<br />
F) Placed on ice for 5 minutes.<br />
G) Added 0.4 mL of room temperature SOC medium.<br />
H) Shook at 225 rpm for 1 hour.<br />
I) Diluted control cells 1:100 with SOC medium.<br />
J) Spread 100(&micro;L) of this dilution on LB-Amp agar plates<br />
K) Spread 50 and 250(&micro;L) of experimental cells on LB-Cam agar plates. <br />
L) Incubated overnight at 37<sup>o</sup>C<br />
<br />
==<font color="white">Aug 12, 2010==<br />
<br />
(In Lab: JV)<br><br />
<br />
<b>Objective:</b> Determine if Adam's colony PCRs' from Aug. 11, 2010 worked. <br><br />
<b>Method:</b> Samples were run on a 2.5% agarose gel (1X TAE) for 1 hour at 100V. <br><br />
<br />
GEL PICTURE!<br />
<br />
<b>Results:</b> Lanes 2, 3, 4 and 8 showed PCR amplification. Colonies chosen don't show the correct insert size. <br><br />
[[image:Lethbridge_100812 JV AS Colony PCR Pfu.JPG|200px]]<br />
<br />
----<br />
(In Lab: JV)<br><br />
<b>Objective:</b> Screen for colonies with the correct insert from Aug. 4, 2010 transformations. <br><br />
<br />
<b>Method:</b> Colony PCR. Changes - Used pipette tip instead of toothpick. Put colony in 20(&micro;L) autoclaved Milli-Q water. <br><br />
<u>PCR:</u> Thermocycler set to iGEM PFUTEST<br><br />
<br />
1. pLacI-sRBS: A (11 - 17) <br />
2. pBAD-sRBS: B (11 - 17) <br />
3. dT-pTet: C (11 - 17) <br />
4. pLacI-mRBS: D (11-17)<br />
5. pBAD-mRBS: E (11-17)<br />
6. mRBS-TetR: F (11-17) <br />
<br />
Controls - mRBS, pTet, sRBS<br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x47)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>9.8<td>460.6<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>94<br />
<tr><td>dNTPs<td>2<td>94<br />
<tr><td>Forward Primer (VF2)<td>2<td>94<br />
<tr><td>Reverse Primer (VR)<td>2<td>94<br />
<tr><td>Template DNA (Cell Lysate)<td>2<td>94<br />
<tr><td>Pfu polymerase<td>0.2<td>9.4<br />
</table><br><br />
<br />
Added 18&micro;L Master Mix to each reaction tube.<br><br />
Analyzed PCR products on 2.5% TAE gel.<br><br />
<b>Results:</b><br><br />
[[image:Lethbridge_100812ASJVMassColonyPCRLargeGel (1).JPG|200px]]<br><br><br />
[[image:Lethbridge_100812ASJVMassColonyPCRSmallGel.JPG|200px]]<br />
<br />
----<br />
(In Lab: ADS, KG)<br><br />
<b>Objective:</b> Perform PCR on lumazine, mms6, xylE plasmids with prefix and suffix primers (these will tell us exact size without subtracting VF2/VR regions). If right will ligate into pET28a plasmids. <br><br />
<br />
<b>Method:</b> Plasmids used included: 6 mms6 maxipreps. 4 lumazine maxipreps. 5 xylE maxipreps. 1 mRBS maxiprep<br><br />
<u>PCR:</u> Thermocycler set to iGEM Program #11 PFU - P/S<br><br />
<br />
PCR- Conditions:<br />
1. Initial Denaturation 95<sup>o</sup>C for 3 min<br />
2. Denaturation 95<sup>o</sup>C for 30 sec<br />
3. Anneal (54<sup>o</sup>C) for 30 sec<br />
4. Extend 72<sup>o</sup>C for 3 min<br />
5. Final Extend 72<sup>o</sup>C for 15 min<br />
6. Held 4<sup>o</sup>C infinitely<br />
(25 cycles)<br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x16.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>9.8<td>161.7<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>33<br />
<tr><td>dNTPs<td>2<td>33<br />
<tr><td>Forward Primer (Prefix)<td>2<td>33<br />
<tr><td>Reverse Primer (Suffix Antisense)<td>2<td>33<br />
<tr><td>Template DNA (Cell Lysate)<td>2<td><br />
<tr><td>Pfu polymerase<td>0.2<td>3.3<br />
</table><br><br />
<br />
Added 18&micro;L Master Mix to each reaction tube.<br><br />
Analyzed PCR products on 2.5% TAE gel run at 100 V for 35 minutes.<br><br />
[[image:Lethbridge_100813ADS Awesome PCR.JPG|200px]]<br />
<br />
==<font color="white">Aug 13, 2010==<br />
<br />
(In Lab: AS)<br><br />
<br />
<b>Objective:</b> PCR amplify minipreps prepared on Aug 9/2010 to screen for properly assembled BioBricks. <br><br />
<br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x12.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>41.85<td>523.1<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>62.5<br />
<tr><td>dNTPs<td>1<td>12.5<br />
<tr><td>Forward Primer (VF2)<td>0.5<td>6.25<br />
<tr><td>Reverse Primer (VR)<td>0.5<td>6.25<br />
<tr><td>Template DNA<td>1<td><br />
<tr><td>Pfu polymerase<td>0.15<td>1.888<br />
</table><br><br />
<br />
Added 49&micro;L Master Mix to each reaction tube.<br><br />
<br />
<br />
----<br />
(In Lab: AS)<br><br />
<br />
<b>Objective:</b> PCR amplify pSB1A3, pSB1T3 and pSB1C3 for use in future 3 part assembly and subsequent growth for glycerol stock. <br><br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x13.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>14.9<td>52.15<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>7<br />
<tr><td>dNTPs<td>1<td>3.5<br />
<tr><td>Forward Primer (SB-prep-2)<td>0.7<td>2.45<br />
<tr><td>Reverse Primer (SB-prep-3p)<td>0.7<td>2.45<br />
<tr><td>Template DNA<td>0.5<td><br />
<tr><td>Pfu polymerase<td>0.2<td>0.7<br />
</table><br><br />
<br />
PCR- Conditions:<br />
1. 94<sup>o</sup>C for 30 sec<br />
2. 94<sup>o</sup>C for 30 sec<br />
3. 55<sup>o</sup>C for 30 sec<br />
4. 68<sup>o</sup>C for 3 min<br />
5. 68<sup>o</sup>C for 10 min<br />
6. 4<sup>o</sup>C infinitely<br />
(36 cycles)<br />
<br />
Added 19.5&micro;L Master Mix to each reaction tube.<br><br />
Analyzed products on 1% TAE agarose gel which ran for 60 minutes at 100 V.<br> <br />
<br />
<b>Results:</b><br><br />
[[image:Lethbridge_100814PlasmidBackbones.JPG|100px]]<br />
<br />
==<font color="white">Aug 14, 2010==<br />
<br />
(In Lab: AS)<br><br />
<br />
<u>2.5% agarose gel(1x TAE)</u><br><br />
<table border ="3"><br />
<tr><td>lane<td><b>contents</b><br />
<tr><td>1<td>pBAD-mRBS 1<br />
<tr><td>2<td>pBAD-mRBS 2<br />
<tr><td>3<td>pBAD-sRBS 1<br />
<tr><td>4<td>pBAD-sRBS 2<br />
<tr><td>5<td>mRBS-TetR 1<br />
<tr><td>6<td>mRBS-TetR 3<br />
<tr><td>7<td>50 bp Ladder<br />
<tr><td>8<td>dT-pTet 1<br />
<tr><td>9<td>dT-pTet 3<br />
<tr><td>10<td>pLacI-mRBS 1<br />
<tr><td>11<td>pLacI-mRBS 2<br />
<tr><td>12<td>pLacI-sRBS 2<br />
<tr><td>13<td>pLacI-sRBS 3<br />
<tr><td>14<td>MT<br />
<tr><td>15<td>MT<br />
<tr><td>16<td>MT<br />
<tr><td>17<td>MT<br />
<tr><td>18<td>MT<br />
<tr><td>19<td>MT<br />
<tr><td>20<td>MT<br />
</table><br />
<br />
<table border ="3"><br />
<tr><td>lane<td><b>contents</b><br />
<tr><td>1<td>K249001<br />
<tr><td>2<td>K249004<br />
<tr><td>3<td>K249005<br />
<tr><td>4<td>K249006<br />
<tr><td>5<td>MT<br />
<tr><td>6<td>K249008<br />
<tr><td>7<td>K249008 (Qiagen)<br />
<tr><td>8<td>K249014<br />
<tr><td>9<td>K249017<br />
<tr><td>10<td>50 bp Ladder<br />
<tr><td>11<td>1<br />
<tr><td>12<td>2<br />
<tr><td>13<td>3<br />
<tr><td>14<td>4<br />
<tr><td>15<td>5<br />
<tr><td>16<td>xylE-dT<br />
<tr><td>17<td>Lumazine-dT<br />
<tr><td>18<td>pLacI-sRBS<br />
<tr><td>19<td>MT<br />
<tr><td>20<td>MT<br />
<tr><td>21<td>MT<br />
<tr><td>22<td>MT<br />
<tr><td>23<td>MT<br />
<tr><td>24<td>MT<br />
<tr><td>25<td>MT<br />
<tr><td>26<td>MT<br />
<tr><td>27<td>MT<br />
</table><br />
<br />
[[image:Lethbridge_100815MiniprepPCR.JPG|200px]]<br />
<br />
----<br />
(In Lab: AS)<br><br />
<br />
<b>Objective:</b> Insert mms6 and lumazine into pET28a using NotI restriction site. <br><br />
<br />
<b>Method:</b> 1. Reserve 1(&micro;L) of dirty PCR product for analysis. 2. Clean up PCR products using Qiagen prep. 3. Restrict 4 mms6 maxipreps and 4 lumazine maxipreps.<br><br />
<br />
<u>Restriction Reactions:</u><br />
<br />
For lumazine and mms6 - <br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x8.5)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>7.8<td>66.30<br />
<tr><td>Orange Buffer (10x)</td><td>2<td>17<br />
<tr><td>pDNA</td><td>10<td><br />
<tr><td>NotI</td><td>0.2<td>1.7<br />
</table><br />
<br />
Added 10 (&micro;L) to each tube.<br />
<br />
For pET28a - <br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>Reaction Mix(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>4.8<br />
<tr><td>Orange Buffer (10x)</td><td>5<br />
<tr><td>pDNA</td><td>40<br />
<tr><td>NotI</td><td>0.2<br />
</table><br />
<br />
Incubated at 37<sup>o</sup>C.<br />
Analyzed results on 2.5% TAE agarose gel which ran at 100 V for 50 minutes.<br><br />
[[image:Lethbridge_100814ColonyPCRRetryandStartofassembly.JPG|200px]]<br />
<br />
<b>Results:</b> Lost all the DNA in the column clean-up step and will have to re-do.<br><br />
<br />
==<font color="white">Aug 14, 2010 Evening==<br />
<br />
(In Lab: AS)<br><br />
<br />
<b>Objective:</b> Assemble mms6-dT and lumazine-dT using three antibiotic assembly. <br><br />
<br />
<b>Method:</b> <br />
#PCR amplify BioBricks (Prefix/Suffix) <br />
#Restrict BioBricks <br />
#Ligate BioBricks into psB1C3 <br />
#Confirm ligation by PCR analysis (VF2/VR) <br />
#Transform ligation mixes <br />
#Screen colonies with Colony PCR <br />
<br />
<u>PCR:</u> Thermocycler set to iGEM program 11<br><br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x10.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>10.8<td>113.4<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>21<br />
<tr><td>dNTPs<td>2<td>21<br />
<tr><td>Forward Primer (Prefix)<td>2<td>21<br />
<tr><td>Reverse Primers (Suffix Antisense)<td>2<td>21<br />
<tr><td>Template DNA<td>1<td><br />
<tr><td>Pfu polymerase<td>0.2<td>2.1<br />
</table><br><br />
<br />
Added 19 (&micro;L) to each tube. <br />
<br />
<u>Restriction Reactions:</u><br />
<br />
For lumazine and mms6 - <br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x5.5)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>11.6<td>63.8<br />
<tr><td>Red Buffer (10x)</td><td>2<td>11<br />
<tr><td>pDNA</td><td>6<td><br />
<tr><td>EcoRI</td><td>0.2<td>1.1<br />
<tr><td>SpeI</td><td>0.2<td>1.1<br />
</table><br />
<br />
Added 14 (&micro;L) to each tube.<br />
<br />
For dT - <br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>Reaction Mix(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>58<br />
<tr><td>Tango Buffer (10x)</td><td>10<br />
<tr><td>pDNA</td><td>30<br />
<tr><td>XbaI</td><td>1<br />
<tr><td>PstI</td><td>1<br />
</table><br />
<br />
For pSB1C3 - <br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>Reaction Mix(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>70<br />
<tr><td>Orange Buffer (10x)</td><td>10<br />
<tr><td>pDNA</td><td>30<br />
<tr><td>EcoRI</td><td>1<br />
<tr><td>PstI</td><td>1<br />
<tr><td>DpnI</td><td>1<br />
</table><br />
<br />
Also cut lumazine, mms6 and dT with one enzyme for two part, PCR amplification and subsequent ligation into pSB1X3. <br />
<br />
For lumazine and mms6, CUT with SpeI - <br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x5.5)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>15.8<td>86.9<br />
<tr><td>Tango Buffer (10x)</td><td>2<td>11<br />
<tr><td>pDNA</td><td>2<td><br />
<tr><td>SpeI</td><td>0.2<td>1.1<br />
</table><br />
<br />
Added 18 (&micro;L) to each reaction.<br />
<br />
For dT, CUT with XbaI- <br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>Reaction Mix(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>79<br />
<tr><td>Tango Buffer (10x)</td><td>10<br />
<tr><td>pDNA</td><td>10<br />
<tr><td>XbaI</td><td>1<br />
</table><br />
<br />
Incubated at 37<sup>o</sup>C for 1.5 hours. Heat killed enzymes for 20 minutes at 80<sup>o</sup>C.<br />
<br />
<u>Ligation Reactions:</u><br />
<br />
3 Part: Lumazine/mms6 + dT + psB1C3<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x5.5)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>11.0<td>64.9<br />
<tr><td>T4 Ligase Buffer (10x)</td><td>2<td>11<br />
<tr><td>Plasmid (psB1C3)</td><td>2<td>11<br />
<tr><td>Part 1 (Lumazine/mms6)</td><td>2<td><br />
<tr><td>Part 2 (dT)</td><td>2<td>11<br />
<tr><td>T4 DNA Ligase</td><td>0.2<td>1.1<br />
</table><br />
<br />
2 Part: Lumazine/mms6 + dT <br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x5.5)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>13.8<td>75.9<br />
<tr><td>T4 Ligase Buffer (10x)</td><td>2<td>11<br />
<tr><td>Part 1 (Lumazine/mms6)</td><td>2<td><br />
<tr><td>Part 2 (dT)</td><td>2<td>11<br />
<tr><td>T4 DNA Ligase</td><td>0.2<td>1.1<br />
</table><br />
<br />
Added 18(&micro;L) to each rxn tube. Incubated 1 hour and overnight at room temperature ( 25<sup>o</sup>C).<br />
<br />
<br />
<u>Screening via PCR amplification :</u> Thermocycler set to iGEM program 11<br><br />
3 Part: Lum/mms6 + dT + pSB1C3<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x5.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>33.8<td>185.9<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>27.5<br />
<tr><td>dNTPs<td>2<td>11<br />
<tr><td>VF2 Primer<td>2<td>11<br />
<tr><td>VR Primer<td>2<td>11<br />
<tr><td>Template DNA<td>5<td><br />
<tr><td>Pfu polymerase<td>0.2<td>1.1<br />
</table><br><br />
<br />
Added 45 (&micro;L) MM to each tube. <br />
<br />
2 Part: Lum/mms6 + dT <br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x5.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>6.8<td>37.4<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>11<br />
<tr><td>dNTPs<td>2<td>11<br />
<tr><td>Prefix Primer<td>2<td>11<br />
<tr><td>Suffix Antisense Primer<td>2<td>11<br />
<tr><td>Template DNA<td>5<td><br />
<tr><td>Pfu polymerase<td>0.2<td>1.1<br />
</table><br><br />
<br />
Added 15 (&micro;L) MM to each tube.<br><br />
<b>Results:</b><br><br />
<br />
==<font color="white">Aug 15, 2010==<br />
(In Lab: ADS)<br><br />
<br />
<b>Objective:</b> Insert mms6 and lumazine into pET28a using NotI restriction site. <br><br />
<br />
<b>Method:</b> 1. Reserve 1(&micro;L) of dirty PCR product for analysis. 2. Clean up PCR products using Qiagen prep. 3. Restrict 3 mms6 maxipreps and 2 lumazine maxipreps.<br><br />
<br />
<u>Restriction Reactions:</u><br />
<br />
For lumazine and mms6 - <br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x10.5)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>11.8<td>123.9<br />
<tr><td>Orange Buffer (10x)</td><td>2<td>21<br />
<tr><td>pDNA</td><td>6<td><br />
<tr><td>NotI</td><td>0.2<td>2.1<br />
</table><br />
<br />
Added 14 (&micro;L) to each tube.<br />
<br />
For pET28a - <br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>Reaction Mix(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>4.8<br />
<tr><td>Orange Buffer (10x)</td><td>5<br />
<tr><td>pDNA</td><td>40<br />
<tr><td>NotI</td><td>0.2<br />
</table><br />
<br />
Incubated at 37<sup>o</sup>C for 1.5 hours. Heat killed enzymes at 80<sup>o</sup>C for 20 minutes.<br />
<br />
<u>Ligation Reactions:</u><br />
<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x5.5)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>13.9<td>75.9<br />
<tr><td>T4 Ligase Buffer (10x)</td><td>2<td>11<br />
<tr><td>Plasmid (pET28a)</td><td>2<td>11<br />
<tr><td>Cut out part (Lumazine/mms6)</td><td>2<td><br />
<tr><td>T4 DNA Ligase</td><td>0.2<td>1.1<br />
</table> <br />
<br />
Added 18(&micro;L) to each tube. Incubated 1 hour and overnight at room temperature. <br />
<br />
==<font color="white">Aug 15, 2010 Evening==<br />
<br />
(In Lab: AS)<br><br />
<br />
<b>Objective:</b> Assemble Lum-dT & mms6-dT using BioBrick standard assembly. <br><br />
<br />
<b>Method:</b> Obtain plasmid DNA from maxipreps. <br><br />
<br />
<u>Restriction Reactions:</u><br />
<br />
For lumazine and mms6 - <br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x5.5)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>7.6<td>41.8<br />
<tr><td>Red Buffer (10x)</td><td>2<td>11<br />
<tr><td>pDNA</td><td>10<td><br />
<tr><td>EcoRI</td><td>0.2<td>1.1<br />
<tr><td>SpeI</td><td>0.2<td>1.1<br />
</table><br />
<br />
For dT - <br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>Reaction Mix(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>38<br />
<tr><td>Orange Buffer (10x)</td><td>10<br />
<tr><td>pDNA</td><td>50<br />
<tr><td>XbaI</td><td>1<br />
<tr><td>EcoRI</td><td>1<br />
</table><br />
<br />
Incubated at 37<sup>o</sup>C for 1.5 hours. Heat killed enzymes for 20 minutes at 80<sup>o</sup>C.<br />
<br />
<u>Ligation Reactions:</u><br />
<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x5.5)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>13.8<td>75.9<br />
<tr><td>T4 Ligase Buffer (10x)</td><td>2<td>11<br />
<tr><td>Plasmid (dT)</td><td>2<td>11<br />
<tr><td>Cut out part (Lumazine/mms6)</td><td>2<td><br />
<tr><td>T4 DNA Ligase</td><td>0.2<td>1.1<br />
</table> <br />
<br />
Added 18(&micro;L) MM to each rxn tube. Incubated at one hour and overnight at room temperature. <br />
<br />
<u>Screening via PCR amplification :</u> Thermocycler set to iGEM program 11<br><br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x5.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>36.8<td>185.9<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>27.5<br />
<tr><td>dNTPs<td>2<td>11<br />
<tr><td>VF2 Primer<td>2<td>11<br />
<tr><td>VR Primer<td>2<td>11<br />
<tr><td>Template DNA<td>2<td><br />
<tr><td>Pfu polymerase<td>0.2<td>1.1<br />
</table><br><br />
<br />
Added 45 (&micro;L) MM to each tube.<br />
<br />
Analyzed PCR products of BioBrick standard assembly; 3 part (or 3 antibiotic) assembly; and 3 part (3AB) Intermediate/2 part assembly on a 2% TAE agarose gel. <br><br />
<b>Results:</b><br><br />
[[image:Lethbridge_100815PCRAssembly.JPG|200px]]<br />
<br />
<br />
----<br />
(In Lab: ADS)<br><br />
<br />
<b>Objective:</b> Produce large quantities of pSB1A3, pSB1C3 and pSB1T3.<br><br />
<br />
<b>Method:</b><br><br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x3.2)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>75<td>240<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>10<td>32<br />
<tr><td>dNTPs<td>5<td>16<br />
<tr><td>Primer (SB-prep-2Ea)<td>3.5<td>11.2<br />
<tr><td>Primer (SB-prep-3P)<td>3.5<td>11.2<br />
<tr><td>Template DNA<td>2<td><br />
<tr><td>Pfu polymerase<td>1<td>3.2<br />
</table><br><br />
<br />
Added 98(&micro;L) to each tube. Used PLASRB PCR Protocol from Aug. 13, 2010.<br />
<br />
==<font color="white">Aug 16, 2010 ==<br />
<br />
(In Lab: KG)<br><br />
<br />
<b>Objective:</b> Confirm overnight ligations done on August 15, 2010. <br><br />
<br />
<b>Method:</b><br><br />
<br />
<u>Screening via PCR amplification :</u> Thermocycler set to iGEM program 4<br><br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>33.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<br />
<tr><td>dNTPs<td>2<br />
<tr><td>VF2 Primer<td>2<br />
<tr><td>VR Primer<td>2<br />
<tr><td>Template DNA<td>5<br />
<tr><td>Pfu polymerase<td>0.2<br />
</table><br><br />
<br />
3AB Master Mix<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>Master Mix(x5.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>185.9<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>27.5<br />
<tr><td>dNTPs<td>11<br />
<tr><td>Prefix Primer<td>11<br />
<tr><td>Suffix Primer<td>11<br />
<tr><td>Template DNA<td>2<br />
<tr><td>Pfu polymerase<td>1.1<br />
</table><br><br />
<br />
BBS/pSB1C3 Master Mix<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>Master Mix(x11)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>371.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>55<br />
<tr><td>dNTPs<td>22<br />
<tr><td>VF2 Primer<td>22<br />
<tr><td>VR Primer<td>22<br />
<tr><td>Template DNA<td><br />
<tr><td>Pfu polymerase<td>2.2<br />
</table><br><br />
<br />
Analyzed PCR products of overnight BioBrick standard assembly; 3 part (or 3 antibiotic) assembly; and 3 part (3AB) Intermediate/2 part assembly on a 2% TAE agarose gel. <br><br />
<b>Results:</b><br><br />
[[image:Lethbridge_100816PostLigationPCRScreen.JPG|200px]]<br />
<br />
==<font color="white">Aug 16, 2010 Evening ==<br />
<br />
(In Lab: KG, AS)<br><br />
<br />
<b>Objective:</b> Restriction of PCR Products (mms6-dT, lumazine-dT). Restriction is necessary for ligation into plasmid backbone pSB1C3 <br><br />
<br />
<b>Method:</b><br><br />
<br />
<u>Restriction Reactions:</u><br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x5.5)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>7.6<td>41.8<br />
<tr><td>Orange Buffer (10x)</td><td>2<td>11<br />
<tr><td>pDNA</td><td>10<td><br />
<tr><td>PstI</td><td>0.2<td>1.1<br />
<tr><td>SpeI</td><td>0.2<td>1.1<br />
</table><br />
<br />
Incubated at 37<sup>o</sup>C for 1.5 hours. Heat killed enzymes for 2 minutes at 80<sup>o</sup>C.<br />
<br />
<u>Ligation Reactions:</u><br />
<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x5.5)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>13.8<td>75.9<br />
<tr><td>T4 Ligase Buffer (10x)</td><td>2<td>11<br />
<tr><td>Plasmid Backbone (pSB1C3)</td><td>2<td>11<br />
<tr><td>pDNA</td><td>2<td><br />
<tr><td>T4 DNA Ligase</td><td>0.2<td>1.1<br />
</table><br />
<br />
----<br />
(In Lab: KG)<br><br />
<br />
<b>Objective:</b> Transformations of insertions of mms6 or lumazine into pET28a. <br><br />
<br />
<b>Method:</b> used [[Team:Lethbridge/Notebook/Protocols|Competent Cell Transformation]] protocol<br />
* changes:<br />
**used 50&micro;L aliquottes of DH5&alpha<br />
**did not pipette up and down once, the cells were just swirled 3 times<br />
**added 400&micro;L SOC media, shoock at 37<sup>0</sup>C for 90 min<br />
**platted 250&micro;L and 150&micro;L<br><br />
<br />
<table border ="3"><br />
<td><b>results</b><br />
<tr><td>contents<td><b>&250&micro;L</b><td><b>150&micro;L</b><br />
<tr><td>+ control(pUC19)<td>good<td>good<br />
<tr><td>mms6<td>good<td>good<br />
<tr><td>mms6-2<td>good<td>good<br />
<tr><td>mms6<td>good<td>x<br />
<tr><td>Lumazine<td>good<td>x<br />
<tr><td>Lumazine<td>good<td>x<br />
</table><br><br />
<br />
==<font color="white">Aug 17, 2010 ==<br />
<br />
(In Lab: JV)<br><br />
<br />
<b>Objective:</b> Confirm ligations done on August 16, 2010. <br><br />
<br />
<b>Method:</b><br><br />
<u>Screening via PCR amplification :</u> Thermocycler set to iGEM program 4<br><br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x5.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>12.8<td>70.4<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>11<br />
<tr><td>dNTPs<td>1<td>5.5<br />
<tr><td>VF2 Primer<td>1<td>5.5<br />
<tr><td>VR Primer<td>1<td>5.5<br />
<tr><td>Template DNA<td>2<td><br />
<tr><td>Pfu polymerase<td>0.2<td>1.1<br />
</table><br><br />
<br />
Ran a 2% Agarose gel in 1X TAE buffer for 65 minutes at 100V.<br><br />
[[image:Lethbridge_100817PostLigationPCRTest.JPG|100px]]<br />
<br />
==<font color="white">Aug 17, 2010 Evening ==<br />
<br />
(In Lab: AS)<br><br />
<br />
<b>Objective:</b> Repeat ligation of mms6-dT and lumazine-dT to pSB1C3. <br><br />
<b>Method:</b> Use already restricted mms6-dT and lumazine-dT. Restrict pSB1C3 PCR product with EcoRI and PstI.<br><br />
<br />
<u>Restriction Reactions:</u><br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>28.6<br />
<tr><td>Orange Buffer (10x)</td><td>6<br />
<tr><td>pDNA (pSB1C3)</td><td>25<br />
<tr><td>PstI</td><td>0.2<br />
<tr><td>EcoRI</td><td>0.2<br />
</table><br />
<br />
Incubated at 37<sup>o</sup>C for 1.5 hours. Heat killed enzymes for 20 minutes at 80<sup>o</sup>C.<br />
<br />
<u>Ligation Reactions:</u><br />
<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x5.5)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>13.8<td>75.9<br />
<tr><td>T4 Ligase Buffer (10x)</td><td>2<td>11<br />
<tr><td>Part 2 (pSB1C3)</td><td>2<td>11<br />
<tr><td>Part 1 (mms6-dT/Lum-dT)</td><td>2<br />
<tr><td>T4 DNA Ligase</td><td>0.2<td>1.1<br />
</table><br />
<br />
Added 18(&micro;L) MM to each rxn tube. Incubated overnight at room temperature.<br />
<br />
----<br />
<b>Objective:</b> Ligation confirmation by PCR. 2 different PCR reaction conditions were utilized. Believe PstI is not being heat inactivated. <br><br />
<br />
<b>Method:</b><br><br />
PCR 1 - Show complete insertion of mms6-dT, lumazine-dT into pSB1C3. Both EcoRI and PstI ligations occurred.<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x11)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>33.8<td>371.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>55<br />
<tr><td>dNTPs<td>2<td>22<br />
<tr><td>Forward VF2 Primer<td>2<td>22<br />
<tr><td>Reverse VR Primer<td>2<td>22<br />
<tr><td>Template DNA<td>5<td><br />
<tr><td>Pfu polymerase<td>0.2<td>2.2<br />
</table><br><br />
<br />
Added 45(&micro;L) MM to each rxn tube.<br />
<br />
PCR 2 - Show PstI is not heat killed and only EcoRI ligation occurred.<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x16)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>33.8<td>540.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>80<br />
<tr><td>dNTPs<td>2<td>32<br />
<tr><td>Forward VF2 Primer<td>2<td>32<br />
<tr><td>Reverse VR Primer<td>2<td>32<br />
<tr><td>Template DNA<td>5<td><br />
<tr><td>Pfu polymerase<td>0.2<td>3.2<br />
</table><br><br />
<br />
Added 45(&micro;L) MM to each rxn tube.<br />
----<br />
<b>Objective:</b> Analyze tendency of PstI to be heat killed. <br><br />
<b>Hypothesis:</b> Ligation will be counteracted by PstI digestion even following 20 minute heat kill at 80<sup>o</sup>C .<br><br />
<b>Method:</b> Restrict plasmid DNA with PstI (and EcoRI control). Reactions stopped by heat killing. Plasmids re-ligated with T4 DNA Ligase. Ligation confirmed with PCR. If PCR product is present then enzyme heat killed but if there is no PCR Product then enzyme was not heat killed. <br><br />
<br />
<u>Restriction Reactions:</u><br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>12.8<br />
<tr><td>Orange Buffer (10x)</td><td>2<br />
<tr><td>pDNA (dT)</td><td>5<br />
<tr><td>PstI</td><td>0.2<br />
<tr><td>EcoRI (control only)</td><td>0.2<br />
</table><br />
<br />
Incubated at 37<sup>o</sup>C for 1.5 hours. Heat killed enzymes for 20 minutes at 80<sup>o</sup>C.<br />
<br />
<u>Ligation Reactions:</u><br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>15.8<br />
<tr><td>T4 Ligase Buffer (10x)</td><td>2<br />
<tr><td>Restriction Mix</td><td>2<br />
<tr><td>T4 DNA Ligase</td><td>0.2<br />
</table><br />
<br />
Incubated at room temperature overnight. <br />
<br />
PCR to confirm Ligation<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x2.2)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>33.8<td>74.36<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>11<br />
<tr><td>dNTPs<td>2<td>4.4<br />
<tr><td>Forward VF2 Primer<td>2<td>4.4<br />
<tr><td>Reverse VR Primer<td>2<td>4.4<br />
<tr><td>Template DNA<td>5<td><br />
<tr><td>Pfu polymerase<td>0.2<td>0.44<br />
</table><br><br />
<br />
Added 45(&micro;L) to each reaction tube.<br />
<br />
==<font color="white">Aug 18, 2010 ==<br />
<br />
(In Lab: JV)<br><br />
<b>Objective:</b> Confirmation of ADS' analysis of PstI's tendency to be heat killed. <br><br />
<b>Method:</b> Will PCR ligation reactions from different assembly methods and EcoRI and PstI controls. <br><br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>33.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<br />
<tr><td>dNTPs<td>2<br />
<tr><td>Forward VF2 Primer<td>2<br />
<tr><td>Reverse VR Primer<td>2<br />
<tr><td>Template DNA<td>5<br />
<tr><td>Pfu polymerase<td>0.2<br />
</table><br><br />
Ran on cycle 4 of thermocycler. <br />
<br />
Viewed PCRs on 2% TAE agarose gel that ran at 110 V for 30 minutes.<br><br />
[[image:Lethbridge_100818AssemblyandHeatKillPCR.JPG|200px]]<br />
----<br />
(In Lab: HB)<br><br />
<b>Objective:</b> Analyze tendency of PstI to be heat killed. <br><br />
<b>Method:</b> Restrict plasmid DNA with PstI (and EcoRI control). Reactions stopped by heat killing. Plasmids re-ligated with T4 DNA Ligase. Ligation confirmed with PCR. If PCR product is present then enzyme heat killed but if there is no PCR Product then enzyme was not heat killed. <br><br />
<br />
<u>Restriction Reactions:</u><br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>12.8<br />
<tr><td>Orange Buffer (10x)</td><td>2<br />
<tr><td>pDNA (dT)</td><td>5<br />
<tr><td>PstI</td><td>0.2<br />
<tr><td>EcoRI (control only)</td><td>0.2<br />
</table><br />
<br />
Incubated at 37<sup>o</sup>C for 1.5 hours. Heat killed enzymes for 20 minutes at 80<sup>o</sup>C.<br />
<br />
<u>Ligation Reactions:</u><br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>15.8<br />
<tr><td>T4 Ligase Buffer (10x)</td><td>2<br />
<tr><td>Restriction Mix</td><td>2<br />
<tr><td>T4 DNA Ligase</td><td>0.2<br />
</table><br />
<br />
Incubated at room temperature overnight. <br />
<br />
PCR to Confirm Ligation<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x6.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>33.8<td>219.7<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>32.5<br />
<tr><td>dNTPs<td>2<td>13<br />
<tr><td>Forward VF2 Primer<td>2<td>13<br />
<tr><td>Reverse VR Primer<td>2<td>13<br />
<tr><td>Template DNA<td>5<td><br />
<tr><td>Pfu polymerase<td>0.2<td>1.3<br />
</table><br><br />
<br />
Added 45(&micro;L) to each reaction tube.<br />
Used thermocycler iGEM Program 4. <br />
<br />
3 different treatments: 1. Restricted, heat killed and ligated. 2. Restricted and heat killed. 3. Restricted and not heat killed.<br />
<br />
==<font color="white">Aug 19, 2010 ==<br />
<br />
(In Lab: JV)<br><br />
<br />
<b>Objective:</b> Determine if any transformations from Aug 16, 2010 have the correct insert. <br><br />
<b>Method:</b> Pick colonies and incubate at 37<sup>o</sup>C in LB Media with Kan overnight. Use QIAGEN method to extract plasmid DNA. Restrict plasmid DNA to determine if mms6 or lumazine has correctly ligated into pET-28(A). <br><br />
<br />
<u>Restriction Reactions:</u><br />
<br />
mms6 RESTRICTED - <br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x31)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>15.75<td>488.25<br />
<tr><td>Red Buffer (10x)</td><td>2<td>62<br />
<tr><td>Template DNA</td><td><td><br />
<tr><td>Enzyme (EcoRV)</td><td>0.25<td>7.75<br />
</table><br />
<br />
mms6 UNRESTRICTED - <br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x31)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>16<td>496<br />
<tr><td>Red Buffer (10x)</td><td>2<td>62<br />
<tr><td>Template DNA</td><td><td><br />
</table><br />
<br />
lumazine RESTRICTED - <br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x31)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>15.75<td>488.25<br />
<tr><td>Tango Buffer (10x)</td><td>2<td>62<br />
<tr><td>Template DNA</td><td><td><br />
<tr><td>Enzyme (EcoRV)</td><td>.25<td>7.75<br />
</table><br />
<br />
lumazine UNRESTRICTED - <br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x31)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>16<td>496<br />
<tr><td>Tango Buffer (10x)</td><td>2<td>62<br />
<tr><td>Template DNA</td><td><td><br />
</table><br />
<br />
Added 18(&micro;L) to each restriction digest reaction. Incubated at 37<sup>o</sup>C for 1.5 hours. Heat killed enzymes for 20 minutes at 80<sup>o</sup>C.<br />
<br />
Samples were run on a 2% agarose gel in 1X TAE Buffer. <br />
<br />
----<br />
(In Lab: HB)<br><br />
<br />
<b>Objective:</b> Run a PCR testing VF2/Suffix and Prefix/VR. A colony PCR of lumazine and mms6 in pET28a. A PCR of 15 ligation tests. <br><br />
(In Lab: JV)<br><br />
<b>Objective:</b> Screen for colonies with the correct insert from Aug. 4, 2010 transformations. <br><br />
<br />
<b>Method:</b> Colony PCR. Changes - Used pipette tip instead of toothpick. Put colony in 20(&micro;L) autoclaved Milli-Q water. <br><br />
<br />
<u>PCR:</u> Thermocycler set to iGEM Program 4<br> <br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>9.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<br />
<tr><td>dNTPs<td>2<br />
<tr><td>Forward Primer (VF2)<td>2<br />
<tr><td>Reverse Primer (VR)<td>2<br />
<tr><td>Template DNA (Cell Lysate)<td>2<br />
<tr><td>Pfu polymerase<td>0.2<br />
</table><br><br />
<br />
Added 18&micro;L Master Mix to each reaction tube.<br />
<br />
<b>Method:</b> Control test of primer combinations. <br><br />
<br />
Combination 1 - VF2/Suffix<br />
Combination 2 - Prefix/VR<br />
Combination 3 - Prefix/Suffix<br />
dT maxiprep used as known template DNA source.<br />
<br />
<u>PCR Combination 1:</u><br> <br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>9.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<br />
<tr><td>dNTPs<td>2<br />
<tr><td>Forward Primer (Prefix)<td>2<br />
<tr><td>Reverse Primer (Suffix)<td>2<br />
<tr><td>Template DNA (dT)<td>2<br />
<tr><td>Pfu polymerase<td>0.2<br />
</table><br><br />
<br />
<u>PCR Combination 2:</u><br> <br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>9.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<br />
<tr><td>dNTPs<td>2<br />
<tr><td>Forward Primer (Prefix)<td>2<br />
<tr><td>Reverse Primer (VR)<td>2<br />
<tr><td>Template DNA (dT)<td>2<br />
<tr><td>Pfu polymerase<td>0.2<br />
</table><br><br />
<br />
<u>PCR Combination 3:</u><br> <br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>9.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<br />
<tr><td>dNTPs<td>2<br />
<tr><td>Forward Primer (VF2)<td>2<br />
<tr><td>Reverse Primer (suffix)<td>2<br />
<tr><td>Template DNA (dT)<td>2<br />
<tr><td>Pfu polymerase<td>0.2<br />
</table><br><br />
<br />
<b>Method:</b> PCR of 15 ligation tests. <br><br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x19.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>9.8<td>191.1<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>39<br />
<tr><td>dNTPs<td>2<td>39<br />
<tr><td>Forward VF2 Primer<td>2<td>39<br />
<tr><td>Reverse VR Primer<td>2<td>39<br />
<tr><td>Template DNA<td>2<td><br />
<tr><td>Pfu polymerase<td>0.2<td>3.9<br />
</table><br><br />
<br />
Added 18(&micro;L) to each tube. <br />
----<br />
(In Lab: JV)<br><br />
Analyzed HB's PCR products on 2% TAE gel which ran for 60 minutes at 100 V.<br><br />
[[image:Lethbridge_100819PCRMania.JPG|200px]]<br />
<br />
==<font color="white">Aug 20, 2010==<br />
<br />
(In Lab: JV)<br><br />
<br />
<b>Objective:</b> Determine if attempts to PCR amplify plasmid backbone were successful. <br><br />
<br />
<b>Method:</b> Ran samples on 1% agarose gel with 1X TAE buffer for 50 minutes at 100 V. <br><br />
<br />
<b>Results:</b> DNA concentration was good, however there was no evidence of an insert into pET-28(A). <br><br />
<br />
[[image:Lethbridge_100819pET28aTransformScreenGel1.JPG|200px]]<br><br><br />
[[image:Lethbridge_100819pET28aTransformScreenGel2.JPG|200px]]<br><br><br />
[[image:Lethbridge_100819pET28aTransformScreenGel3.JPG|200px]]<br><br><br />
[[image:Lethbridge_100819pET28aTransformScreenGel4.JPG|200px]]<br><br><br />
<br />
==<font color="white">Aug 23, 2010==<br />
<br />
(In Lab: JV)<br><br />
<br />
<b>Objective:</b> Obtained part <partinfo>BBa_K118021</partinfo> and <partinfo>BBa_I716462</partinfo>. <br><br />
<br />
<b>Method:</b> Used competent cell transformation protocol. <br><br />
<br />
----<br />
(In Lab: HB)<br><br />
<br />
<b>Objective:</b> Restrict 18 maxiprepped parts to quantify DNA. <br><br />
<br />
<b>Method:</b> Restrict all 18 parts and run on a 1% agarose gel with unrestricted parts. <br><br />
<br />
<u>Restriction Reactions:</u><br />
<br />
Restriction Mix -<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x19)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>12.8<td>243.2<br />
<tr><td>Orange Buffer (10x)</td><td>2<td>38<br />
<tr><td>Template DNA</td><td>5<td><br />
<tr><td>EcoRI</td><td>0.2<td>3.8<br />
</table><br />
<br />
Unrestricted Mix -<br />
<table><table border ="3"><br />
<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x19)(&micro;L)</b><br />
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>13<td>247<br />
<tr><td>Orange Buffer (10x)</td><td>2<td>38<br />
<tr><td>Template DNA</td><td>5<td><br />
</table><br />
<br />
15(&micro;L) added to each rxn tube.<br><br />
Incubated at 37<sup>o</sup>C for 1.5 hours. <br><br />
<br />
[[image:Lethbridge_100823HBMaxipreps.jpg|200px]]<br />
----<br />
(In Lab: AV)<br><br />
<b>Objective:</b> To PCR amplify pSB1T3, pSB1C3, pSB1A3 and run on 1% agarose gel. <br><br />
<br />
<b>Method:</b><br><br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x6.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>14.9<td>96.85<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>13<br />
<tr><td>dNTPs<td>1<td>6.5<br />
<tr><td>SB-prep-2 Primer<td>0.7<td>4.55<br />
<tr><td>SB-prep-3p Primer<td>0.7<td>4.55<br />
<tr><td>Template DNA<td>0.5<td><br />
<tr><td>Pfu polymerase<td>0.2<td>1.3<br />
</table><br><br />
<br />
Added 19.5(&micro;L) to each tube. <br />
Ran on program 6 of thermocycler.<br />
<br />
<b>Results:</b> Nothing visible on gel, therefore will have to try loading a larger volume of PCR product.<br><br />
[[image:Lethbridge_100823AVBackbonePCR.jpg|100px]]<br />
<br />
==<font color="white">Aug 24, 2010==<br />
<br />
(In Lab: AV)<br><br />
<b>Objective:</b> To re-run a 1% agarose gel of PCR products from Aug 23, 2010. <br><br />
<b>Results:</b> Nothing appeared on gel, therefore the PCR was unsuccessful. <br><br />
<br />
==<font color="white">Aug 25, 2010==<br />
<br />
(In Lab: JV)<br><br />
<b>Objective:</b> Create amounts of pSB1C3. <br><br />
<br />
<b>Method:</b><br><br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x6.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>14.9<td>96.85<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>13<br />
<tr><td>dNTPs<td>1<td>6.5<br />
<tr><td>SB-prep-2 Primer<td>0.7<td>4.55<br />
<tr><td>SB-prep-3p Primer<td>0.7<td>4.55<br />
<tr><td>Template DNA<td>0.5<td><br />
<tr><td>Pfu polymerase<td>0.2<td>1.3<br />
</table><br><br />
<br />
Added 19.5(&micro;L) to each tube.<br />
<br />
PCR- Conditions:<br />
1. 95<sup>o</sup>C for 5 min<br />
2. 94<sup>o</sup>C for 30 sec<br />
3. 55<sup>o</sup>C for 30 sec<br />
4. 68<sup>o</sup>C for 4 min<br />
5. 68<sup>o</sup>C for 10 min<br />
6. 4<sup>o</sup>C infinitely<br />
(36 cycles)<br />
<br />
==<font color="white">Aug 25, 2010==<br />
(In Lab: FM)<br><br />
<b>Objective:</b> Gradient PCR of xylE. <br><br />
<br />
<b>Method:</b><br><br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x10)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>5.8<td>58<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>1<td>10<br />
<tr><td>dNTPs<td>1<td>10<br />
<tr><td>Standard Prefix or Fusion Prefix Primer<td>0.5<td>5<br />
<tr><td>Standard Suffix or Fusion Suffix Primer<td>0.5<td>5<br />
<tr><td>Template DNA<td>1<td>10<br />
<tr><td>Pfu polymerase<td>0.2<td>2<br />
</table><br><br />
<br />
Gradient Temperatures:<br />
58.5<sup>o</sup>C, 60.5<sup>o</sup>C, 62.3<sup>o</sup>C, 64.1<sup>o</sup>C, 65.9<sup>o</sup>C, 67.7<sup>o</sup>C, 69.4<sup>o</sup>C, 71.1<sup>o</sup>C<br />
-----<br />
(In Lab: JS)<br><br />
<b>Objective:</b> Run a 2% agarose gel of gradient PCR of xylE. <br><br />
<br />
[[image:Lethbridge_100826 xylE PCR Fix.jpg|200px]]<br />
<br />
==<font color="white">Aug 26, 2010==<br />
(In Lab: KG)<br><br />
<b>Objective:</b> Do PCR from Aug 25, 2010 to compare PCR of part from registry and our pSB1C3. <br><br />
<br />
<b>Method:</b><br><br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x2)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>14.9<td>29.8<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>4<br />
<tr><td>dNTPs<td>1<td>2<br />
<tr><td>SB-prep-26 Primer<td>0.7<td>1.4<br />
<tr><td>SB-prep-3P Primer<td>0.7<td>1.4<br />
<tr><td>Template DNA<td>0.5<td><br />
<tr><td>Pfu polymerase<td>0.2<td><br />
</table><br><br />
<br />
PCR- Conditions:<br />
1. 95<sup>o</sup>C for 5 min<br />
2. 94<sup>o</sup>C for 30 sec<br />
3. 55<sup>o</sup>C for 30 sec<br />
4. 68<sup>o</sup>C for 4 min<br />
5. 68<sup>o</sup>C for 10 min<br />
6. 4<sup>o</sup>C infinitely<br />
(36 cycles)<br />
<br />
Ran a 1% agarose gel of gradient PCR of xylE.<br />
Was stained in ethidium bromide for too long.<br />
<br />
==<font color="white">Aug 31, 2010==<br />
(In Lab: DM)<br><br />
<b>Objective:</b> Overexpression test of xylE part (BBa_K118021) in DH5alpha cells in m9 minimal media. <br><br />
<b>Method:</b> [[Team:Lethbridge/Notebook/Protocols|Overexpression]]<br />
<br />
0.5 M Catechol was made, to allow for the addition of 1(&micro;L) of this stock solution to be added to the samples taken during the overexpression.<br />
Upon addition of catechol, the solutions turned yellow!<br />
1 mL samples were taken for SDS-PAGE analysis. <br />
Optical density readings at 600 nm and absorbance readings at 375 and 275 nm were recorded for flasks containing either glucose or sucrose.<br />
----<br />
(In Lab: ADS)<br><br />
<b>Objective:</b> PCR amplify plasmid backbones (pSB1C3, pSB1A3 and pSB1T3). <br><br />
<b>Method:</b> Use PCR product from Aug 13, 2010 as template DNA.<br />
<br />
<table border ="3"><br />
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x3.5)(&micro;L)</b><br />
<tr><td>Milli-Q H<sub>2</sub>O<td>14.4<td>52.4<br />
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>7<br />
<tr><td>dNTPs<td>1<td>3.5<br />
<tr><td>SB-prep-26 Primer<td>0.7<td>2.45<br />
<tr><td>SB-prep-3P Primer<td>0.7<td>2.45<br />
<tr><td>Template DNA<td>1<td><br />
<tr><td>Pfu polymerase<td>0.2<td>0.7<br />
</table><br><br />
<br />
Added 19(&micro:L) to each tube. <br />
Phusion polymerase was used and apparently in the other previously unsuccessful PCRs, instead of Pfu polymerase. <br />
<br />
PCR- Conditions:<br />
1. 95<sup>o</sup>C for 5 min<br />
2. 94<sup>o</sup>C for 30 sec<br />
3. 55<sup>o</sup>C for 30 sec<br />
4. 68<sup>o</sup>C for 4 min<br />
5. 68<sup>o</sup>C for 10 min<br />
6. 4<sup>o</sup>C infinitely<br />
(36 cycles)<br />
<br />
Analyzed PCR on 1% TAE agarose gel which ran for 60 min at 100 V. <br />
----<br />
<b>Objective:</b> Obtain new sources of pSB1T3, pSB1C3 and pSB1A3 plasmid backbone. Backbone from registry used up. <br><br />
<br />
<b>Method:</b> pSB1A3 can be obtained from anything team already possesses. pSB1C3 can be obtained from BBa_J04450 (RFP) in kit. Don't have tetracycline plates so cannot obtain pSB1T3. Used competent cell transformation protocol. <br><br />
<br />
<b>Method:</b> pSB1A3 can be obtained from anything team already possesses. pSB1C3 can be obtained from BBa_J04450 (RFP) in kit. Don't have tetracycline plates so cannot obtain pSB1T3. Used [[Team:Lethbridge/Notebook/Protocols|Competent Cell Transformation]] protocol. <br><br />
<br />
* changes:<br />
**used 50&micro;L aliquottes of DH5&alpha<br />
**did not pipette up and down once, the cells were just swirled 3 times<br />
**added 500&micro;L SOC media, shoock at 37<sup>0</sup>C for 90 min<br />
**platted 250&micro;L and 150&micro;L<br><br />
<br />
<table border ="3"><br />
<td><b>Results</b><br />
<tr><td>contents<td><b>&250&micro;L</b><td><b>150&micro;L</b><br />
<tr><td>+ control(pUC19)<td>good<td>good<br />
<tr><td>J04450<td>X (TMTC)<td>X(TMTC)<br />
</table><br><br />
<br />
Prepared overnight cultures for minipreps. Added 5(&micro;L) of 35 mg/mL Chloramphenicl to 5 mL of LB Media. Inoculated media with cells from single colony picked from transformation plate. Incubated overnight at 37<sup>o</sup>C with shaking.<br />
<br><br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/JulyTeam:Lethbridge/Notebook/Lab Work/July2010-10-27T19:16:09Z<p>Liszabruder: </p>
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<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/April"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8a/UofLapril.jpg" width="60"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/May"><br />
<img src="https://static.igem.org/mediawiki/2010/7/7b/UofLmaybutton.jpg" width="60"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/June"><br />
<img src="https://static.igem.org/mediawiki/2010/8/80/UofLjunebutton.jpg" width="60"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/July"><br />
<img src="https://static.igem.org/mediawiki/2010/5/53/UofLjulybutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/August"><br />
<img src="https://static.igem.org/mediawiki/2010/1/15/UofLaugustbutton.jpg" width="60"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/September"><br />
<img src="https://static.igem.org/mediawiki/2010/4/4d/UofLseptemberbutton.jpg" width="60"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/October"><br />
<img src="https://static.igem.org/mediawiki/2010/4/4e/UofLoctoberbutton.jpg" width="60"/><br />
</a><br />
</th><br />
<br />
<br />
<br />
<tr><br />
</table><br />
</center><br />
</body><br />
</html><br />
<hr><br />
<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">July 2010=<br />
==<font color="white">July 3/2010==<br />
(In Lab: JS)<br><br />
<br />
<b>Objective:</b> To restrict and ligate pBAD-TetR and YEPs into pSB1C3 backbone.<br><br />
<br />
<b>Method:</b> Used [[Team:Lethbridge/Notebook/Protocols|Restriction of Plasmid DNA]] protocol and ligated the parts into pSB1C3.<br><br />
<br />
==<font color="white">July 5/2010==<br />
(In Lab: JV, AV, HB)<br><br />
<br />
<b>Objective:</b> Run a 1% agarose gel of purified PCR samples from June 24/10<br><br />
<br />
<b>Method:</b><br><br />
<br />
<table><table border ="3"><br />
<tr><td><b>Lane</b></td><td><b>Sample</b></td><td><b>Components (&micro;L)</b></td></tr><br />
<tr><td>1</td><td>1kb Ladder</td><td>0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>2</td><td>1 - pBAD (A4)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>3</td><td>2 - pBAD (A5)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>4</td><td>3 - SRBS (A6)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>5</td><td>4 - SRBS (A7)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>6</td><td>5 - CFP Complete (A8)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>7</td><td>6 - SRBS (A10)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>8</td><td>7 - EYFP (B1)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>9</td><td>8 - N term tag (B2)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>10</td><td>9 - pSB NEYFP (B4)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>11</td><td>10 - pSB NEYFP (B5)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>12</td><td>11 - CFP (B6)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>13</td><td>12 - pBAD-TetR (B10)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>14</td><td>13 - D3</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>15</td><td>14 - C term (D4)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>16</td><td>15 - C term (D5)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>17</td><td>16 - pLacI (D6)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>18</td><td>17 - NEYFP (E2)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>19</td><td>18 - CEYFP (E6)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>20</td><td>19 - CEYFP (E7)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>1</td><td>1kb ladder</td><td>0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>2</td><td>20 - EYFP (E8)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>3</td><td>21 - EYFP (E9)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>4</td><td>22 - EYFP (E10)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>5</td><td>23 - ECFP (F1)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>6</td><td>24 - ECFP (F2)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>7</td><td>25 - ECFP (F3)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>8</td><td>26 - pBAD-TetR (F4)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>9</td><td>27 - pBAD-TetR (F5)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>10</td><td>28 - EYFP (G1)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>11</td><td>29 - pSB CEYFP (G4)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>12</td><td>30 - pBAD (1) (G6)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>13</td><td>31 - pBAD (2) (G7)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>14</td><td>32 - N term tag (G8)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
<tr><td>15</td><td>33 - lumazine (G9)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr><br />
</table><br><br />
<br />
Ran gel at 100V for 45 minutes.<br><br />
<br />
<b>Results:</b><br />
[[image:Lethbridge_100705AVPCR.JPG|200px]]<br><br />
<br />
==<font color="white">July 5/2010 Evening==<br />
<br />
<b>Objective:</b> To over-express CFP complete in DH5&alpha;<br><br />
<br />
<b>Method:</b><br><br />
<br />
1) Inoculated 6mL culture with ampicillin and glycerol stock A6-May 13,2010 CFP complete<br />
2) Went in the shaker at 6:50pm<br />
<br />
==<font color="white">July 6/2010==<br />
(In lab: JV, AV, HB)<br><br />
<b>Objective:</b> To continue the over-expression of CFP complete in DH5&alpha;<br><br />
<br />
<b>Method:</b><br><br />
1) Put both cultures (taken out of shaker at 9:15am) and put them in 500mL of LB w/ Amp.<br><br />
2) initial OD was 0.071 (600&lambda;)<br><br />
<br />
Issue:<br><br />
*After checking the the sequencing it was evident that our promotor is always off. It is turned off by the product of the gene TetR. Which is not part of our construct. <br><br />
<br />
<table><table border ="3"><br />
<tr><td><b>Time (hours)</b></td><td><b>OD (600&lambda;)</b></td></tr><br />
<tr><td>0</td><td>0.071</td></tr><br />
<tr><td>1</td><td>0.390</td></tr><br />
<tr><td>1.5(T<sub>0</sub>)</td><td>0.606</td></tr><br />
<tr><td>2.5 (T<sub>1</sub>)</td><td>1.250</td></tr><br />
<tr><td>3.5(T<sub>2</sub>)</td><td>3.04</td></tr><br />
<tr><td>4.5(T<sub>3</sub>)</td><td>2.75</td></tr><br />
</table><br><br />
<br />
<b>Results:</b> Ran samples on a 15% SDS-page. The gel did not show any signs of over-expression.<br><br />
<br />
<br />
<b>Objective:</b> To determine if maxipreps were successful via restriction by NotI and running on 1% Agarose gel<br><br />
<br />
<b>Method:</b><br><br />
1) Restriction:<br><br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td></tr><br />
<tr><td>DNA</td><td>0.071</td></tr><br />
<tr><td>Buffer Orange</td><td>0.390</td></tr><br />
<tr><td>NotI</td><td>0.606</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>O</td><td>1.250</td></tr><br />
</table><br><br />
<br />
Incubated for 1 hour at 37<sup>o</sup>C.<br />
Heat killed on heat block at 80<sup>o</sup>C for 20 mins.<br><br />
<br />
2) 1% Agarose gel<br><br />
<table><table border ="3"><br />
<tr><td><b>Lane</b></td><td><b>Sample</b></td><td><b>Volume (&micro;L)</b></td></tr><br />
<tr><td>1</td><td>1 kb Ladder</td><td>0.5 Ladder + 2 Loading dye (6x) + 7.5 MilliQ H<sub>2</sub>O</td></tr><br />
<tr><td>2</td><td>PET28a restricted</td><td>8 DNA + 2 Loading dye (6x)</td></tr><br />
<tr><td>3</td><td>PET28a</td><td>8 DNA + 2 Loading dye (6x)</td></tr><br />
<tr><td>4</td><td>Lumazine restricted</td><td>8 DNA + 2 Loading dye (6x)</td></tr><br />
<tr><td>5</td><td>Lumazine</td><td>8 DNA + 2 Loading dye (6x)</td></tr><br />
<tr><td>6</td><td>mms6 restricted</td><td>8 DNA + 2 Loading dye (6x)</td></tr><br />
<tr><td>7</td><td>mms6</td><td>8 DNA + 2 Loading dye (6x)</td></tr><br />
<tr><td>8</td><td>xylE restricted</td><td>8 DNA + 2 Loading dye (6x)</td></tr><br />
<tr><td>9</td><td>xylE</td><td>8 DNA + 2 Loading dye (6x)</td></tr><br />
<tr><td>10</td><td>Empty</td><td>Empty</td></tr><br />
</table><br><br />
<br />
Ran at 100V for 80 mins.<br><br />
<br />
<b>Results:</b><br />
[[image:Lethbridge_100705AV BW (2).jpg|200px]]<br><br />
<br />
<font color ="Red">More to fill in, but Anthony does not understand the stuff written in the lab book</font><br><br />
<br />
==<font color="white">July 8/2010==<br />
(In lab: JV, AV, HB, HS)<br><br />
<b>Objective:</b><br><br />
<br />
<font color ="red"> Anthony does not understand the stuff written in the lab book.</font><br />
<br />
==<font color="white">July 8/2010 - Evening==<br />
(In lab: KG)<br><br />
<b>Objective:</b>To transform TetR in pSB1A2 plasmid (BBa_C0040 - 2010 iGEM Distribution Kit Plate Well 4A) and pTetR in pSB1A2 plasmid (BBa_R0040 - 2010 iGEM Distribution Kit Plate Well 6) into DH5&alpha;.<br><br />
<br />
<b>Method:</b>Used the Competent Cell Transformation Protocol as well as transformed pUC19 as a positive control.<br><br />
<br />
<b>Results:</b><br />
<table><table border ="3"><br />
<tr><td><b>Plate</b></td><td><b>Number of Colonies</b></td></tr><br />
<tr><td>50 &micro;L TetR</td><td>0</td></tr><br />
<tr><td>200 &micro;L TetR</td><td>5</td></tr><br />
<tr><td>50 &micro;L pTetR</td><td>4</td></tr><br />
<tr><td>200 &micro;L pTetR</td><td>34</td></tr><br />
<tr><td>50 &micro;L pUC19</td><td>3</td></tr><br />
<tr><td>200 &micro;L pUC19</td><td>4</td></tr><br />
</table><br><br />
<br />
==<font color="white">July 9/2010==<br />
(In lab: JV)<br><br />
<b>Objective:</b>To overexpress pLacI-mRBS-mms6-dT construct.<br><br />
<br />
<b>Method:</b>Used the Overexpression Protocol.<br><br />
<table><table border ="3"><br />
<tr><td><b>Time (hours)</b></td><td><b>OD (600&lambda;)</b></td></tr><br />
<tr><td>0</td><td>0.052</td></tr><br />
<tr><td>1</td><td>0.111</td></tr><br />
<tr><td>2</td><td>0.315</td></tr><br />
<tr><td>2.5</td><td>0.449</td></tr><br />
<tr><td>3(T<sub>0</sub>)</td><td>0.772</td></tr><br />
<tr><td>4(T<sub>1</sub>)</td><td>2.22</td></tr><br />
<tr><td>5(T<sub>2</sub>)</td><td>2.06</td></tr><br />
<tr><td>6(T<sub>3</sub>)</td><td>2.50</td></tr><br />
</table><br><br />
<br />
Samples were run on a 15% SDS PAGE for 20 minutes at 80V and 1 hour at 200V.<br><br />
<br />
<font color ="red">SDS PAGE picture!!!!!!!!!!!</font><br />
<br />
==<font color="white">July 10/2010==<br />
(In lab: JV)<br><br />
<b>Objective:</b>To determine if maxipreps finished on July 9th and 10th have significant concentrations of DNA.<br><br />
<br />
<b>Method:</b>Run 1% Agarose gel<br><br />
<br />
<table><table border ="3"><br />
<tr><td><b>Lane</b></td><td><b>Sample</b></td><td><b>Components (&micro;L)</b></td></tr><br />
<tr><td>1<td>dT</td><td>2 DNA + 2 loading dye (6x) + 6 MilliQ H<sub>2</sub>O</td></tr><br />
<tr><td>2<td>mms6 (1)</td><td>2 DNA + 2 loading dye (6x) + 6 MilliQ H<sub>2</sub>O</td></tr><br />
<tr><td>3<td>mms6 (2)</td><td>2 DNA + 2 loading dye (6x) + 6 MilliQ H<sub>2</sub>O</td></tr><br />
<tr><td>4<td>pBAD-TetR (1)</td><td>2 DNA + 2 loading dye (6x) + 6 MilliQ H<sub>2</sub>O</td></tr><br />
<tr><td>5<td>pBAD-TetR (2)</td><td>2 DNA + 2 loading dye (6x) + 6 MilliQ H<sub>2</sub>O</td></tr><br />
<tr><td>6<td>mRBS</td><td>2 DNA + 2 loading dye (6x) + 6 MilliQ H<sub>2</sub>O</td></tr><br />
<tr><td>7<td>pLacI</td><td>2 DNA + 2 loading dye (6x) + 6 MilliQ H<sub>2</sub>O</td></tr><br />
<tr><td>8<td>sRBS</td><td>2 DNA + 2 loading dye (6x) + 6 MilliQ H<sub>2</sub>O</td></tr><br />
<tr><td>9<td>1 kb Ladder</td><td>0.5 ladder + 2 loading dye (6x) + 7.5 MilliQ H<sub>2</sub>O</td></tr><br />
<tr><td>10<td>Empty</td><td>Empty</td></tr><br />
</table><br><br />
<br />
Ran at 100V for 40 minutes. Strained in EtBr for 10 minutes.<br><br />
<br />
<b>Results:</b><br />
[[image:Lethbridge_100710JV BW.JPG|200px]]<br />
<br />
==<font color="white">July 12/2010==<br />
(In lab: AV,HB,JV)<br><br />
<b>Objective:</b> Maxiprep pLacI (A2) from glycerol stock.<br><br />
<br />
<b>Method:</b> Used Maxiprep Protocol. Cell pellet weighed 1.02g.<br><br />
<br />
<b>Objective:</b> Add dT to the end of mms6, xylE, and lumazine.<br />
<br />
<b>Method:</b><br><br />
1) Restrict "Part 1" BioBricks: mms6, xylE, and lumazine with EcoRI and SpeI.<br><br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td></tr><br />
<tr><td>pDNA</td><td>2</td></tr><br />
<tr><td>Red Buffer</td><td>2</td></tr><br />
<tr><td>EcoRI</td><td>0.25</td></tr><br />
<tr><td>SpeI</td><td>0.25</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>O</td><td>15.5</td></tr><br />
</table><br><br />
2) Restrict "Part 2" BioBrick: dT with EcoRI and XbaI.<br><br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td><td><b>2.5x Volume (&micro;L)</b></td></tr><br />
<tr><td>pDNA</td><td>2</td><td>5</td></tr><br />
<tr><td>Orange Buffer</td><td>2</td><td>5</td></tr><br />
<tr><td>EcoRI</td><td>0.25</td><td>0.625</td></tr><br />
<tr><td>XbaI</td><td>0.25</td><td>0.625</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>O</td><td>15.5</td><td>38.75</td></tr><br />
</table><br><br />
3) 1% Agarose gel (1x TAE) of restricted DNA<br />
<table><table border ="3"><br />
<tr><td><b>Lane</b></td><td><b>Sample</b></td><td><b>Component (&micro;L)</b></td></tr><br />
<tr><td>1</td><td>1 kb Ladder</td><td>0.5 ladder + 2 loading dye (6x) + 7.5 MilliQ H<sub>2</sub>O</td></tr><br />
<tr><td>2</td><td>dT</td><td>8 DNA + 2 loading dye (6x)</td></tr><br />
<tr><td>3</td><td>dT restricted</td><td>8 DNA + 2 loading dye (6x)</td></tr><br />
<tr><td>4</td><td>mms6</td><td>8 DNA + 2 loading dye (6x)</td></tr><br />
<tr><td>5</td><td>mms6 restricted</td><td>8 DNA + 2 loading dye (6x)</td></tr><br />
<tr><td>6</td><td>xylE</td><td>8 DNA + 2 loading dye (6x)</td></tr><br />
<tr><td>7</td><td>xylE restricted</td><td>8 DNA + 2 loading dye (6x)</td></tr><br />
<tr><td>8</td><td>Lumazine</td><td>8 DNA + 2 loading dye (6x)</td></tr><br />
<tr><td>9</td><td>Lumazine restricted</td><td>8 DNA + 2 loading dye (6x)</td></tr><br />
<tr><td>10</td><td>Empty</td><td>Empty</td></tr><br />
</table><br><br />
Ran at 100V for 43 minutes. Stained in EtBr for 10 minutes.<br><br />
<b>Results:</b><br />
[[image:Lethbridge_100712HB REstriction.JPG|200px]]<br><br />
<br />
4) Ligate restricted dT to the ends of the "Part 1" Biobricks.<br><br />
<br />
==<font color="white">July 12/2010 - Evening==<br />
(In lab: KG,TF)<br><br />
<b>Objective:</b> Continue with addition of dT to the ends of mms6, xylE, and lumazine.<br><br />
<br />
<b>Method:</b><br><br />
<br />
4) Ligate restricted dT to the ends of the "Part 1" Biobricks.<br><br />
dT to mms6:<br><br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td></tr><br />
<tr><td>T4 DNA Ligase</td><td>0.25</td></tr><br />
<tr><td>10x T4 Ligation Buffer</td><td>2</td></tr><br />
<tr><td>dT</td><td>8.3</td></tr><br />
<tr><td>mms6</td><td>9.2</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>O</td><td>0.25</td></tr><br />
</table><br><br />
dT to xylE:<br><br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td></tr><br />
<tr><td>T4 DNA Ligase</td><td>0.25</td></tr><br />
<tr><td>10x T4 Ligation Buffer</td><td>2</td></tr><br />
<tr><td>dT</td><td>8.3</td></tr><br />
<tr><td>xylE</td><td>3.4</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>O</td><td>6.05</td></tr><br />
</table><br><br />
dT to lumazine:<br><br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td></tr><br />
<tr><td>T4 DNA Ligase</td><td>0.25</td></tr><br />
<tr><td>10x T4 Ligation Buffer</td><td>2</td></tr><br />
<tr><td>dT</td><td>8.3</td></tr><br />
<tr><td>lumazine</td><td>3.35</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>O</td><td>6.1</td></tr><br />
</table><br><br />
Ligations were incubated at room temperature overnight.<br><br />
<br />
<br />
<b>Objective:</b> Prepare mastermix for four PCR reactions for following day.<br><br />
<br />
<b>Method:</b><br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td><td><b>5x Volume (&micro;L)</b></td></tr><br />
<tr><td>10mM dNTPs</td><td>1</td><td>5</td></tr><br />
<tr><td>5x Phusion Buffer</td><td>4</td><td>20</td></tr><br />
<tr><td>Forward Primer (VF2)</td><td>1</td><td>5</td></tr><br />
<tr><td>Reverse Primer (VR)</td><td>1</td><td>5</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>O</td><td>10.8</td><td>54</td></tr><br />
</table><br><br />
<br />
==<font color="white">July 13/2010==<br />
(in lab: JV)<br><br />
<b>Objective:</b> To determine if ligations of previous day (July 12/2010) were successful.<br><br />
<b>Method:</b><br><br />
1) Use prepared mastermix to run a PCR of ligated mms6-dT, ligated xylE-dT, ligated lumazine-dT, and control-dT<br><br />
- To each PCR tube, add 17.8&mirco;L of mastermix, 0.2&micro;L of Phusion DNA polymerase, and 2&micro;L of template DNA.<br><br />
- Ran PCR's for 36 cycles using the iGEM preset.<br><br />
<br />
2) 2% Agarose gel<br><br />
<table><table border ="3"><br />
<tr><td><b>Lane</b></td><td><b>Sample</b></td><td><b>Components (&micro;L)</b></td></tr><br />
<tr><td>1</td><td>50 bp Ladder</td><td>1 ladder + 2 loading dye (6x) + 7 MilliQ H<sub>2</sub>O</td></tr><br />
<tr><td>2</td><td>mms6-dT</td><td>8 PCR product + 2 loading dye (6x)</td></tr><br />
<tr><td>3</td><td>xylE-dT</td><td>8 PCR product + 2 loading dye (6x)</td></tr><br />
<tr><td>4</td><td>lumazine-dT</td><td>8 PCR product + 2 loading dye (6x)</td></tr><br />
<tr><td>5</td><td>dT</td><td>8 PCR product + 2 loading dye (6x)</td></tr><br />
<tr><td>6</td><td>Empty</td><td>Empty</td></tr><br />
<tr><td>7</td><td>Empty</td><td>Empty</td></tr><br />
<tr><td>8</td><td>Empty</td><td>Empty</td></tr><br />
<tr><td>9</td><td>Empty</td><td>Empty</td></tr><br />
<tr><td>10</td><td>Empty</td><td>Empty</td></tr><br />
</table><br><br />
Ran at 100V for __ minutes. Stained in EtBr for 10 minutes.<br><br />
<br />
<b>Results:</b><br />
[[image:Lethbridge_100713JV PCR.JPG|200px]]<br><br />
<br />
==<font color="white">July 14/2010==<br />
(in lab:J.S, K.G )<br><br />
<b>Objective:</b> Inoculate culture for maxi prep with placI<br />
<br />
<b>Method:</b><br />
To a 450mL solution of LB media 4.5(&micro;L) of ampicillin was added with glycerol placI aseptically.<br />
----<br />
(in lab:J.S, K.G )<br><br />
<b>Objective:</b> Restriction of dT and mms6<br />
<br />
<b>protocol</b><br />
<br />
1)<br />
<br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td></tr><br />
<tr><td>Buffer&*</td><td>2</td></tr><br />
<tr><td>10x T4 Milli Q H<sub>2</sub>O</td><td>15.5</td></tr><br />
<tr><td>pDNA**</td><td>2</td></tr><br />
<tr><td>Restriction enzymes***</td><td>0.25</td></tr><br />
</table><br><br />
<br />
<b>*</b> Orange buffer was used for dT, and Red buffer was used for mms6<br />
<br />
<b>**</b>pDNA was dT and mms6<br />
<br />
<b>***</b>Restriction enzymes for dT were XbalI and EcoRI, and for mms6 SpeI and EcoRI<br />
<br />
2)<br />
Reaction was incubated at 37<sup>0</sup>C for 1 hour. Start 8:50pm till 9:50pm<br />
After incubation reaction was heat shocked at 80<sup>0</sup>C for 20 minutes<br />
<br />
----<br />
(in lab:J.S, K.G )<br><br />
<b>Objective:</b> Ligation of dT to each of mms6, xylE and lumazine.<br />
<br />
<b>protocol</b><br />
<br />
1)<br />
<br />
dT to mms6:<br><br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td></tr><br />
<tr><td>T4 DNA Ligase</td><td>0.25</td></tr><br />
<tr><td>10x T4 Ligation Buffer</td><td>2</td></tr><br />
<tr><td>dT</td><td>8.3</td></tr><br />
<tr><td>mms6</td><td>9.2</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>O</td><td>0.25</td></tr><br />
</table><br><br />
dT to xylE:<br><br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td></tr><br />
<tr><td>T4 DNA Ligase</td><td>0.25</td></tr><br />
<tr><td>10x T4 Ligation Buffer</td><td>2</td></tr><br />
<tr><td>dT</td><td>8.3</td></tr><br />
<tr><td>xylE</td><td>3.4</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>O</td><td>6.05</td></tr><br />
</table><br><br />
dT to lumazine:<br><br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td></tr><br />
<tr><td>T4 DNA Ligase</td><td>0.25</td></tr><br />
<tr><td>10x T4 Ligation Buffer</td><td>2</td></tr><br />
<tr><td>dT</td><td>8.3</td></tr><br />
<tr><td>lumazine</td><td>3.35</td></tr><br />
<tr><td>MilliQ H<sub>2</sub>O</td><td>6.1</td></tr><br />
</table><br><br />
<br />
2)<br />
Incubated reaction overnight at room temperature<br><br />
<br />
==<font color="white">July 15/2010==<br />
(in lab: AV)<br><br />
<b>Objective:</b> Maxiprep pLacI and mms6.<br><br />
<br />
<table><table border ="3"><br />
<tr><td><b>component</b></td><td><b>pallet weight (g)</b></td></tr><br />
<tr><td>mms6</td><td>1.02</td></tr><br />
<tr><td>pLacI</td><td>1.54</td></tr><br />
</table><br><br />
<br />
==<font color="white">July 15/2010 Evening==<br />
(in lab: AV)<br><br />
<b>Objective:</b> transform ligations from July 14 and July 12,2010;xylE/dt, mms6/dt lumazine/dt into DH5&alpha. <br />
Also to transform mms6 from July 6 and July 10 into Bl21(DE3)<br />
<br />
<b>Protocol:</b><br />
to transform competent cells see protocol:[https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Competent_Cell_Transformation]<br />
<br />
<b>*</b> cells were incubated on ice for 30 minutes started at 7:00-7:30. <br />
<b>**</b>incubated for 1 hour from 8:00 till 9:00<br />
<br />
<table><table border ="3"><br />
<tr><td><b>'''Results'''</b></tr><br />
<tr><td><b>plate</b></td><td><b># of colonies</b></td><td><b>Components (&micro;L)</b></td></tr><br />
<tr><td>1</td><td>200&micro;L xylE-dt July 12</td><td>0</td></tr><br />
<tr><td>2</td><td>200&micro;L mms6-dt July 12</td><td>1</td></tr><br />
<tr><td>3</td><td>200&micro;L lumazine-dt</td><td>1</td></tr><br />
<tr><td>4</td><td>200&micro;L mms6 maxiprep July 6</td><td>Lawn</td></tr><br />
<tr><td>5</td><td>200&micro;L positve control puC19 into BL21(DE3)</td><td>Lawn</td></tr><br />
<tr><td>6</td><td>200&micro;L xylE-dt July 14</td><td>0</td></tr><br />
<tr><td>7</td><td>200&micro;L mms6-dt July 14</td><td>120</td></tr><br />
<tr><td>8</td><td>200&micro;L lumazine-dt July14</td><td>0</td></tr><br />
<tr><td>9</td><td>200&micro;L mms6 maxiprep July 10</td><td>.</td></tr><br />
</table><br><br />
<br />
<b>*</b>because of a shortage of plates not all transformations were plated at 50&micro;L and 200&micro;L<br />
<br />
==<font color="white">July 16,2010==<br />
(in lab: M.C, D.M)<br><br />
<b>Objective:</b> PCR amplify mms6-dT, xylE-dT, lumazine-dT legations along with dT maxi prep for comparison<br />
<br />
<b>Protocol:</b><br />
<br />
Master Mix<br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td></tr><br />
<tr><td>Milli-Q H<sub>2</sub>O<</td><td>54</td></tr><br />
<tr><td>Pfu Buffer + MgSO<sub>4</sub></td><td>20</td></tr><br />
<tr><td>10 mM dMTPs</td><td>5</td></tr><br />
<tr><td>forward primer</td><td>5</td></tr><br />
<tr><td>reverse primer</td><td>5</td></tr><br />
</table><br><br />
89 &micro;L TOTAL--> 17.8 into each PCR reaction<br />
<br />
+ 2 &micro;L ligation<br />
+ 2 &micro;L Pfu polymerase <br />
<br />
Ran iGem-ligTest in thermocycler<br />
<br />
<br />
==<font color="white">July 19, 2010==<br />
(in lab: A.V, H.B)<br><br />
<b>Objective:</b> Purification pf pLacI and mms6 maxipreps done on July 14 and 16, 2010 using the biobasic protocol for purification of PCR products. <br />
<br />
<b>Objective:</b> Add pLacI to sRBS and add dT to xylE and lumazine<br />
<br />
<b>Method:</b><br />
<br />
Restrict pLacI, xylE and lumazine with SpeI and EcoRI and restrict dT and sRBS with EcorI and XbaI<br />
<br />
1)Restrictions:<br />
<br />
pLacI, xylE, and Lumazine <br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td></tr><br />
<tr><td>Milli-Q H<sub>2</sub>O</td><td>15.5</td></tr><br />
<tr><td>Red Buffer<sub>4</sub></td><td>2</td></tr><br />
<tr><td>SpeI</td><td>0.25</td></tr><br />
<tr><td>EcoRI</td><td>0.25</td></tr><br />
<tr><td>pDNA</td><td>2</td></tr><br />
</table><br><br />
<br />
dT<br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td></tr><br />
<tr><td>Milli-Q H<sub>2</sub>O</td><td>38.75</td></tr><br />
<tr><td>Orange Buffer<sub>4</sub></td><td>5</td></tr><br />
<tr><td>XbaI</td><td>0.625</td></tr><br />
<tr><td>EcoRI</td><td>0.625</td></tr><br />
<tr><td>pDNA</td><td>5</td></tr><br />
</table><br><br />
<br />
sRBS<br />
<table><table border ="3"><br />
<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td></tr><br />
<tr><td>Milli-Q H<sub>2</sub>O</td><td>15.5</td></tr><br />
<tr><td>Red Buffer<sub>4</sub></td><td>2</td></tr><br />
<tr><td>xBal</td><td>0.25</td></tr><br />
<tr><td>EcoRI</td><td>0.25</td></tr><br />
<tr><td>pDNA</td><td>2</td></tr><br />
</table><br><br />
<br />
Restriction incubation at 37.5<sup>0</sup>C started at 11:55am. Ended at 12:55pm<br />
Heat killed enzymes at 80<sup>0</sup>C for 20 minutes<br />
<br />
2)Restrictions were run on a 1% agarose gel (1 X TAE)<br />
1% Agarose gel<br><br />
<table><table border ="3"><br />
<tr><td><b>Lane</b></td><td><b>Sample</b></td><td><b>Components (&micro;L)</b></td></tr><br />
<tr><td>1</td><td>1kB Ladder</td><td>0.5 ladder + 2 loading dye (5x) + 5.5 MilliQ H<sub>2</sub>O</td></tr><br />
<tr><td>2</td><td>pLacI</td><td>6 pDNA + 2 loading dye (5x)</td></tr><br />
<tr><td>3</td><td>pLacI restriction digest</td><td>6 pDNA + 2 loading dye (5x)</td></tr><br />
<tr><td>4</td><td>sRBS</td><td>6 pDNA + 2 loading dye (5x)</td></tr><br />
<tr><td>5</td><td>sRBS restriction digest</td><td>6 pDNA + 2 loading dye (5x)</td></tr><br />
<tr><td>6</td><td>xylE</td><td>6 pDNA + 2 loading dye (5x)</td></tr><br />
<tr><td>7</td><td>xylE restriction digest</td><td>6 pDNA + 2 loading dye (5x)</td></tr><br />
<tr><td>8</td><td>lumazine</td><td>6 pDNA + 2 loading dye (5x)</td></tr><br />
<tr><td>9</td><td>lumazine restriction digest</td><td>6 pDNA + 2 loading dye (5x)</td></tr><br />
<tr><td>10</td><td>dT</td><td>6 pDNA + 2 loading dye (5x)</td></tr><br />
<tr><td>11</td><td>dT restriction digest</td><td>6 pDNA + 2 loading dye (5x)</td></tr><br />
</table><br><br />
<br />
Gel ran for 70 minutes at 100V and was stained in EtBr for 10 minutes<br />
<br />
<b>Results:</b><br />
[[image:Lethbridge_100719HBAVDigest.JPG|200px]]<br><br />
<br />
Results after quantifying restriction digest <br />
<table><table border ="3"><br />
<tr><td><b>Lane</b></td><td><b>Content</b></td><td><b> Quantity of DNA (ng/&micro;L)</b></td></tr><br />
<tr><td>1</td><td>1kB Ladder</td><td>12.5</td></tr><br />
<tr><td>3</td><td>pLacI restriction digest</td><td>5.21</td></tr><br />
<tr><td>5</td><td>sRBS restriction digest</td><td>3.72</td></tr><br />
<tr><td>7</td><td>xylErestriction digest</td><td>3.36</td></tr><br />
<tr><td>9</td><td>Lumazine restriction digest</td><td>2.63</td></tr><br />
<tr><td>11</td><td>dT restriction digest</td><td>2.98</td></tr><br />
</table><br><br />
<br />
==<font color="white">July 19, 2010 Evening==<br />
(K.G)<br><br />
<b>Objective:</b> Ligate together rbs-xylE and dT, lumazine and dT, also pLacI and sRBS.<br><br />
<br />
<b>Method:</b> All ligation mixes had:<br><br />
*10X T4 Ligation Buffer<br />
*Milli-Q H<sub>2</sub>O to fill to 20&micro;L<br />
*T4 DNA Ligase<br />
*20ng plasmid DNA<br><br />
<br />
Ligations were left over-night at room temperature. <br><br />
<br />
(J.V.)<br><br />
<b>Objective:</b>Determine the results of the transformations done by K.G. on July 15/2010.<br><br />
<br />
<b>Method:</b> Inoculate 5mL LB media w/Amp and colony. Incubated over night at 37<sup>o</sup>C.<br><br />
<br />
<b>Results:</b> Lumazine Synthase with dT ligated onto it grew.<br><br />
<br />
==<font color="white">July 20, 2010==<br />
(AV, HB)<br><br />
<b>Objective:</b>Miniprep lumazine-dt and 4 N-terminus tags and analyze.<br><br />
<br />
<b>Method:</b><br><br />
<br />
*Use [[Team:Lethbridge/Notebook/Protocols|boiling lysis miniprep]] to isolate plasmid DNA.<br />
*PCR amplify BioBrick part to determine if the correct DNA was isolated.<br />
*Ran a 1% Agarose gel to visualize PCR products.<br />
<br />
<b><font color="white">Results:</font></b><br />
Agarose gel did not show any bands.<br><br />
<br />
==<font color="white">July 20, 2010 Evening==<br />
(AV, HB)<br><br />
<b>Objective:</b>Transform ligation done on July 19, 2010 in to competent DH5&alpha; cells.<br><br />
* xylE-dT<br />
*lumazine synthase-dT<br />
*pLacI-sRBS<br />
*EYFP and ECFP<br><br />
<br />
<b>Method:</b><br><br />
*Use [[Team:Lethbridge/Notebook/Protocols|Competent Cell Transformation]]<br />
<br />
<b>Results:</b><br><br />
*xylE-dT no colonies<br />
*pLacI-sRBS<br />
*EYFP<br />
*lumazine-dT<br />
*ECFP<br />
<br />
==<font color="white">July 21, 2010==<br />
(JV)<Br><br />
<b>Objective:</b> Isolate plasmid DNA from pTet, TetR, pET-28(a).<br />
<br />
<b>Method:</b><br><br />
*Used [[Team:Lethbridge/Notebook/Protocols|Maxiprep]] protocol.<br />
<br />
(JV)<br><br />
<br />
<b>Objective:</b> To induce over-expression of pLacI-RBS-Mms6-dt in BL21(DE3) cells.<br><br />
<br />
<b>Method:</b> Used [[Team:Lethbridge/Notebook/Protocols|Overexpression]].<br><br />
Ran SDS gel for 78 minutes at 200V<br><br />
<br />
==<font color="white">July 21, 2010 Evening==<br />
(TF, AS)<br><br />
<b>Objective:</b> Colony PCR to test for insertions of BioBrick construction, and for quality control of parts received from the Registry. Also to prepare DNA to be sent away for sequencing.<br><br />
<b>Method:</b><br><br />
-Ran PCR's<br><br />
-Ran 2% Agarose gel for:.<br><br />
*K249001<br><br />
*K249004<br><br />
*K249005<br><br />
*K249006<br><br />
*K249008<br><br />
*K249014<br><br />
*K249017<br><br />
*mms6-dt<br><br />
*lumazine-dt<br><br />
*ECFP<br><br />
*EYFP<br><br />
*N-term tag<br><br />
*xylE-dt<br><br />
*SRBS-pLacI<br><br />
*dt<br><br />
*pTet maxiprep<br><br />
*pET-28(a) maxiprep<br><br />
*TetR maxiprep<br><br />
Gel ran at 100V for 60minutes.<br><br />
<b>Results:</b> The PCR's did not work. However, the maxipreps show DNA<br><br />
[[image:Lethbridge_100722 JV BW.jpg|200px]]<br />
<br />
==<font color="white">July 23, 2010==<br />
(JV)<br><br />
<b>Objective:</b> Insert xylE, Mms6, and lumazine synthase into pET-28(a).<br><br />
<b>Method:</b> A restriction digest was performed on xylE and Mms6 and ran for 90 minutes at 37 C<br><br />
<b>Results:</b>Did not see any cut out biobricks.<br><br />
[[image:Lethbridge_100723MaxiprepRestrictions.jpg|200px]]<br />
<br />
==<font color="white">July 27, 2010==<br />
(in lab: JV, AV, HB)<br><br />
<br />
<b>Objective:</b> To miniprep the overnight cultures of the parts ordered from the Parts Registry and to test the efficiency of the Qiagen Miniprep Kit.<br><br />
<br />
<b>Method:</b> <br><br />
The following parts were miniprep'd using [[Team:Lethbridge/Notebook/Protocols|Boiling Lysis Plasmid Preparation]]:<br><br />
*dT (control)<br />
*E0020 (ECFP)<br />
*E0030 (EYFP)<br />
*K249001<br />
*K249004<br />
*K249005<br />
*K249006<br />
*K249008<br />
*K249014<br />
*K249017<br />
<br />
The following parts were miniprep'd using Qiagen Miniprep Kit:<br><br />
*K249005<br />
*K249008<br />
<br />
<b>Results:</b> Qiagen Miniprep Kit produced a comparable quantity of DNA to [[Team:Lethbridge/Notebook/Protocols|Boiling Lysis Plasmid Preparation]]. Since the Qiagen Miniprep Kit is faster and easier, all minipreps will be done using the Qiagen Miniprep Kit.<br />
<br />
==<font color="white">July 28, 2010==<br />
(in lab: JV)<br><br />
<br />
<b>Objective:</b> To create a large quantity of pSB1C3 plasmid.<br><br />
<br />
<b>Method:</b> PCR amplified pSB1C3 received from iGEM headquarters.<br />
<br />
<br />
<br />
==<font color="white">July 29, 2010==<br />
(in lab: HB, AV)<br><br />
<br />
<b>Objective:</b> To PCR amplify the minipreps on July 27, 2010.<br><br />
<br />
<b>Method:</b> PCR amplified the 11 minipreps and dT control.<br><br />
<br />
<b>Results:</b> PCR amplification was successful and 0.2 &micro;L of polymerase will be used per PCR reaction in the future.<br><br />
<br />
<br><br><br />
<b>Objective:</b> To maxiprep EYFP (E0030), ECFP (E0020), and Lumazine Synthase (K249002).<br><br />
<br />
<b>Method:</b> Used [[Team:Lethbridge/Notebook/Protocols|Maxiprep]]<br><br />
<br />
<table><table border="3"><br />
<tr><td><b>Cell Pellet</b></td><td><b>Weight (g)</b></td></tr><br />
<tr><td>ECFP</td><td>2.32</td></tr><br />
<tr><td>EYFP</td><td>2.00</td></tr><br />
<tr><td>Lumazine Synthase</td><td>2.66</td></tr></table><br />
<br />
==<font color="white">July 29, 2010 Evening==<br />
(in lab: KG)<br><br />
<br />
<b>Objective:</b> Run PCR products from the morning on a 2% agarose gel.<br><br />
<br />
<b>Method:</b> Ran 2% agarose gel of the 11 minipreps and dT control and stained in EtBr for 17 minutes.<br><br />
<b>Results:</b>PCR amplification of last year's parts not consistent with sizes listed on registry.<br><br />
[[image:Lethbridge_100729 Registry Parts PCR KG.jpg|200px]]<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T19:12:34Z<p>Liszabruder: </p>
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<BLOCKQUOTE><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
<br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br />
<br><br />
<br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br />
[[image:UofLmms6growth.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/Parts/Characterized_PartsTeam:Lethbridge/Parts/Characterized Parts2010-10-27T19:10:34Z<p>Liszabruder: </p>
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<img src="https://static.igem.org/mediawiki/2010/0/0a/UofLArt.jpg" width="80"/><br />
</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/News"><br />
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</a><br />
</th><br />
</table><br />
</body><br />
</html><br />
<hr><br />
<html><br />
<center><br />
<font color="white">Check out the parts we have submitted and the parts we have characterized! You can also access our <a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results page here</font></a>!<br />
</center><br />
</html><br />
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<html><br />
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<hr><br />
<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. The parts we have created so far are for the characterization of the microcompartment system.<br />
<br />
==<font color="white">Tet Repressible N-terminal Arg Tagged EYFP (no terminator)==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00">BBa_K331030</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
==<font color="white">Tet Repressible C-terminal Arg Tagged EYFP (no terminator)==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00">BBa_K331031</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
==<font color="white">Tet Repressible C-terminal Arg Tagged ECFP (no terminator)==<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/Parts/Characterized_Existing_PartsTeam:Lethbridge/Parts/Characterized Existing Parts2010-10-27T19:08:36Z<p>Liszabruder: </p>
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<a href="https://2010.igem.org/Team:Lethbridge"><br />
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</a><br />
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</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Team"><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Project"><br />
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</a><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work"><br />
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</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Parts"><br />
<img src="https://static.igem.org/mediawiki/2010/8/84/UofLPartsSubmittedToTheRegistrybutton.jpg" width="80"/><br />
</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Modeling"><br />
<img src="https://static.igem.org/mediawiki/2010/e/e1/UofLModelingbutton.jpg" width="80"/><br />
</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><br />
<img src="https://static.igem.org/mediawiki/2010/2/26/UofLEthicsbutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Safety"><br />
<img src="https://static.igem.org/mediawiki/2010/0/00/UofLSafetybutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Art"><br />
<img src="https://static.igem.org/mediawiki/2010/0/0a/UofLArt.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/News"><br />
<img src="https://static.igem.org/mediawiki/2010/c/c3/UofLNewsButton.jpg" width="80"/><br />
</a><br />
</th><br />
</table><br />
</body><br />
</html><br />
<hr><br />
<html><br />
<center><br />
<font color="white">Check out the parts we have submitted and the parts we have characterized! You can also access our <a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results page here</font></a>!<br />
</center><br />
</html><br />
<br />
<html><br />
<body><br />
<center><br />
<table border="0" width="28%" style="background-color:#000000"><br />
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<div class="miniContainer"><br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Parts"><br />
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</a><br />
</th><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized Parts"><br />
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</a><br />
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<th><a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized Existing Parts"><br />
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</th><br />
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</table><br />
</center><br />
</body><br />
</html><br />
<hr><br />
<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathways within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so the system can be optimized. These parts we have received directly from the Registry and were further characterized for future use in characterization of the microcompartment system.<br />
<br />
==<font color="white">N-terminal Arginine Fusion Vector==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Placement_of_Oligoarginine_Tail_on_Proteins"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
==<font color="white">C-terminal Arginine Fusion Vector==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00">BBa_K249005</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Placement_of_Oligoarginine_Tail_on_Proteins"><font color="#00DC00">Results</font></a></html> page.<br />
<br><br><br />
<br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br />
==<font color="white">Mms6 (Nanoparticles)==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results#Characterization_of_Mms6"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/Parts/Characterized_Existing_PartsTeam:Lethbridge/Parts/Characterized Existing Parts2010-10-27T19:06:34Z<p>Liszabruder: </p>
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<th><br />
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<a href="https://2010.igem.org/Team:Lethbridge"><br />
<img src="https://static.igem.org/mediawiki/2010/2/22/UofLHome.jpg" width="80"/><br />
</a><br />
<br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Team"><br />
<img src="https://static.igem.org/mediawiki/2010/0/0d/UofLTeam.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Project"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8d/UofLProjectbutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work"><br />
<img src="https://static.igem.org/mediawiki/2010/7/73/UofLNotebookbutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Parts"><br />
<img src="https://static.igem.org/mediawiki/2010/8/84/UofLPartsSubmittedToTheRegistrybutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Modeling"><br />
<img src="https://static.igem.org/mediawiki/2010/e/e1/UofLModelingbutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Ethics"><br />
<img src="https://static.igem.org/mediawiki/2010/2/26/UofLEthicsbutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Safety"><br />
<img src="https://static.igem.org/mediawiki/2010/0/00/UofLSafetybutton.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Art"><br />
<img src="https://static.igem.org/mediawiki/2010/0/0a/UofLArt.jpg" width="80"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/News"><br />
<img src="https://static.igem.org/mediawiki/2010/c/c3/UofLNewsButton.jpg" width="80"/><br />
</a><br />
</th><br />
</table><br />
</body><br />
</html><br />
<hr><br />
<html><br />
<center><br />
<font color="white">Check out the parts we have submitted and the parts we have characterized! You can also access our <a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results page here</font></a>!<br />
</center><br />
</html><br />
<br />
<html><br />
<body><br />
<center><br />
<table border="0" width="28%" style="background-color:#000000"><br />
<br />
<tr><br />
<th><br />
<div class="miniBar"><br />
<div class="countdown"><object type="application/x-shockwave-flash" data="http://www.oneplusyou.com/bb/files/countdown/countdown.swf?co=FFFFFF&bgcolor=000000&date_month=10&date_day=26&date_year=0&un=PARTS SUBMISSION DEADLINE&size=normal&mo=10&da=26&yr=2010" width="300" height="100"><param name="movie" value="http://www.oneplusyou.com/bb/files/countdown/countdown.swf?co=FFFFFF&bgcolor=000000&date_month=10&date_day=26&date_year=0&un=PARTS SUBMISSION DEADLINE&size=normal&mo=10&da=26&yr=2010" /><param name="bgcolor" value="#000000" /></object><img src="http://www.oneplusyou.com/q/img/bb_badges/countdown.jpg" alt="" style="display: none;" height="1" width="1" /></div><br />
<div class="miniContainer"><br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Parts"><br />
<img src="https://static.igem.org/mediawiki/2010/d/d4/UofLsubmittedparts.jpg" width="60"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized Parts"><br />
<img src="https://static.igem.org/mediawiki/2010/5/55/UofLcharacterizedparts.jpg" width="60"/><br />
</a><br />
</th><br />
<br />
<th><a href="https://2010.igem.org/Team:Lethbridge/Parts/Characterized Existing Parts"><br />
<img src="https://static.igem.org/mediawiki/2010/c/ce/UofLcharacterizedexistingparts.jpg" width="80"/><br />
</a><br />
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</table><br />
</center><br />
</body><br />
</html><br />
<hr><br />
<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathways within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so the system can be optimized. These parts we have received directly from the Registry and were further characterized for future use in characterization of the microcompartment system.<br />
<br />
==<font color="white">N-terminal Arginine Fusion Vector==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
==<font color="white">C-terminal Arginine Fusion Vector==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00">BBa_K249005</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> page.<br />
<br><br><br />
<br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br />
==<font color="white">Mms6 (Nanoparticles)==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/Parts/Characterized_PartsTeam:Lethbridge/Parts/Characterized Parts2010-10-27T18:59:09Z<p>Liszabruder: </p>
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<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. The parts we have created so far are for the characterization of the microcompartment system.<br />
<br />
==<font color="white">Tet Repressible N-terminal Arg Tagged EYFP (no terminator)==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00">BBa_K331030</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
==<font color="white">Tet Repressible C-terminal Arg Tagged EYFP (no terminator)==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00">BBa_K331031</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
==<font color="white">RBS with C-terminal Oligo Arginine - ECFP Fusion==<br />
<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
==<font color="white">Tet Repressible C-terminal Arg Tagged ECFP (no terminator)==<br />
You can view <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> on the registry and find the experimental details on our <html><a href="https://2010.igem.org/Team:Lethbridge/Results"><font color="#00DC00">Results</font></a></html> page.<br />
<br />
<br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T18:41:52Z<p>Liszabruder: </p>
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<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br><br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br />
[[image:UofLmms6growth.jpg|450px|center]]<br />
<br><br />
<br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T18:40:57Z<p>Liszabruder: /* References */</p>
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<br />
<BLOCKQUOTE><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
<br><br><br />
<br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br><br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br />
[[image:UofLmms6growth.jpg|450px|center]]<br />
<br><br><br />
<br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br><br />
<br></div>Liszabruderhttp://2010.igem.org/File:UofLmms6growth.jpgFile:UofLmms6growth.jpg2010-10-27T18:40:28Z<p>Liszabruder: </p>
<hr />
<div></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T18:39:57Z<p>Liszabruder: /* Results */</p>
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<BLOCKQUOTE><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
<br><br><br />
<br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br><br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br />
[[image:UofLmms6growth.jpg|450px|center]]<br />
<br><br><br />
<br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T18:38:57Z<p>Liszabruder: /* Future Directions */</p>
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<BLOCKQUOTE><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
<br><br><br />
<br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
<hr><br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br><br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br><br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T18:38:29Z<p>Liszabruder: /* Results */</p>
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<BLOCKQUOTE><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br><br />
[[image:UofLcfpfigureblack.jpg|450px|center]]<br />
<br><br><br />
<br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br><br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br><br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br></div>Liszabruderhttp://2010.igem.org/File:UofLcfpfigureblack.jpgFile:UofLcfpfigureblack.jpg2010-10-27T18:36:30Z<p>Liszabruder: </p>
<hr />
<div></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T18:34:58Z<p>Liszabruder: /* Method */</p>
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<BLOCKQUOTE><br />
<br />
=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
<br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br />
[[image:UofL.cfpfigureblackjpg|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br><br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (<html><a href="BBa_R0010" target="new"><font color="#00DC00"> BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
<br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
<br><br><br />
===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
<br><br><br />
===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br></div>Liszabruderhttp://2010.igem.org/Team:Lethbridge/ResultsTeam:Lethbridge/Results2010-10-27T18:34:15Z<p>Liszabruder: /* Method */</p>
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<BLOCKQUOTE><br />
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=<font color="white">Compartmentalization Parts=<br />
<br />
One of the sub-projects for the bioremediation of the tailings ponds is to create synthetic <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartments</font></a></html> that we can then use to isolate various pathway within an <i>Escherichia coli</i> cell. To do this we need to have a microcompartment as well as a means to characterize the compartment so that the system can be optimized. Here are the experiments we have performed so far towards the characterization of the microcomparments.<br />
<br><br><br />
==<font color="white">Placement of Oligoarginine Tail on Proteins</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00" size="+1">BBa_K249004</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005"target="new"><font color="#00DC00" size="+1">BBa_K249005</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030"target="new"><font color="#00DC00" size="+1">BBa_K331030</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031"target="new"><font color="#00DC00" size="+1">BBa_K331031</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis</font>===<br />
<hr><br />
The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein <i>in vivo</i>.<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> lumazine synthase microcompartment</font></a></html> would cause degradation of our protein before it can be moved into the microcompartment. <br />
<br><br><br />
We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.<br />
<br><br><br />
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===<font color="white">Method</font>===<br />
<hr><br />
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5&alpha; cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.<br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.<br />
[[image:Lethbridge_NvsC-terminalOligoArgBlackfINAL.png|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.<br />
<br><br><br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.<br />
<br><br />
<br><br />
==<font color="white">Characterization of Cyan Fluorescent Protein</font>==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00" size="+1">BBa_K331025</font></a></html><br />
<br><br><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00" size="+1">BBa_K331033</font></a></html><br />
<br><br><br />
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===<font color="white">Hypothesis</font>===<br />
<hr><br />
Light will be observed at 476 nm when cells containing <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> are illuminated with light at 439 nm as a result of the excitation and subsequent fluorescence of Cyan Fluorescent Protein (CFP).<br />
<br><br><br />
<br />
===<font color="white">Introduction</font>===<br />
<hr><br />
Prior to introducing a oligoarginine tail onto the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor. <br />
<br><br><br />
Here we investigate the ability of <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.<br />
<br><br><br />
<br />
===<font color="white">Method</font>===<br />
<hr><br />
In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0034"target="new"><font color="#00DC00">BBa_B0034</font></a></html>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.<br />
<br><br><br />
The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.<br />
<br><br><br />
This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm. <br />
<br><br><br />
<br />
===<font color="white">Results</font>===<br />
<hr><br />
Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br />
[[image:UofL.cfpfigureblackjpg|900px]]<br />
<br><br><br />
===<font color="white">Conclusion</font>===<br />
<hr><br />
The BioBrick <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.<br />
<br><br><br />
<br />
===<font color="white">Future Directions===<br />
We will assembly this into a single biobrick which can differentially express CFP/YFP and lumazine synthase for confirmation (through FRET) that the oligoarginine tail can be utilized to localize proteins within a modified lumazine synthase microcompartment.<br />
<br><br><br />
<br />
===<font color="white">Reference</font>===<br />
<hr><br />
<sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.<br />
<br><br><br />
<sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.<br />
<br><br><br />
=<font color="white">Magnetic Nanoparticles Parts=<br />
One of the sub-projects for the <html><a href="https://2010.igem.org/Team:Lethbridge/Project" target="new"><font color="#00DC00"> bioremediation of the tailings ponds</font></a></html> is to reduce heavy metals to create <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles" target="new"><font color="#00DC00"> magnetic nanoparticles</font></a></html> that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the <i>Escherichia coli</i> cell. He is the work we have accomplished so far to characterize Mms6.<br />
<br><br><br />
==<font color="white">Characterization of Mms6==<br />
===<font color="white">Characterized Parts</font>===<br />
<hr><br />
<html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K249019" target="new"><font color="#00DC00" size="+1">BBa_K249019</font></a></html><br />
<br><br><br />
===<font color="white">Hypothesis===<br />
<hr><br />
Induction of Mms6 protein overexpression in <i>Escherichia coli</i> DH5α cells will cause a change in growth. <br />
<br><br><br />
<br />
===<font color="white">Introduction=== <br />
<hr><br />
A parallel project we are undertaking in <html><a href="https://2010.igem.org/Team:Lethbridge/Project"><font color="#00DC00">oilsands remediation</font></a></html> is in the clean-up of heavy metal contaminants utilizing the Mms6 protein expressed by <i>Magnetiospirilum magneticum</i><sup>1</sup>. The Mms6 protein is capable of reducing aqueous iron into <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00">magnetite nanoparticles</font></a></html>. The magnetite nanoparticles can be removed with much greater ease than aqueous iron. <br />
<br><br><br />
We chose to investigate how the presence of the Mms6 protein affected <i>Escherichia coli</i> DH5α, its bacterial host cell. <br />
<br><br><br />
===<font color="white">Method===<br />
<hr><br />
In order to characterize the effect of Mms6 production on <i>Escherichia coli</i> DH5α cells, we utilized BioBrick <html><a href=" http://partsregistry.org/Part:BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html>, a part submitted by the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00">2009 Lethbridge iGEM</font></a></html> team. <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> is the Mms6 coding region (<html><a href="http://partsregistry.org/Part: BBa_K249016" target="new"><font color="#00DC00">BBa_K249016</font></a></html>) with a ribosomal binding side (<html><a href="http://partsregistry.org/Part: BBa_B0030" target="new"><font color="#00DC00">BBa_B0030</font></a></html>) and a double terminator (<html><a href="http://partsregistry.org/Part: BBa_B0015" target="new"><font color="#00DC00">BBa_B0015</font></a></html>) under the control of a lactose inducible promoter (BBa_R0010</font></a></html>). <br />
<br><br><br />
We inoculated two 500 mL cultures of LB media with <i>Escherichia coli</i> DH5α cells containing <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> and grew the cells until they reached approximately 0.6 OD<sub>600</sub> units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.<br />
<br><br><br />
===<font color="white">Results===<br />
<hr><br />
The addition of IPTG to cells containing the <html><a href="http://partsregistry.org/Part: BBa_K249019" target="new"><font color="#00DC00">BBa_K249019</font></a></html> BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.<br />
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===<font color="white">Conclusion===<br />
<hr><br />
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors. <br />
<br><br><br />
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight. <br />
<br><br><br />
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the <html><a href=" https://2010.igem.org/Team:Calgary" target="new"><font color="#00DC00">Calgary team</font></a></html> by using their troubleshooting kit.<br />
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===<font color="white">References===<br />
<hr><br />
<sup>1</sup>Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. <i>Journal of Colloid and Interface Science</i>. 343:65-60.<br />
<br><br></div>Liszabruder