Team:Lethbridge

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<a href="https://2010.igem.org/Team:Lethbridge">
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<th><a href="https://2010.igem.org/Team:Lethbridge/Modeling">
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<th><a href="https://2010.igem.org/Team:Lethbridge/Ethics">
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<th><a href="https://2010.igem.org/Team:Lethbridge/Safety">
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Check out these important project links!
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<p>University of Lethbridge IGEM team</p>
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<center>
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<p>&nbsp;</p>
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    <li class="TabbedPanelsTab" tabindex="0">Main</li>
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    <li class="TabbedPanelsTab" tabindex="0">Team</li>
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    <li class="TabbedPanelsTab" tabindex="0">Projects</li>
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    <li class="TabbedPanelsTab" tabindex="0">Notebook</li>
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    <li class="TabbedPanelsTab" tabindex="0">Meetings</li>
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    <li class="TabbedPanelsTab" tabindex="0">Parts</li>
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    <li class="TabbedPanelsTab" tabindex="0">Collaborations</li>
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    <div class="TabbedPanelsContent">blah balh balh</div>
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      <p>Our team is based yada yada</p>
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        <ul class="TabbedPanelsTabGroup">
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          <li class="TabbedPanelsTab" tabindex="0">Team Pics</li>
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          <li class="TabbedPanelsTab" tabindex="0">Tab 3</li>
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          <li class="TabbedPanelsTab" tabindex="0">Team bios</li>
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        <div class="TabbedPanelsContentGroup">
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          <div class="TabbedPanelsContent">Team pictures</div>
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          <div class="TabbedPanelsContent">Content 3</div>
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          <div class="TabbedPanelsContent">Pics of us</div>
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      <p>&nbsp;</p>
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      <p>&nbsp;</p>
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      <p>&nbsp;</p>
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      <p>&nbsp;</p>
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      <p>&nbsp;</p>
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    <div class="TabbedPanelsContent">
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      <p>Overview</p>
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        <ul class="TabbedPanelsTabGroup">
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          <li class="TabbedPanelsTab" tabindex="0">Project 1</li>
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          <li class="TabbedPanelsTab" tabindex="0">Project 2</li>
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          <div class="TabbedPanelsContent">Content 1</div>
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          <div class="TabbedPanelsContent">Content 2</div>
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      <p>&nbsp;</p>
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    <div class="TabbedPanelsContent"><p>&nbsp;</p>
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          <li class="TabbedPanelsTab" tabindex="0">Lab Work</li>
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          <li class="TabbedPanelsTab" tabindex="0">Common Protocols</li>
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          <li class="TabbedPanelsTab" tabindex="0">Working Plasmids Box</li>
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          <li class="TabbedPanelsTab" tabindex="0">Working Glycerol Stocks</li>
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<b><font size=+1>Jump to Month:<br>
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<th><a href="https://2010.igem.org/Team:Lethbridge/Judging">
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<a href=#april>April</a><br>
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<img src="https://static.igem.org/mediawiki/2010/f/fd/UofLjudgingbutton.jpg" width="60"/>
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<a href=#may>May</a><br></b><br>
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</a>
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<a name="april"></a></font>
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<b><font size=+2>April 13/2010</font> (In the Lab: JV, AS)</b><br>
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<b>Objective:</b> Test Restriction Endonucleases for Activity<br>
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<th><a href="https://2010.igem.org/Team:Lethbridge/Results">
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<b>Relevant Information:</b><br>
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<img src="https://static.igem.org/mediawiki/2010/3/3f/UofLresultsbutton.jpg" width="60"/>
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Endonucleases available
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</a>
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<table><table border="3">
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</th>
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<tr><td>Endonuclease</td><td>Optimal Buffer**</td><td>Other Buffers</td></tr>
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<tr><td>EcoRV</td><td>None</td><td>2xT(100%); O,G(50-100%)</td></tr>
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<th><a href="https://2010.igem.org/Team:Lethbridge/Collaboration">
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<tr><td>EcoRI</td><td>Red</td><td>O(100%);R(100%)*;2xT(100%)</td></tr>
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<img src="https://static.igem.org/mediawiki/2010/b/b2/UofLcollaborationbutton.png" width="60"/>
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<tr><td>BcuI/SpeI</td><td>Tango</td><td>B(50-100%);G(50-100%)</td></tr>
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</a>
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<tr><td>XbaI</td><td>Tango</td><td>B,G,2xT(50-100%)</td></tr>
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</th>
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<tr><td>PstI</td><td>Orange</td><td>R(100%); B,G,T,2xT(50-100%)</td></tr>
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<tr><td>DpnI</td><td>Tango</td><td>B,G(100%): O,R,2xT(50-100%)</td></tr>
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<tr>
</table>
</table>
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*Star Activity<br>
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</center>
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**Optimal Buffer from Fermentas<br><br>
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</body>
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Use pUC19 plasmid as test, it has cut sites for EcoRI, PstI, XbaI (unsure about BcuI/SpeI, DpnI but will try anyways), and none for EcoRV <br>
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</html>
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<u>Red Buffer:</u> EcoRI, PstI, Control (No Enzyme)<br>
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<hr>
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<u>Tango Buffer:</u> BcuI/SpeI, XbaI, DpnI, Control (No Enzyme><br><br>
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<u>Methods:</u>
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Set up Master Mixes:
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<table><table border="3">
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<tr><td><b>Red MM</b></td><td>per tube (&micro;L)</td><td>Total (&micro;L)</td></tr>
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<tr><td>MilliQ H<sub>2</sub>0</td><td>13.75</td><td>55</td></tr>
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<tr><td>Red Buffer (10x)</td><td>2</td><td>7</td></tr>
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<tr><td>pUC19 (10pg/&micro;L)</td><td>2</td><td>7</td></tr>
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<tr><td><b>Total</b></td><td>19.75</td><td>69</td></tr>
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</table><br>
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<table><table border="3">
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<tr><td><b>Tango MM</b></td><td>per tube (&micro;L)</td><td>Total (&micro;L)</td></tr>
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<tr><td>MilliQ H<sub>2</sub>0</td><td>13.75</td><td>55</td></tr>
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<tr><td>Tango Buffer (10x)</td><td>2</td><td>7</td></tr>
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<tr><td>pUC19 (10pg/&micro;L)</td><td>2</td><td>7</td></tr>
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<tr><td><b>Total</b></td><td>19.75</td><td>69</td></tr>
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</table><br>
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To each tube, add <b>19.75&micro;L</b> of master mix and <b>0.25&micro;L</b> of enzyme<br>
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Incubated reaction mixes at 37<sup>o</sup>C (Start:7:00pm; End:7:45pm)<br>
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Add 3.3&micro;L of 6x loading dye to each reaction mixture and load 10&micro;L final volume onto a 1% agarose (in TAE) gel.<br>
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Add 1&micro;L of 6x loading dye to 1&micro;L of GeneRuler 1kb ladder (at 0.5&micro;g/&micro;L)<br>
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Gel loading order as follows:<br>
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<table><table border="3">
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<tr><td><b>Lane</b></td><td><b>Sample</b></td></tr>
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<tr><td>1</td><td>1kb Ladder</td></tr>
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<tr><td>2</td><td>Tango Control</td></tr>
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<tr><td>3</td><td>DpnI (Tango)</td></tr>
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<tr><td>4</td><td>BcuI/SpeI (Tango)</td></tr>
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<tr><td>5</td><td>XbaI (Tango)</td></tr>
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<tr><td>6</td><td>EcoRI (Red)</td></tr>
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<tr><td>7</td><td>PstI (Red)</td></tr>
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<tr><td>8</td><td>Red Control</td></tr>
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<tr><td>9</td><td>Empty</td></tr>
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<tr><td>10</td><td>Empty</td></tr>
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</table><br>
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Ran gel at 100V for 1 hour<br>
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<b>Results:</b> pUC19 plasmid DNA not present at a high enough concentration to visualize by ethidium bromide staining (1kb ladder did stain). <br>
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<b>Conclusion:</b> Will have to re-run experiment with DNA that is present at high enough concentrations to visualize by ethidium bromide staining<br><br><br>
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<BLOCKQUOTE>
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=<font color="white">Project Description=
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<a name="may"></a>
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The tailings ponds that result from the extraction of heavy crude oil and bitumen (used to make synthetic crude oil) from the oil sands have used up vast amounts of fresh water and contain substantial quantities of useable organic matter. While most of the bitumen is extracted from the oil sands, some is left behind and added to the tailings ponds. The residual hydrocarbon compounds can be potentially extracted from the ponds and utilized as another source of fuel. Consequently, cleaning the tailings ponds and increasing efficiency of extraction from the oil sands.
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<b><font size=+2>May 5/2010</font>(in the lab: JV)</b><br>
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<br>
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<b>Objective:</b> Test Restriction Endonucleases for activity (take 2)<br>
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<b>Relevant Information:</b><br>
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We wish to develop and characterize a BioBrick, that can breakdown some of the more prominent toxic organic compounds found in the tailing ponds to a more useable form.  We are currently targeting catechol, a aromatic compound shown to be degraded by bacteria living in the tailings ponds (Kato <i>et al.</i>, 2001).  Catechol is being converted into 2-hydroxymuconic semialdehyde, which we later hope to further convert into a useful hydrocarbon compound.
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Plasmid DNA used here will be "ES-pSB-CEYFP" from last year's plasmid stocks<br>
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<br>
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Prefix Enzymes are: EcoRI and XbaI<br>
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Suffix Enyzmes are: SpeI and PstI<br>
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Additionally, we plan to target our <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol degrading enzyme</font></a></html> into a <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="#00DC00"> microcompartment</font></a></html> which the <html><a href="https://2009.igem.org/Team:Lethbridge" target="new"><font color="#00DC00"> Lethbridge 2009</font></a></html> team began the work on. By compartmentalizing the converted catechol, were trying to develop a way of easily removing the useful hydrocarbon product from the tailings ponds. As a proof of principle, we will target the catechol degradation enzyme into the negatively charged microcompartment by the use of a poly-arginine tag.  Furthermore, to avoid adding a new species into the oil sands environment we plan on using the <html><a href="https://2010.igem.org/Team:Lethbridge/Project/DNA_Degradation"><font color="#00DC00"> DNA digestion part</font></a></html> created by <html><a href="https://2007.igem.org/Berkeley_UC" target="new"><font color="#00DC00"> Berkley in 2007</font></a></html> to render our <i>Escherichia coli </i>cells unable to reproduce or able to horizontally transfer its genes.  
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(JV worked out in lab notebook which buffers would be best for each prefix/suffix enzyme combination)<br>
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<br>
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Reactions will be assembled as follows:<br>
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<table><table border="3">
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Finally, we will be continuing to explore the novel method of the mass production of <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Magnetic_Nanoparticles"><font color="#00DC00"> uniform iron nanoparticles</font></a></html>, which is more efficient and cost effective than current methods (Prozorov <i>et al.</i>, 2007). To optimize the production of nanoparticles we are attaching signal peptide sequences to export the protein to different areas of the cell.  By attaching these signal peptides and having the protein directed to certain areas of the cell we hope to find which area is most productive to produce nanoparticles.
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<tr><td><b>Enzyme</td><td>Buffer</td><td>Volume MM(&micro;L)</td><td>Volume Enzyme(&micro;L)</td></tr></b>
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<br>
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<tr><td>PstI</td><td>Red</td><td>19.75</td><td>.25</td></tr>
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<tr><td>XbaI</td><td>Tango</td><td>19.75</td><td>.25</td></tr>
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<tr><td>SpeI</td><td>Tango</td><td>19.75</td><td>.25</td></tr>
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Reference:<br>
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<tr><td>EcoRI</td><td>Red</td><td>19.75</td><td>.25</td></tr>
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Kato, T., Haruki, M., Imanaka, T., Morikawa, M., and Kanaya, S. (2001). Isolation and characterization of psychrotrophic bacteria from oil-reservoir and oil sands. <i>Applied Microbial Biotechnology.</i> 55, 794-800.
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<tr><td>EcoRI/SpeI</td><td>Red</td><td>19.5</td><td>.25+.25</td></tr>
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<br>
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<tr><td>XbaI/SpeI</td><td>Tango</td><td>19.5</td><td>.25+.25</td></tr>
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Prozorov, T., Mallapragada, S. K., Narasimhan, B., Wang, L., Palo, P., Nilsen-Hamilton, M., Williams, T. J., Bazylinski, D. A., Prozorov, R., and Canfield, P. C. (2007). Protein-mediated synthesis of uniform superparamagnetic magnetite nanocrystals. Adv. Funct. Mater. Advanced Functional Materials. 17, 951-957
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<tr><td>EcoRI/PstI</td><td>Red</td><td>19.5</td><td>.25+.25</td></tr>
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<tr><td>XbaI/PstI</td><td>Tango</td><td>19.5</td><td>.25+.25</td></tr>
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=<font color="white">Sponsors=
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</table><br>
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Make up Master Mixes as follows:<br>
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==<font color="white">Platinum==
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<table><table border="3">
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<tr><td><b>Red MM</td><td>per tube(&micro;L)</td><td>Total*(&micro;L)</td></tr></b>
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===<font color="white">Alberta Innovates Technology Futures===
-
<tr><td>MilliQ H<sub>2</sub>0</td><td>15.75</td><td>86.675</td></tr>
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<html>
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<tr><td>Red Buffer (10x)</td><td>2</td><td>11</td></tr>
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<center>
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<tr><td>pDNA**</td><td>2</td><td>11</td></tr>
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<table>
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</table><br>
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<tr>
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<table><table border="3">
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<th>
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<tr><td><b>Tango MM</td><td>per tube(&micro;L)</td><td>Total*(&micro;L)</td></tr></b>
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<a href="http://www.albertainnovates.ca/technology/introduction" target="_blank">
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<tr><td>MilliQ H<sub>2</sub>0</td><td>15.75</td><td>86.675</td></tr>
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<img src="https://static.igem.org/mediawiki/2010/d/db/UofLAITFlogo.jpg" width="300"/>
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<tr><td>Tango Buffer (10x)</td><td>2</td><td>11</td></tr>
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</a>
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<tr><td>pDNA**</td><td>2</td><td>11</td></tr>
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</th>
 +
</tr>
</table>
</table>
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*Volume per reaction multiplied by 5.5<br>
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</center>
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**Unknown concentration of pDNA<br><br>
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</html>
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Incubated for 70min at 37<sup>o</sup>C (Start-1:05pm; End-2:15pm)<br>
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Added 3.3&micro;L of 6x loading dye to each reaction mixture and loaded 10&micro;L onto a 1% agarose gel (in TAE)<br>
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===<font color="white">Oil Sands Initiative===
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Added 1&micro;L of 6x loading dye to 2&micro;L of gene ruler 1kb ladder<br>
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<b>https://2010.igem.org/Oil_Sands</b>
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Load order as follows:<br>
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<table><table border="3">
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<tr><td><b>Lane</td><td>Sample</td><td>Volume Loaded (&micro;L)</td></tr></b>
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<tr><td>1</td><td>pSB-CEYFP/PstI</td><td>10</td></tr>
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<tr><td>2</td><td>pSB-CEYFP/EcoRI</td><td>10</td></tr>
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<tr><td>3</td><td>pSB-CEYFP/EcoRI/PstI</td><td>10</td></tr>
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<tr><td>4</td><td>pSB-CEYFP/EcoRI/SpeI</td><td>10</td></tr>
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<tr><td>5</td><td>pSB-CEYFP/XbaI/PstI</td><td>10</td></tr>
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<tr><td>6</td><td>pSB-CEYFP/XbaI</td><td>10</td></tr>
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<tr><td>7</td><td>pSB-CEYFP/SpeI</td><td>10</td></tr>
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<tr><td>8</td><td>pSB-CEYFP/XbaI/SpeI</td><td>10</td></tr>
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<tr><td>9</td><td>pSB-CEYFP/Red Master Mix Control</td><td>10</td></tr>
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<tr><td>10</td><td>pSB-CEYFP/Tango Master Mix Control</td><td>10</td></tr>
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<tr><td>11</td><td>pSB-CEYFP/MilliQ H<sub>2</sub>0 Control</td><td>10</td></tr>
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<tr><td>12</td><td>Ladder</td><td>4</td></tr>
+
-
</table><br>
+
-
Ran gel at 100V for 1 hour<br><br>
+
-
<b>Results:</b><br>
+
-
<a href="https://static.igem.org/mediawiki/2010/c/c3/100505JV-EnzymeTest1Cropped.jpg"><img src="https://static.igem.org/mediawiki/2010/c/c3/100505JV-EnzymeTest1Cropped.jpg"  width="225" height="151"/></a><br>
+
-
This gel shows that SpeI does not cut on its own, and does not cut when combined with other enzymes<br>
+
-
<b>Conclusion:</b> Test other source of SpeI to see if it has any activity.<br><br>
+
-
<b><font size=+2>May 6/2010</font>(in the lab:KG, AS)</b><br>
+
<html>
-
<b>Objective:</b> To check if the old SpeI enzyme (exp date: March 2011) will cleave plasmid DNA, since we believe the newer SpeI enzyme (exp date: 2012) does not.<br>
+
<center>
-
<b>Method:</b><br>
+
<table border="0" cellpadding="5" cellspacing="5" width="40%">
-
<table><table border ="3">
+
<tr>
-
<tr><td><b>Red Master Mix</b></td><td>per tube (&micro;L)</td><td>Total Volume*</td></tr>
+
<th>
-
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>15.75</td><td>63</td></tr>
+
<a href="http://www.see.ualberta.ca/OSRIN.cfm" target="_blank">
-
<tr><td>Red Buffer (10x)</td><td>2</td><td>8</td></tr>
+
<img src="https://static.igem.org/mediawiki/2010/a/a1/UofLOSRINlogo.jpg" width="250"/>
-
<tr><td>pDNA**</td><td>2</td><td>8</td></tr>
+
</a>
 +
</th>
 +
<th><a href="http://www.iseee.ca" target="_blank">
 +
<img src="https://static.igem.org/mediawiki/2010/6/6c/UofLISEEElogo.jpg" width="550"/>
 +
</a></th>
</table>
</table>
-
*Volume per tube multiplied by 4<br>
+
</center>
-
**Used pSB NEYFP pDNA from cell E5 in plasmid box<br>
+
</html>
-
Enzymes that will use Red Master Mix are: EcoRI+SpeI (old), EcoRI+SpeI (new)<br>
+
 
-
Add 0.25&micro;L of each enzyme to 19.5&micro;L of master mix<br><br>
+
<html>
-
<table><table border ="3">
+
<center>
-
<tr><td><b>Tango Master Mix</b></td><td>per tube (&micro;L)</td><td>Total Volume*</td></tr>
+
<table border="0" cellpadding="5" cellspacing="5" width="95%">
-
<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>15.75</td><td>94.5</td></tr>
+
<tr>
-
<tr><td>Tango Buffer (10x)</td><td>2</td><td>12</td></tr>
+
<th>
-
<tr><td>pDNA**</td><td>2</td><td>12</td></tr>
+
<a href="http://www.conocophillips.ca" target="_blank">
 +
<img src="https://static.igem.org/mediawiki/2010/4/42/UofLConocoPhillipslogo.jpg" width="160"/>
 +
</a>
 +
</th>
 +
<th><a href="http://www.nexeninc.com" target="_blank">
 +
<img src="https://static.igem.org/mediawiki/2010/2/2e/UofLNexenlogo.jpg" width="120"/>
 +
</a>
 +
</th>
 +
<th><a href="http://www.statoil.com" target="_blank">
 +
<img src="https://static.igem.org/mediawiki/2010/4/45/UofLStatoillogo.jpg" width="90"/>
 +
</a>
 +
</th>
 +
<th><a href="http://www.suncor.com" target="_blank">
 +
<img src="https://static.igem.org/mediawiki/2010/c/c3/UofLSuncorlogo.jpg" width="130"/>
 +
</a>
 +
</th>
 +
<th><a href="http://www.total-ep-canada.com" target="_blank">
 +
<img src="https://static.igem.org/mediawiki/2010/e/ed/UofLTotallogo.jpg" width="75"/>
 +
</a>
 +
</th>
</table>
</table>
-
*Volume per tube multiplied by 6<br>
+
</center>
-
**Used pSB NEYFP pDNA from cell E5 in plasmid box<br>
+
</html>
-
Enzymes that will use Tango Master Mix are: SpeI (old), SpeI (new), XbaI+SpeI (old), XbaI+SpeI (new)<br>
+
 
-
Add 0.25&micro;L of each enzyme to 19.5&micro;L of master mix<br><br>
+
==<font color="white">Gold==
-
Incubated all reactions at 37<sup>o</sup>C for 1h (Start-8:30pm; End-9:30pm)<br>
+
-
Will not be able to run on agarose gel tonight, will label them so JV can run them in the morning<br>
+
-
<u>Tube Names:</u><br>
+
-
Master Mix 1 Control (Red Buffer)<br>
+
-
Master Mix 2 Control (Tango Buffer)<br>
+
-
E+S(N); EcoRI + SpeI(N)<br>
+
-
E+S(O); EcoRI + SpeI(O)<br>
+
-
X+S(N); XbaI + SpeI(N)<br>
+
-
X+S(O); XbaI + SpeI(O)<br>
+
-
S(N); SpeI(N)<br>
+
-
S(O); SpeI(O)<br><br>
+
-
Placed in -20<sup>o</sup>C freezer of later analysis by agarose electrophoresis<br><br>
+
-
<b><font size=+2>May 10/2010</font>(in the lab:JV)</b><br>
+
===<font color="white">Autodesk===
-
<b>Objective:</b> To analyze the restriction test done by KG and AS on May 6/2010 by agarose electrophoresis<br><br>
+
<html>
-
<b>Method:</b><br>
+
<center>
-
<table><table border="3">
+
<table>
-
<tr><td><b>Lane</b></td><td>Sample</td><td>Quantity Loaded (&micro;L)</td></tr>
+
<tr>
-
<tr><td>1</td><td>MM1 Control</td><td>10</td></tr>
+
<th>
-
<tr><td>2</td><td>MM2 Control</td><td>10</td></tr>
+
<a href="http://www.autodeskresearch.com/" target="_blank">
-
<tr><td>3</td><td>EcoRI+SpeI(N)</td><td>10</td></tr>
+
<img src="https://static.igem.org/mediawiki/2010/6/67/UofLAutodesklogo.jpg"/>
-
<tr><td>4</td><td>EcoRI+SpeI(O)</td><td>10</td></tr>
+
</a>
-
<tr><td>5</td><td>SpeI(N)</td><td>10</td></tr>
+
</th>
-
<tr><td>6</td><td>SpeI(O)</td><td>10</td></tr>
+
</tr>
-
<tr><td>7</td><td>XbaI+SpeI(N)</td><td>10</td></tr>
+
-
<tr><td>8</td><td>XbaI+SpeI(O)</td><td>10</td></tr>
+
-
<tr><td>9</td><td>1kb Ladder</td><td>5</td></tr>
+
</table>
</table>
-
Run gel for 60min at 100V<br><br>
+
</center>
-
<b>Results:</b><br>
+
</html>
-
<a href="https://static.igem.org/mediawiki/2010/b/b0/100510JRV-EnzymeTest1Cropped.jpg"><img src="https://static.igem.org/mediawiki/2010/b/b0/100510JRV-EnzymeTest1Cropped.jpg"  width="225" height="151"/></a><br>
+
-
It appears as though both SpeI enzymes are working properly here. We will utilize the newer batch of SpeI (expires 2012) from this point forward.<br><br>
+
-
<b><font size=+2>May 10/2010</font>(in the lab:JV, KG, AV)</b><br>
+
==<font color="white">Silver==
-
<b>Objective:</b>Make 24 LB agar plates with 100&micro;g/mL ampicillin antibiotic.<br>
+
-
<b>Method:</b>Make 2L of LB media with agar<br>
+
-
2x10g Tryptone<br>
+
-
2X2.5g Yeast Extract<br>
+
-
2x5g NaCl<br>
+
-
2x10g Agar<br><br>
+
-
Continued <b> May 11/2010<br></b>
+
-
(Stock Ampicillin solution is 100mg/mL)<br>
+
-
Have 4x500mL of LB with Agar<br>
+
-
Add 500&micro;L of stock ampicillin to 500mL of media<br><br>
+
-
<b><font size=+2>May 11/2010 Evening </font> (in the lab: KG, AV, MC, TF, JV, JS)<br></b>
+
-
<b>Objective:</b> To transform the following plasmids into DH5&alpha; <i>E.coli</i> cells.
+
-
<table><table border="3">
+
-
<tr><td><b>Construct Name (2009)</td></b><td><b>Construct Location (2009)</b></td></tr>
+
-
<tr><td>Lumazine</td><td>J4</td></tr>
+
-
<tr><td>Lumazine-dT</td><td>J5,J6</td></tr>
+
-
<tr><td>sRBS-Lumazine-dT</td><td>J7,J8</td></tr>
+
-
<tr><td>pBAD-TetR</td><td>I4</td></tr>
+
-
<tr><td>pBAD</td><td>A5,F10</td></tr>
+
-
<tr><td>sRBS</td><td>D5,E10</td></tr>
+
-
<tr><td>pSB-CEYFP</td><td>E5,D6</td></tr>
+
-
<tr><td>pSB-NEYFP</td><td>F5,C6</td></tr>
+
-
<tr><td>C-term Tag</td><td>C10</td></tr>
+
-
<tr><td>N-term Tag</td><td>D9,D10</td></tr>
+
-
<tr><td>pTet</td><td>E4</td></tr>
+
-
<tr><td>EYFP</td><td>A4</td></tr>
+
-
<tr><td>CFP Complete</td><td>D4</td></tr>
+
-
</table><br>
+
-
<b>Method: </b>Followed <a href="https://2010.igem.org/Team:Lethbridge#transformation">"Competent Cell Transformation"</a> protocol in Common Protocols section and plated on LB agar supplemented with ampicillin.<br>
+
-
<b>Results: </b>The following plasmids were successfully transformed and formed colonies:<br>
+
-
<ul>
+
-
<li>Lumazine (J4)</li>
+
-
<li>sRBS-Lumazine-dT (J7)</li>
+
-
<li>sRBS-Lumazine-dT (J8)</li>
+
-
<li>pBAD (A5) </li>
+
-
<li>pBAD (F10) </li>
+
-
<li>pSB-CEYFP </li>
+
-
<li>pSB-NEYFP </li>
+
-
<li>N-term tag </li>
+
-
<li>EYFP (A4)</li>
+
-
<li>CFP Complete (D4)</li></ul><br>
+
-
<b>Conclusion:</b> Need another attempt to transform the following plasmids:<br>
+
-
<ul>
+
-
<li>Lumazine-dT (J5,J6)</li>
+
-
<li>pBAD-TetR </li>
+
-
<li>sRBS (D5,E10)</li>
+
-
<li>C-Term tag</li>
+
-
<li>pTet</li></ul><br>
+
-
<br><b><font size=+2>May 12/2010</font>(in the lab: JV)</b><br>
+
 
-
<b>Objective: Miniprep of plasmid DNA from transformed cells</b>(JV, AV, HB)<br>
+
===<font color="white">MathWorks===
-
<b>Method:</b><br>  
+
<html>
-
<ul><li>Inoculate 5mL of LB liquid media (with 100&micro;L/mL Ampicillin) with cells from competent cells plates (picked with sterile toothpick).</li>
+
<center>
-
<li>Allow cells in liquid culture to grow overnight in 37<sup>o</sup>C shaking incubator (300RPM)
+
<table>
-
Purify plasmid DNA from cells by using "Boiling Lysis Plasmid Preparation" protocol in Common Protocols Section.</li>
+
<tr>
-
<li>CHANGE: Step 14, used MilliQ H<sub>2</sub>O (with 20ng/&micro;L RNase A) instead of TE buffer.</li></ul><br>
+
<th>
-
Plasmids were transferred to the "iGEM 2010 - Working Plasmid DNA" box in the -20<sup>o</sup>C freezer in the iGEM lab. Plasmids were placed in the following cells:
+
<a href="http://www.mathworks.com/" target="_blank">
-
<table><table border="3">
+
<img src="https://static.igem.org/mediawiki/2010/c/c3/UofLMathWorkslogo.jpg" width="300" border="0"/>
-
<tr><td>Construct</td><td>Cell in Working Plasmid Box (2010)</td><td>Original Cell in Old Box</td></tr>
+
</a>
-
<tr><td>sRBS-Lumazine-dT</td><td>A1</td><td>J7</td></tr>
+
</th>
-
<tr><td>sRNS-Lumazine-dT</td><td>A2</td><td>J8</td></tr>
+
</tr>
-
<tr><td>CFP Complete</td><td>B6</td><td>D4</td></tr>
+
-
<tr><td>Lumazine</td><td>A3</td><td>J4</td></tr>
+
-
<tr><td>pBAD</td><td>A4</td><td>A5</td></tr>
+
-
<tr><td>pBAD</td><td>A5</td><td>F10</td></tr>
+
-
<tr><td>pSB-CEYFP</td><td>B5</td><td></td></tr>
+
-
<tr><td>pSB-NEYFP</td><td>B4</td><td></td></tr>
+
-
<tr><td>EYFP</td><td>B1</td><td>A4</td></tr>
+
-
<tr><td>N-term tag</td><td>B2</td><td></td></tr>
+
-
</table><br>
+
-
Also generated sterile glycerol stocks and placed in -80<sup>o</sup>C freezer in the 2010 iGEM box as follows:
+
-
<table><table border="3">
+
-
<tr><td>Construct</td><td>Cell Working Glycerol Stock Box (2010)</td></tr>
+
-
<tr><td>sRBS-Lumazine-dT (J7)</td><td>B2,C4,D2</td></tr>
+
-
<tr><td>sRNS-Lumazine-dT (J8)</td><td>C6</td></tr>
+
-
<tr><td>CFP Complete</td><td>A10, C8</td></tr>
+
-
<tr><td>Lumazine</td><td>A8,B10</td></tr>
+
-
<tr><td>pBAD (from A5)</td><td>B5,B9</td></tr>
+
-
<tr><td>pBAD (from F10)</td><td>B3,B7</td></tr>
+
-
<tr><td>pSB-CEYFP</td><td>C3,B5</td></tr>
+
-
<tr><td>pSB-NEYFP</td><td>B6,C1</td></tr>
+
-
<tr><td>EYFP</td><td>C7,B8</td></tr>
+
-
<tr><td>N-term tag</td><td>C2,D4</td></tr>
+
-
</table><br>
+
-
<b>Objective:</b> Restrict plasmid DNA with restriction endonucleases (JV)<br>
+
-
<b>Method:</b><br>
+
-
Have:
+
-
10 lanes of restricted plasmid DNA <br>
+
-
10 lanes of unrestricted plasmid DNA <br>
+
-
1 lane of buffer control <br>
+
-
Use EcoRI (prefix cutter) and PstI (suffix cutter) <br><br>
+
-
Pipetting Scheme for Restriction Tubes:
+
-
<table><table border="3">
+
-
<tr><td><b>Ingredient</b></td><td>Volume/tube (&micro;L)</td><td>Total Volume*</td></tr>
+
-
<tr><td>MilliQ H<sub>2</sup>O</td><td>15.5</td><td>155</td></tr>
+
-
<tr><td>Red Buffer (10X)</td><td>2</td><td>20</td></tr>
+
-
<tr><td>EcoRI</td><td>0.25</td><td>2.5</td></tr>
+
-
<tr><td>PstI</td><td>0.25</td><td>2.5</td></tr>
+
</table>
</table>
-
*Amount per tube multiplied by 10<br>
+
</center>
-
Pipetting Scheme for Unrestricted reactions:
+
</html>
-
<table><table border="3">
+
 
-
<tr><td><b>Ingredient</b></td><td>Volume/tube (&micro;L)</td><td>Total Volume*</td></tr>
+
 
-
<tr><td>MilliQ H<sub>2</sup>O</td><td>16</td><td>160</td></tr>
+
===<font color="white">Geneious===
-
<tr><td>Red Buffer (10X)</td><td>2</td><td>20</td></tr>
+
<html>
 +
<center>
 +
<table>
 +
<tr>
 +
<th>
 +
<a href="http://www.geneious.com/" target="_blank">
 +
<img src="https://static.igem.org/mediawiki/2010/3/30/UofLGeneiouslogo.jpg" width="300"/>
 +
</a>
 +
</th>
 +
</tr>
</table>
</table>
-
*Amount per tube multiplied by 10<br>
+
</center>
-
Buffer Control will be 18&micro;L MilliQ H<sub>2</sub>O + 2&micro;L 10x Red Buffer.<br>
+
</html>
-
Place in 37<sup>o</sup>C water bath at 2:55pm and removed at 4:57pm for a 2 hour incubation.<br>
+
-
Analyzed restriction digests on a 1% agarose gel (large gel apparatus ~70mL)<br>
+
-
Added 1&micro;L of 6x DNA loading dye to 5&micro;L of sample<br>
+
-
Added 2&micro;L of 6x DNA loading dye to 6&micro;L of TAE buffer and 2&micro;L of 1kb DNA mass ladder.<br>
+
-
Loaded samples as follows:<br>
+
-
<table><table border="3">
+
-
<tr><td><b>Lane</td><td>Sample</td><td>Volume Loaded (&micro;L)</b></td></tr>
+
-
<tr><td>1</td><td>1 kb Ladder</td><td>5</td></tr>
+
-
<tr><td>2</td><td>Buffer Control</td><td>5</td></tr>
+
-
<tr><td>3</td><td>pSB-NEYFP</td><td>5</td></tr>
+
-
<tr><td>4</td><td>Restricted Lumazine</td><td>5</td></tr>
+
-
<tr><td>5</td><td>Lumazine</td><td>5</td></tr>
+
-
<tr><td>6</td><td>Restricted pSB-NEYFP</td><td>5</td></tr>
+
-
<tr><td>7</td><td>pSB-CEYFP</td><td>5</td></tr>
+
-
<tr><td>8</td><td>Restricted pSB-CEYFP</td><td>5</td></tr>
+
-
<tr><td>9</td><td>pBAD</td><td>5</td></tr>
+
-
<tr><td>10</td><td>Restricted pBAD</td><td>5</td></tr>
+
-
<tr><td>11</td><td>EYFP</td><td>5</td></tr>
+
-
<tr><td>12</td><td>Restricted EYFP</td><td>5</td></tr>
+
-
<tr><td>13</td><td>CFP Complete</td><td>5</td></tr>
+
-
<tr><td>14</td><td>Restricted CFP Complete</td><td>5</td></tr>
+
-
<tr><td>15</td><td>sRBS-Lumazine-dT (J7)</td><td>5</td></tr>
+
-
<tr><td>16</td><td>Restricted sRBS-Lumazine-dT (J7)</td><td>5</td></tr>
+
-
<tr><td>17</td><td>N-term Tag</td><td>5</td></tr>
+
-
<tr><td>18</td><td>Restricted N-term Tag</td><td>5</td></tr>
+
-
<tr><td>19</td><td>sRBS-Lumazine-dT (J8)</td><td>5</td></tr>
+
-
<tr><td>20</td><td>Restricted sRBS-Lumazine-dT (J8)</td><td>5</td></tr>
+
-
</table><br>
+
-
Ran gel at 100V for 90 minutes (Start-9:50pm; End-11:20pm)<br>
+
-
Stained with ethidium bromide for 20 minutes.<br>
+
-
<b>Results:</b><br>
 
-
<a href="https://static.igem.org/mediawiki/2010/7/79/100513TF.KG.JS-Plasmids_for_Seq.Cropped.jpg"><img src="https://static.igem.org/mediawiki/2010/7/79/100513TF.KG.JS-Plasmids_for_Seq.Cropped.jpg"  width="225" height="151"/></a><br><br>
 
-
<b><font size=+2>May 13/2010 Evening</font>(in lab: AS,TF,KG,JS,MC)</b><br>
 
-
<b>Objective:</b> To make a second attempt at transforming plasmids that didn't transform the first time. These plasmids are:<br>
 
-
<ul>
 
-
<li>Lumazine-dT (J5,J6)</li>
 
-
<li>pBad-TetR</li>
 
-
<li>sRBS (D5,E10)</li>
 
-
<li>C-term tag</li>
 
-
<li>pTet</li></ul><br>
 
-
All DH5&alpha; cells were used up in the last transformation, had to aliquot an additional 50x 20&micro;L aliquots (MC,TF)<br>
 
-
Transform plasmid DNA (Using "Competent Cell Transformation" Protocol) into newly aliquotted DH5&alpha; cells. (KG,JS)<br>
 
-
NOTES:<br>
 
-
AS concerned that there is something not quite right with LB liquid media added to transformed cells, but continued anyways (JV informed AS the next day that the LB liquid media had not been sterilized).<br>
 
-
Plated all 250&micro;L of culture.<br>
 
-
<b>Results:</b><br>
 
-
<table><table border="3">
 
-
<tr><td><b>Construct</td><td>Result</b></td></tr>
 
-
<tr><td>Lumazine-dT(1)</td><td>Growth present</td></tr>
 
-
<tr><td>sRBS-Lumazine-dT</td><td>Growth present</td></tr>
 
-
<tr><td>sRBS (D5)</td><td>Growth present</td></tr>
 
-
<tr><td>sRBS (E10)</td><td>Growth present</td></tr>
 
-
<tr><td>C-term tag</td><td>No growth present</td></tr>
 
-
<tr><td>pTet</td><td>No growth present</td></tr>
 
-
</table><br>
 
-
<b>Next Steps:</b><br>
 
-
Make another attempt to transform the C-term tag and pTet constructs.<br>
 
-
Start overnight cultures of cells that grew for plasmid prep and sequencing.<br><br>
 
-
<b><font size=+2>May 14/2010</font>(in the lab: JV)</b><br>
+
==<font color="white">Bronze==
-
<b>Objective:</b> Quantify pDNA concentration in order to ensure sufficient material for sequence analysis.<br>
+
-
<b>Method:</b> Measure absorbance of samples at 260nm.<br>
+
-
<b>Results:</b><br>
+
-
<table><table border="3">
+
-
<tr><td><b>Sample</td><td>Absorbance at 260nm</b></td></tr>
+
-
<tr><td>sRBS-Lumazine-dT (J7)</td><td>0.311</td></tr>
+
-
<tr><td>sRBS-Lumazine-dT (J8)</td><td>0.309</td></tr>
+
-
<tr><td>CFP complete</td><td>0.316</td></tr>
+
-
<tr><td>N-term tag</td><td>0.290</td></tr>
+
-
<tr><td>pSB-CEYFP</td><td>0.338</td></tr>
+
-
<tr><td>pSB-NEYFP</td><td>0.403</td></tr>
+
-
<tr><td>pBAD (A5)</td><td>0.282</td></tr>
+
-
<tr><td>pBAD (F10)</td><td>0.562</td></tr>
+
-
<tr><td>EYFP</td><td>0.389</td></tr>
+
-
<tr><td>Lumazine</td><td>0.221</td></tr>
+
-
</table><br>
+
-
<b>Conclusion:</b> All plasmids present in sufficient concentrations for sequence analysis.<br>
+
-
<b>Objective:</b> Purify plasmid DNA from cells recently transformed.<br>
+
===<font color="white">Integrated DNA Technologies===
-
<b>Method:</b>  
+
<html>
-
<ul>
+
<center>
-
<li>Inoculate 5mL of sterile LB liquid media (with 100&micro;g/mL ampicillin) with cells picked from colonies of transformation plates, including the following:<br>
+
<table>
-
Lumazine-dT (J5)<br>
+
<tr>
-
pBad-TetR<br>
+
<th>
-
sRBS (D5,E10)<br></li>
+
<a href="http://www.idtdna.com/" target="_blank">
-
<li>NOTE: Lumazine-dT did NOT grow overnight</li>
+
<img src="https://static.igem.org/mediawiki/2010/4/43/UofLIDTlogo.jpg"/>
-
<li>Followed "Boiling Lysis Plasmid Preparation (Miniprep)" protocol. <b>(May 15/2010; JV,TF)</b><br>
+
</a>
-
NOTE: Added 50&micro;L of MilliQ H<sub>2</sub>O (with RNase A at a concentration of 20ng/&micro;L) to dissolve pDNA instead of TE buffer.<br><br></li></ul>
+
</th>
-
<b>Objective:</b> Perform restriction digest on the above prepared plasmid DNA.<br>
+
</tr>
-
<b>Method:</b><br>
+
-
Used EcoRI as prefix cutter and PstI as suffix cutter.<br>
+
-
Pipetting Scheme for Restriction Tubes:
+
-
<table><table border="3">
+
-
<tr><td><b>Ingredient</b></td><td>Volume/tube (&micro;L)</td><td>Total Volume*</td></tr>
+
-
<tr><td>MilliQ H<sub>2</sup>O</td><td>16</td><td>56</td></tr>
+
-
<tr><td>Red Buffer (10X)</td><td>2</td><td>7</td></tr>
+
-
<tr><td>EcoRI</td><td>0.25</td><td>0.875</td></tr>
+
-
<tr><td>PstI</td><td>0.25</td><td>0.875</td></tr>
+
</table>
</table>
-
*Amount per tube multiplied by 3.5<br>
+
</center>
-
Add 18&micro;L master mix to each plasmid DNA sample<br>
+
</html>
-
Pipetting Scheme for Unrestricted reactions:
+
 
-
<table><table border="3">
+
 
-
<tr><td><b>Ingredient</b></td><td>Volume/tube (&micro;L)</td><td>Total Volume*</td></tr>
+
===<font color="white">GeneArt & Mr. Gene===
-
<tr><td>MilliQ H<sub>2</sup>O</td><td>16</td><td>56</td></tr>
+
<html>
-
<tr><td>Red Buffer (10X)</td><td>2</td><td>7</td></tr>
+
<center>
 +
<align="left">
 +
<table border="0" cellpadding="5" cellspacing="5" width="40%">
 +
<tr>
 +
<th>
 +
<a href="http://www.geneart.com/" target="_blank">
 +
<img src="https://static.igem.org/mediawiki/2010/e/ed/UofLGENEARTlogo.jpg"/>
 +
</a>
 +
</th>
 +
<th><a href="http://mrgene.com/desktopdefault.aspx/tabid-2/" target="_blank">
 +
<img src="https://static.igem.org/mediawiki/2010/2/26/UofLMRGENElogo.jpg"/>
 +
</a></th>
</table>
</table>
-
*Amount per tube multiplied by 3.5<br>
+
</center>
-
Add 18&micro;L master mix to each plasmid DNA sample<br>
+
</html>
-
Buffer Control will be 18&micro;L MilliQ H<sub>2</sub>O + 2&micro;L 10x Red Buffer.<br>
+
 
-
Place in 37<sup>o</sup>C water bath at 12:37pm and removed at 1:55pm for approximately 1 hour incubation.<br>
+
 
-
Analyze samples on a 1% agarose gel (small gel apparatus).<br>
+
===<font color="white">Macrogen===
-
Add 3.3&micro;L of 6x DNA loading dye to each reaction mixture and load.<br>
+
<html>
-
<table><table border="3">
+
<center>
-
<tr><td><b>Lane</td><td>Sample</td><td>Volume Loaded (&micro;L)</td></b></tr>
+
<table>
-
<tr><td>1</td><td>1 kb Ladder</td><td>4</td></tr>
+
<tr>
-
<tr><td>2</td><td>Restricted sRBS (E10)</td><td>10</td></tr>
+
<th>
-
<tr><td>3</td><td>sRBS (E10)</td><td>10</td></tr>
+
<a href="http://www.macrogen.com/eng/macrogen/macrogen_main.jsp" target="_blank">
-
<tr><td>4</td><td>Restricted sRBS (D5)</td><td>10</td></tr>
+
<img src="https://static.igem.org/mediawiki/2010/a/a7/UofLMacrogenlogo.jpg"/>
-
<tr><td>5</td><td>sRBS (D5)</td><td>10</td></tr>
+
</a>
-
<tr><td>6</td><td>Restricted sRBS-Lumazine-dT</td><td>10</td></tr>
+
</th>
-
<tr><td>7</td><td>sRBS-Lumazine-dT</td><td>10</td></tr>
+
</tr>
-
<tr><td>8</td><td>Red Buffer Control</td><td>10</td></tr>
+
</table>
</table>
-
Ran gel at 100V for 75 minutes (Start-2:30pm; End-3:45pm)<br>
+
</center>
-
Stained in ethidium bromide for 10 minutes<br><br>
+
</html>
-
<b>Results:</b><br>
+
-
<b>Picture to come.....</b><br>
+
-
There is plasmid DNA in each sample which, when cut with both the prefix and suffix enzyme, yields a band approximately 2000bp (size of pSB1A3 is 2157bp).<br>
+
 +
==<font color="white">Sponsorship Breakdown==
 +
===<font color="white">The University of Lethbridge iGEM Team requests your support in the following ways:===
 +
a) $2000 scholarship of one team member – It is our hope that every team member will be able to travel to the iGEM competition taking place in November 2010.  However, without enough funding, this will not be the situation.  By sponsoring one student at a time, we can work towards reaching our goal of the entire U of L team traveling to MIT and provide every student with the possibility to meet and talk to some of the brightest and inspiring minds of synthetic biology.
-
</div>
+
b) Monetary donation towards lab supplies, travel arrangements and iGEM competition registration fees.
-
<div class="TabbedPanelsContent">
+
-
<b><font size=+2>Common Protocols:</b></font><br>
+
-
<ol><li><a href=#transformation>Competent Cell Transformation</a></li>
+
-
<li><a href=#miniprep>Boiling Lysis Plasmid Preparation (Miniprep)<br>
+
-
</ol>
+
-
<a name="transformation"></a>
+
-
<b>Competent Cell Transformation</b><br>
+
-
<ol>
+
-
<li>Thaw 20&micro;L of aliquotted cells (DH5&alpha; of BL21(DE3)) on ice.</li>
+
-
<li>Gently pipet 2.0&micro;L of DNA into competent cells<br>
+
-
ATTENTION: <br>
+
-
Do not perform any additional mixing<br>
+
-
Never use more DNA that <u>10% of the volume of the competent cells </u>otherwise the cells get destroyed by osmotic shock</li>
+
-
<li>Incubate the cells on ice for 30 minutes.</li>
+
-
<li>Heat shock the cells in a water bath at <u>42<sup>o</sup>C for EXACTLY 45 seconds.</u></li>
+
-
<li>Incubate the cells on ice for 1 minute.</li>
+
-
<li>Add 250&micro;L sterile media to the cells and incubate at 37<sup>o</sup>C for 1 hour with shaking (200RPM).</li>
+
-
<li>Plate 100&micro;L and 50&micro;L on prewarmed LB agar plate containing the appropriate antibiotic.<br>
+
-
For ligations, plate all 250&micro;L.</li>
+
-
<li>Leave plate for 10-15 minutes to soak the cell suspension into the agar.</li>
+
-
<li>Flip plate over (agar on top)</li>
+
-
<li>Incubate the plates in the 37<sup>o</sup>C incubator overnight</li>
+
-
</ol><br>
+
-
<a name="miniprep"></a>
+
-
<b>Boiling Lysis Plasmid Preparation (Miniprep)</b><br>
+
-
<ol>
+
-
<li>Aseptically transfer 1.5mL of each overnight culture to a 1.5mL microcentrifuge tube (MCT) and pellet the cells by centrifugation in a benchtop microcentrifuge (2min at 13000RPM)</li>
+
-
<li>Remove and discard as much of the supernatant as possible by aspiration (e.g with a Pasteur Pipette). Do not suck up the cell pellet!!</li>
+
-
<li>Rinse the cell pellet by washing 1.0mL of sterile MilliQ H<sub>2</sub>O gently down the inside wall of the MCT. This removes any traces of the supernatant adhering to the MCT wall while minimizing the disturbance to the cell pellet. (/li>
+
-
<li>Resuspend the cell pellet in 350&micro;L of STET. </li>
+
-
<li>Add 25&micro;L of the Lysozyme solution and mix by inversion.</li>
+
-
<li>Place the MCT in the bioling water bath for EXACTLY 35 seconds, remove and incubate on ice for 5 minutes.</li>
+
-
<li>Pellet the cellular debris by centrifugation at 13000RPM for 15 minutes. Transfer the supernatant to a fresh MCT and discard the pellet.</li>
+
-
<li>Precipitate the plasmid DNA by adding 40&micro;L of 3.0M sodium acetate (pH 5.2) and 420&micro;L isopropanol. Mix by inversion. Mix by inversion and incubate for 5 minutes at room temperature.</li>
+
-
<li>Pellet the plasmid DNA by centrifugation at 13000RPM for 10 minutes at 4<sup>o</sup>C. A pellet of plasmid DNA should be visible at the base of the MCT when complete.</li>
+
-
<li>Being careful not to disturb the pellet, discard the supernatant and rinse the pellet with 500&micro;L of ice cold ethanol.</li>
+
-
<li>Repeat above step.</li>
+
-
<li>Invert and tap the open MCT several times against a piece of paper towel on your bench to remove as much ethanol as possible.</li>
+
-
<li>Store the open MCT at room temperature for approximately 10 minutes to allow all remaining traces of ethanol to evaporate</li>
+
-
<li>Add 50&micro;L of TE (pH 8.0) containing RNase A and resuspend the plasmid DNA by flicking the base of the MCT with your finger. The plasmid DNA is ready for use or can be stored long term at -20<sup>o</sup>C.</ol>
+
-
</div>
+
-
<div class="TabbedPanelsContent">
+
-
<b>Updated May 19/2010</b><br><br>
+
-
<b>Row A</b>
+
-
<table><table border="3">
+
-
<tr><td>Cell</td><td>Common Name</td><td>Standard Registry of Parts ID</td><td>Date Entered</td></tr>
+
-
<tr><td>A1</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A2</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A3</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A4</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A5</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A6</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A7</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A8</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A9</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A10</td><td></td><td></td><td></td></tr>
+
-
</table><br>
+
-
<b>Row A</b>
+
-
<table><table border="3">
+
-
<tr><td>Cell</td><td>Common Name</td><td>Standard Registry of Parts ID</td><td>Date Entered</td></tr>
+
-
<tr><td>A1</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A2</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A3</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A4</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A5</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A6</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A7</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A8</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A9</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A10</td><td></td><td></td><td></td></tr>
+
-
</table><br>
+
-
<b>Row A</b>
+
-
<table><table border="3">
+
-
<tr><td>Cell</td><td>Common Name</td><td>Standard Registry of Parts ID</td><td>Date Entered</td></tr>
+
-
<tr><td>A1</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A2</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A3</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A4</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A5</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A6</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A7</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A8</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A9</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A10</td><td></td><td></td><td></td></tr>
+
-
</table><br>
+
-
<b>Row A</b>
+
-
<table><table border="3">
+
-
<tr><td>Cell</td><td>Common Name</td><td>Standard Registry of Parts ID</td><td>Date Entered</td></tr>
+
-
<tr><td>A1</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A2</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A3</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A4</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A5</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A6</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A7</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A8</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A9</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A10</td><td></td><td></td><td></td></tr>
+
-
</table><br>
+
-
<b>Row A</b>
+
-
<table><table border="3">
+
-
<tr><td>Cell</td><td>Common Name</td><td>Standard Registry of Parts ID</td><td>Date Entered</td></tr>
+
-
<tr><td>A1</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A2</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A3</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A4</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A5</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A6</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A7</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A8</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A9</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A10</td><td></td><td></td><td></td></tr>
+
-
</table><br>
+
-
<b>Row A</b>
+
-
<table><table border="3">
+
-
<tr><td>Cell</td><td>Common Name</td><td>Standard Registry of Parts ID</td><td>Date Entered</td></tr>
+
-
<tr><td>A1</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A2</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A3</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A4</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A5</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A6</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A7</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A8</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A9</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A10</td><td></td><td></td><td></td></tr>
+
-
</table><br>
+
-
<b>Row A</b>
+
-
<table><table border="3">
+
-
<tr><td>Cell</td><td>Common Name</td><td>Standard Registry of Parts ID</td><td>Date Entered</td></tr>
+
-
<tr><td>A1</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A2</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A3</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A4</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A5</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A6</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A7</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A8</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A9</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A10</td><td></td><td></td><td></td></tr>
+
-
</table><br>
+
-
<b>Row A</b>
+
-
<table><table border="3">
+
-
<tr><td>Cell</td><td>Common Name</td><td>Standard Registry of Parts ID</td><td>Date Entered</td></tr>
+
-
<tr><td>A1</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A2</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A3</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A4</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A5</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A6</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A7</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A8</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A9</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A10</td><td></td><td></td><td></td></tr>
+
-
</table><br>
+
-
<b>Row A</b>
+
-
<table><table border="3">
+
-
<tr><td>Cell</td><td>Common Name</td><td>Standard Registry of Parts ID</td><td>Date Entered</td></tr>
+
-
<tr><td>A1</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A2</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A3</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A4</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A5</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A6</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A7</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A8</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A9</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A10</td><td></td><td></td><td></td></tr>
+
-
</table><br>
+
-
<b>Row A</b>
+
-
<table><table border="3">
+
-
<tr><td>Cell</td><td>Common Name</td><td>Standard Registry of Parts ID</td><td>Date Entered</td></tr>
+
-
<tr><td>A1</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A2</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A3</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A4</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A5</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A6</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A7</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A8</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A9</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A10</td><td></td><td></td><td></td></tr>
+
-
</table><br>
+
-
</div>
+
-
<div class="TabbedPanelsContent">
+
-
<b><font color="blue">Updated May 19/2010</b></font>
+
-
<b>Row A</b>
+
-
<table><table border="3">
+
-
<tr><td>Cell</td><td>Common Name</td><td>Standard Registry of Parts ID</td><td>Date Entered</td></tr>
+
-
<tr><td>A1</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A2</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A3</td><td></td><td></td><td></td></tr>
+
-
<tr><td>A4</td><td></td><td></td><td></td></tr>
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<tr><td>A6</td><td></td><td></td><td></td></tr>
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<tr><td>A7</td><td></td><td></td><td></td></tr>
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</table><br>
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<b>Row A</b>
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<table><table border="3">
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<tr><td>Cell</td><td>Common Name</td><td>Standard Registry of Parts ID</td><td>Date Entered</td></tr>
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<tr><td>A1</td><td></td><td></td><td></td></tr>
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<tr><td>A2</td><td></td><td></td><td></td></tr>
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<tr><td>A9</td><td></td><td></td><td></td></tr>
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<tr><td>A10</td><td></td><td></td><td></td></tr>
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</table><br>
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<b>Row A</b>
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<table><table border="3">
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<tr><td>Cell</td><td>Common Name</td><td>Standard Registry of Parts ID</td><td>Date Entered</td></tr>
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<tr><td>A1</td><td></td><td></td><td></td></tr>
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<tr><td>A2</td><td></td><td></td><td></td></tr>
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<tr><td>A3</td><td></td><td></td><td></td></tr>
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<tr><td>A4</td><td></td><td></td><td></td></tr>
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<tr><td>A5</td><td></td><td></td><td></td></tr>
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<tr><td>A6</td><td></td><td></td><td></td></tr>
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<tr><td>A9</td><td></td><td></td><td></td></tr>
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<b>Row A</b>
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<table><table border="3">
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<tr><td>Cell</td><td>Common Name</td><td>Standard Registry of Parts ID</td><td>Date Entered</td></tr>
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<tr><td>Cell</td><td>Common Name</td><td>Standard Registry of Parts ID</td><td>Date Entered</td></tr>
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</div>
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 +
c) Tangible donations of lab supplies or other necessary materials.
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</div>
+
===<font color="white">Donations can be made in two ways:===
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    <div class="TabbedPanelsContent"><iframe src="https://www.google.com/calendar/embed?showPrint=0&amp;height=800&amp;wkst=1&amp;bgcolor=%2333ff33&amp;src=uleth.igem%40gmail.com&amp;color=%2329527A&amp;ctz=America%2FEdmonton" style=" border-width:0 " width="962" height="800" frameborder="0" scrolling="no"></iframe></div>
+
 
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    <div class="TabbedPanelsContent">Content 6</div>
+
1. Donations can be directed towards the U of L iGEM Scholarship account.
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    <div class="TabbedPanelsContent">Content 7</div>
+
 
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  </div>
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2. Donations can be directed towards the U of L iGEM General account and in recognition of your generous support, the U of L iGEM team would like to recognize you and/or your business as outlined in the sponsorship levels.
-
</div>
+
 
-
<p>&nbsp;</p>
+
===<font color="white">Sponsorship Levels===
-
<p>&nbsp; </p>
+
 
-
<p>This site is best viewed with fire fox</p>
+
Platinum - $5000+ or gift in kind <br>
-
<script type="text/javascript">
+
Logo on team shirts, large logo on scientific poster, large logo on team wiki and verbal recognition during team project presentations/media interviews.
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+
 
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Gold - $2000-$4999 or gift in kind<br>
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Logo on team shirts, medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.
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Silver - $1000-$1999 or gift in kind<br>
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+
Medium logo on team shirts, scientific poster, medium logo on team wiki and written recognition at end of team project presentations.
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</script>
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Bronze - <$999 or gift in kind<br>
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</html>
+
Small logo on team shirts, scientific poster, small logo on team wiki and written recognition at end of team project presentations.
 +
<br>
 +
<br>
 +
<br>

Latest revision as of 15:20, 27 October 2010



Check out these important project links!

Contents

Project Description

The tailings ponds that result from the extraction of heavy crude oil and bitumen (used to make synthetic crude oil) from the oil sands have used up vast amounts of fresh water and contain substantial quantities of useable organic matter. While most of the bitumen is extracted from the oil sands, some is left behind and added to the tailings ponds. The residual hydrocarbon compounds can be potentially extracted from the ponds and utilized as another source of fuel. Consequently, cleaning the tailings ponds and increasing efficiency of extraction from the oil sands.

We wish to develop and characterize a BioBrick, that can breakdown some of the more prominent toxic organic compounds found in the tailing ponds to a more useable form. We are currently targeting catechol, a aromatic compound shown to be degraded by bacteria living in the tailings ponds (Kato et al., 2001). Catechol is being converted into 2-hydroxymuconic semialdehyde, which we later hope to further convert into a useful hydrocarbon compound.

Additionally, we plan to target our catechol degrading enzyme into a microcompartment which the Lethbridge 2009 team began the work on. By compartmentalizing the converted catechol, were trying to develop a way of easily removing the useful hydrocarbon product from the tailings ponds. As a proof of principle, we will target the catechol degradation enzyme into the negatively charged microcompartment by the use of a poly-arginine tag. Furthermore, to avoid adding a new species into the oil sands environment we plan on using the DNA digestion part created by Berkley in 2007 to render our Escherichia coli cells unable to reproduce or able to horizontally transfer its genes.

Finally, we will be continuing to explore the novel method of the mass production of uniform iron nanoparticles, which is more efficient and cost effective than current methods (Prozorov et al., 2007). To optimize the production of nanoparticles we are attaching signal peptide sequences to export the protein to different areas of the cell. By attaching these signal peptides and having the protein directed to certain areas of the cell we hope to find which area is most productive to produce nanoparticles.


Reference:
Kato, T., Haruki, M., Imanaka, T., Morikawa, M., and Kanaya, S. (2001). Isolation and characterization of psychrotrophic bacteria from oil-reservoir and oil sands. Applied Microbial Biotechnology. 55, 794-800.
Prozorov, T., Mallapragada, S. K., Narasimhan, B., Wang, L., Palo, P., Nilsen-Hamilton, M., Williams, T. J., Bazylinski, D. A., Prozorov, R., and Canfield, P. C. (2007). Protein-mediated synthesis of uniform superparamagnetic magnetite nanocrystals. Adv. Funct. Mater. Advanced Functional Materials. 17, 951-957

Sponsors

Platinum

Alberta Innovates Technology Futures

Oil Sands Initiative

https://2010.igem.org/Oil_Sands

Gold

Autodesk


Silver

MathWorks


Geneious


Bronze

Integrated DNA Technologies


GeneArt & Mr. Gene


Macrogen

Sponsorship Breakdown

The University of Lethbridge iGEM Team requests your support in the following ways:

a) $2000 scholarship of one team member – It is our hope that every team member will be able to travel to the iGEM competition taking place in November 2010. However, without enough funding, this will not be the situation. By sponsoring one student at a time, we can work towards reaching our goal of the entire U of L team traveling to MIT and provide every student with the possibility to meet and talk to some of the brightest and inspiring minds of synthetic biology.

b) Monetary donation towards lab supplies, travel arrangements and iGEM competition registration fees.

c) Tangible donations of lab supplies or other necessary materials.

Donations can be made in two ways:

1. Donations can be directed towards the U of L iGEM Scholarship account.

2. Donations can be directed towards the U of L iGEM General account and in recognition of your generous support, the U of L iGEM team would like to recognize you and/or your business as outlined in the sponsorship levels.

Sponsorship Levels

Platinum - $5000+ or gift in kind
Logo on team shirts, large logo on scientific poster, large logo on team wiki and verbal recognition during team project presentations/media interviews.

Gold - $2000-$4999 or gift in kind
Logo on team shirts, medium logo on scientific poster, medium logo on team wiki and written recognition at end of team project presentations.

Silver - $1000-$1999 or gift in kind
Medium logo on team shirts, scientific poster, medium logo on team wiki and written recognition at end of team project presentations.

Bronze - <$999 or gift in kind
Small logo on team shirts, scientific poster, small logo on team wiki and written recognition at end of team project presentations.


Retrieved from "http://2010.igem.org/Team:Lethbridge"