http://2013.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=Naruto&year=&month=2013.igem.org - User contributions [en]2024-03-29T02:24:33ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:UCL/Labbook/Week17Team:UCL/Labbook/Week172013-10-05T03:52:17Z<p>Naruto: </p>
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<div class="gap"><br />
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<div class="gap"><br />
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<p class="major_title">Lab Weeks</p><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="full_page"><br />
<p class="body_text"> <a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a> <br />
</p> <br />
</div><br />
<br />
<p class="minor_title">Week 17</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Bacterial Labs</b><br />
</p><br />
<p class="body_text"><br />
<b>Monday 23rd September</b><br />
</p><br />
<p class="body_text"><br />
Nanodrop readings<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Sample</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>MMP9</td><br />
<td>382.5</td><br />
<td>1.86</td><br />
</tr><br />
<tr><br />
<td>ZEC</td><br />
<td>401</td><br />
<td>1.49</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Nanodrops of the 6 colonies</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1x1</td><br />
<td>22.3</td><br />
<td>1.46</td><br />
</tr><br />
<tr><br />
<td>1x2</td><br />
<td>78.5</td><br />
<td>1.45</td><br />
</tr><br />
<tr><br />
<td>1x3</td><br />
<td>11.0</td><br />
<td>1.77</td><br />
</tr><br />
<tr><br />
<td>1x4</td><br />
<td>33.9</td><br />
<td>1.50</td><br />
</tr><br />
<tr><br />
<td>1x5</td><br />
<td>35.2</td><br />
<td>1.55</td><br />
</tr><br />
<tr><br />
<td>5x1</td><br />
<td>28.7</td><br />
<td>1.53</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Tuesday 24th September</b><br />
</p><br />
<p class="body_text"><br />
CMV PCR with Taq polymerase<br />
</p><br />
<p class="body_text"><br />
-10 reactions and 1 control<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Reaction components</th><br />
<th>Tubes 1-10</th><br />
<th>Control (11)</th><br />
</tr><br />
<tr><br />
<td>Taq Buffer</td><br />
<td>5</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>dNTP</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>F Primer</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>R Primer</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Template (pSecTag2A, 1ng/ul)</td><br />
<td>1.5</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Taq polymerase</td><br />
<td>0.25</td><br />
<td>0.25</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>40.25</td><br />
<td>41.75</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Tubes were labelled from 1 to 11.<br />
This was unsuccessful.<br />
</p><br />
<br />
<p class="body_text"><br />
Made new inoculations from 5X CMP and 1X CMP plates for Ligated CMV-MMP9.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Sunday 29th September</b><br />
</p><br />
<p class="body_text"><br />
There were not enough colonies for B3&4. The rest of the samples were <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> minipreped</a><br />
and digested and run on a gel cut.<br />
</p><br />
<p class="body_text"><br />
4 more inoculations in 10 ml LB broth of A2, A5, B2 and C2. <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> Nanodrop</a> readings of these inoculations were poor.<br />
</p><br />
<br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Mammalian Labs</b><br />
</p><br />
<p class="body_text"><br />
<b>Monday 23rd September</b><br />
</p><br />
<p class="body_text"><br />
Seeded 2x105 cells (1x105 cells/ml) per well into 16 wells set in three 6-well plates. Stock Hela passaged (P21).<br />
</p><br />
<br />
<br />
<p class="body_text"><br />
24th September<br />
</p><br />
<p class="body_text"><br />
CMV PCR with Taq polymerase<br />
</p><br />
<p class="body_text"><br />
-10 reactions and 1 control<br />
<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Reaction Components</th><br />
<th>Tubes 1-10</th><br />
<th>Control 11</th><br />
</tr><br />
<tr><br />
<td>Taq buffer</td><br />
<td>5</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>dNTP</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>F primer</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>R primer</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Template (pSecTag2A, 1 ng/ul)</td><br />
<td>1.5</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Taq polymerase</td><br />
<td>0.25</td><br />
<td>0.25</td><br />
</tr><br />
<tr><br />
<td>Water</td><br />
<td>40.25</td><br />
<td>41.25</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>50</td><br />
<td>50</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
This was unsuccessful.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>25th September</b><br />
</p><br />
<p class="body_text"><br />
Aim: To stably transfect Hela cells with ’’Str Ble’’.<br />
</p><br />
<p class="body_text"><br />
Confluency of dishes: 30-50%<br />
<br />
</p><br />
<p class="body_text"><br />
Per well of the 6-well plates: 13 well total.<br />
</p><br />
<p class="body_text"><br />
1. Mass of DNA: 2.0µg<br />
</p><br />
<p class="body_text"><br />
2. Volume of DNA dissolved in TE Buffer: 5.0 µl<br />
</p><br />
<p class="body_text"><br />
3. Final volume of DNA diluted in serum free media: 100 µl<br />
</p><br />
<p class="body_text"><br />
4. Volume of superfect (SF) reagent:10.0 µl<br />
</p><br />
<p class="body_text"><br />
5. Volume of serum-containing media: 600 µl<br />
</p><br />
<p class="body_text"><br />
2x ”mock transfected” wells (No DNA, No SE)<br />
</p><br />
<p class="body_text"><br />
Procedure:<br />
</p><br />
<p class="body_text"><br />
Made a ”mask mix” solution for the 13 well-plates to be <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> transfected</a>, with the following composition :<br />
</p><br />
<p class="body_text"><br />
1. 34 ng<br />
</p><br />
<p class="body_text"><br />
2. 93.5 ul<br />
</p><br />
<p class="body_text"><br />
3. 1870 ul<br />
</p><br />
<p class="body_text"><br />
4. 187 ul<br />
</p><br />
<p class="body_text"><br />
5. 11220 ul<br />
<br />
</p><br />
<p class="body_text"><br />
Transfer DNA-Buffer solution to 15 ml falcon tube. Add non-serum media (1776.5µl), Filter, Add SF, Vortex (10.6), Incubate for 5-10 min at room temperature. Meanwhile aspirate and wash cells with 4ml PBS. Add media with serum (600ml). Pipette up and down x2. Apply to cells(≈700µl). Incubate for 2-3 hours. Change media. Incubate (24 hours)<br />
</p><br />
<p class="body_text"><br />
Note : for characterisation , add 1µl of 100µg/ml zeocin to one plate, 2µl to another, and 3µl to the last to make up the concentrations to those over the page.<br />
</p><br />
<p class="body_text"><br />
<b>26th September</b><br />
</p><br />
<p class="body_text"><br />
Characterisation:<br />
</p><br />
<p class="body_text"><br />
24 hours after exposure:<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Viability (%)</th><br />
<th>50 µg/ml Zec</th><br />
<th>100 µg/ml Zec</th><br />
<th>150 µg/ml Zec</th><br />
</tr><br />
<tr><br />
<td>Control</td><br />
<td>80</td><br />
<td>80</td><br />
<td>70</td><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>80</td><br />
<td>80</td><br />
<td>80</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>75</td><br />
<td>80</td><br />
<td>75</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>75</td><br />
<td>80</td><br />
<td>75</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>70</td><br />
<td>80</td><br />
<td>75</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Sunday 29th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>24 Hours</th><br />
<th>A (%)</th><br />
<th>F (%)</th><br />
<th>48 Hours</th><br />
<th>A (%)</th><br />
<th>F (%)</th><br />
<th>72 Hours</th><br />
<th>A (%)</th><br />
<th>F (%)</th><br />
</tr><br />
<tr><br />
<td>C1</td><br />
<td>0</td><br />
<td>1</td><br />
<td>C1</td><br />
<td>0</td><br />
<td>4</td><br />
<td>C1</td><br />
<td>0</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>C2</td><br />
<td>0</td><br />
<td>1</td><br />
<td>C1</td><br />
<td>0</td><br />
<td>5</td><br />
<td>C1</td><br />
<td>5</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>C3</td><br />
<td>0</td><br />
<td>2</td><br />
<td>C3</td><br />
<td>0</td><br />
<td>4</td><br />
<td>C3</td><br />
<td>1</td><br />
<td>40</td><br />
</tr><br />
<tr><br />
<td>P1</td><br />
<td>0</td><br />
<td>3</td><br />
<td>P1</td><br />
<td>0</td><br />
<td>5</td><br />
<td>P1</td><br />
<td>5</td><br />
<td>50</td><br />
</tr><br />
<tr><br />
<td>P2</td><br />
<td>0</td><br />
<td>2</td><br />
<td>P2</td><br />
<td>0</td><br />
<td>10</td><br />
<td>P2</td><br />
<td>1</td><br />
<td>15</td><br />
</tr><br />
<tr><br />
<td>P3</td><br />
<td>0</td><br />
<td>1</td><br />
<td>P3</td><br />
<td>0</td><br />
<td>10</td><br />
<td>P3</td><br />
<td>1</td><br />
<td>50</td><br />
</tr><br />
</table><br />
<br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/Labbook/Week17Team:UCL/Labbook/Week172013-10-05T03:51:47Z<p>Naruto: </p>
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<p class="major_title">Lab Weeks</p><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="full_page"><br />
<p class="body_text"> <a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a> <br />
</p> <br />
</div><br />
<br />
<p class="minor_title">Week 17</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Bacterial Labs</b><br />
</p><br />
<p class="body_text"><br />
<b>Monday 23rd September</b><br />
</p><br />
<p class="body_text"><br />
Nanodrop readings<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Sample</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>MMP9</td><br />
<td>382.5</td><br />
<td>1.86</td><br />
</tr><br />
<tr><br />
<td>ZEC</td><br />
<td>401</td><br />
<td>1.49</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Nanodrops of the 6 colonies</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1x1</td><br />
<td>22.3</td><br />
<td>1.46</td><br />
</tr><br />
<tr><br />
<td>1x2</td><br />
<td>78.5</td><br />
<td>1.45</td><br />
</tr><br />
<tr><br />
<td>1x3</td><br />
<td>11.0</td><br />
<td>1.77</td><br />
</tr><br />
<tr><br />
<td>1x4</td><br />
<td>33.9</td><br />
<td>1.50</td><br />
</tr><br />
<tr><br />
<td>1x5</td><br />
<td>35.2</td><br />
<td>1.55</td><br />
</tr><br />
<tr><br />
<td>5x1</td><br />
<td>28.7</td><br />
<td>1.53</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Tuesday 24th September</b><br />
</p><br />
<p class="body_text"><br />
CMV PCR with Taq polymerase<br />
</p><br />
<p class="body_text"><br />
-10 reactions and 1 control<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Reaction components</th><br />
<th>Tubes 1-10</th><br />
<th>Control (11)</th><br />
</tr><br />
<tr><br />
<td>Taq Buffer</td><br />
<td>5</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>dNTP</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>F Primer</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>R Primer</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Template (pSecTag2A, 1ng/ul)</td><br />
<td>1.5</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Taq polymerase</td><br />
<td>0.25</td><br />
<td>0.25</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>40.25</td><br />
<td>41.75</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Tubes were labelled from 1 to 11.<br />
This was unsuccessful.<br />
</p><br />
<br />
<p class="body_text"><br />
Made new inoculations from 5X CMP and 1X CMP plates for Ligated CMV-MMP9.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Sunday 29th September</b><br />
</p><br />
<p class="body_text"><br />
There were not enough colonies for B3&4. The rest of the samples were <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> minipreped</a><br />
and digested and run on a gel cut.<br />
</p><br />
<p class="body_text"><br />
4 more inoculations in 10 ml LB broth of A2, A5, B2 and C2. <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> Nanodrop</a> readings of these inoculations were poor.<br />
</p><br />
<br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Mammalian Labs</b><br />
</p><br />
<p class="body_text"><br />
<b>Monday 23rd September</b><br />
</p><br />
<p class="body_text"><br />
Seeded 2x105 cells (1x105 cells/ml) per well into 16 wells set in three 6-well plates. Stock Hela passaged (P21).<br />
</p><br />
<br />
<br />
<p class="body_text"><br />
24th September<br />
</p><br />
<p class="body_text"><br />
CMV PCR with Taq polymerase<br />
</p><br />
<p class="body_text"><br />
-10 reactions and 1 control<br />
<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Reaction Components</th><br />
<th>Tubes 1-10</th><br />
<th>Control 11</th><br />
</tr><br />
<tr><br />
<td>Taq buffer</td><br />
<td>5</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>dNTP</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>F primer</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>R primer</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Template (pSecTag2A, 1 ng/ul)</td><br />
<td>1.5</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Taq polymerase</td><br />
<td>0.25</td><br />
<td>0.25</td><br />
</tr><br />
<tr><br />
<td>Water</td><br />
<td>40.25</td><br />
<td>41.25</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>50</td><br />
<td>50</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
This was unsuccessful.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>25th September</b><br />
</p><br />
<p class="body_text"><br />
<b>Mammalian Lab</b><br />
</p><br />
<p class="body_text"><br />
Aim: To stably transfect Hela cells with ’’Str Ble’’.<br />
</p><br />
<p class="body_text"><br />
Confluency of dishes: 30-50%<br />
<br />
</p><br />
<p class="body_text"><br />
Per well of the 6-well plates: 13 well total.<br />
</p><br />
<p class="body_text"><br />
1. Mass of DNA: 2.0µg<br />
</p><br />
<p class="body_text"><br />
2. Volume of DNA dissolved in TE Buffer: 5.0 µl<br />
</p><br />
<p class="body_text"><br />
3. Final volume of DNA diluted in serum free media: 100 µl<br />
</p><br />
<p class="body_text"><br />
4. Volume of superfect (SF) reagent:10.0 µl<br />
</p><br />
<p class="body_text"><br />
5. Volume of serum-containing media: 600 µl<br />
</p><br />
<p class="body_text"><br />
2x ”mock transfected” wells (No DNA, No SE)<br />
</p><br />
<p class="body_text"><br />
Procedure:<br />
</p><br />
<p class="body_text"><br />
Made a ”mask mix” solution for the 13 well-plates to be <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> transfected</a>, with the following composition :<br />
</p><br />
<p class="body_text"><br />
1. 34 ng<br />
</p><br />
<p class="body_text"><br />
2. 93.5 ul<br />
</p><br />
<p class="body_text"><br />
3. 1870 ul<br />
</p><br />
<p class="body_text"><br />
4. 187 ul<br />
</p><br />
<p class="body_text"><br />
5. 11220 ul<br />
<br />
</p><br />
<p class="body_text"><br />
Transfer DNA-Buffer solution to 15 ml falcon tube. Add non-serum media (1776.5µl), Filter, Add SF, Vortex (10.6), Incubate for 5-10 min at room temperature. Meanwhile aspirate and wash cells with 4ml PBS. Add media with serum (600ml). Pipette up and down x2. Apply to cells(≈700µl). Incubate for 2-3 hours. Change media. Incubate (24 hours)<br />
</p><br />
<p class="body_text"><br />
Note : for characterisation , add 1µl of 100µg/ml zeocin to one plate, 2µl to another, and 3µl to the last to make up the concentrations to those over the page.<br />
</p><br />
<p class="body_text"><br />
<b>26th September</b><br />
</p><br />
<p class="body_text"><br />
<b>Mammalian Lab</b><br />
</p><br />
<p class="body_text"><br />
Characterisation:<br />
</p><br />
<p class="body_text"><br />
24 hours after exposure:<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Viability (%)</th><br />
<th>50 µg/ml Zec</th><br />
<th>100 µg/ml Zec</th><br />
<th>150 µg/ml Zec</th><br />
</tr><br />
<tr><br />
<td>Control</td><br />
<td>80</td><br />
<td>80</td><br />
<td>70</td><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>80</td><br />
<td>80</td><br />
<td>80</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>75</td><br />
<td>80</td><br />
<td>75</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>75</td><br />
<td>80</td><br />
<td>75</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>70</td><br />
<td>80</td><br />
<td>75</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Sunday 29th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>24 Hours</th><br />
<th>A (%)</th><br />
<th>F (%)</th><br />
<th>48 Hours</th><br />
<th>A (%)</th><br />
<th>F (%)</th><br />
<th>72 Hours</th><br />
<th>A (%)</th><br />
<th>F (%)</th><br />
</tr><br />
<tr><br />
<td>C1</td><br />
<td>0</td><br />
<td>1</td><br />
<td>C1</td><br />
<td>0</td><br />
<td>4</td><br />
<td>C1</td><br />
<td>0</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>C2</td><br />
<td>0</td><br />
<td>1</td><br />
<td>C1</td><br />
<td>0</td><br />
<td>5</td><br />
<td>C1</td><br />
<td>5</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>C3</td><br />
<td>0</td><br />
<td>2</td><br />
<td>C3</td><br />
<td>0</td><br />
<td>4</td><br />
<td>C3</td><br />
<td>1</td><br />
<td>40</td><br />
</tr><br />
<tr><br />
<td>P1</td><br />
<td>0</td><br />
<td>3</td><br />
<td>P1</td><br />
<td>0</td><br />
<td>5</td><br />
<td>P1</td><br />
<td>5</td><br />
<td>50</td><br />
</tr><br />
<tr><br />
<td>P2</td><br />
<td>0</td><br />
<td>2</td><br />
<td>P2</td><br />
<td>0</td><br />
<td>10</td><br />
<td>P2</td><br />
<td>1</td><br />
<td>15</td><br />
</tr><br />
<tr><br />
<td>P3</td><br />
<td>0</td><br />
<td>1</td><br />
<td>P3</td><br />
<td>0</td><br />
<td>10</td><br />
<td>P3</td><br />
<td>1</td><br />
<td>50</td><br />
</tr><br />
</table><br />
<br />
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<p class="body_text"> <a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a> <br />
</p> <br />
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<div class="gap"><br />
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<br />
<p class="major_title">October</p><br />
<p class="minor_title">Week 18</p><br />
<div class="full_row"> <br />
<div class="gap"><br />
</div><br />
<p class="body_text"><b>Monday 30th September</b></p><br />
<p class="body_text">Ligated CMV with MMP9 and transformed into Top10 cells. These were subsequently plated on <a href="https://2013.igem.org/Team:UCL/Project/Protocols">chloramphenicol plates</a>.<br />
<br />
<p class="body_text"><b>Tuesday 1st October</b></p><br />
<p class="body_text">Inoculated colonies from plates from September 30th.</p><br />
<br />
<p class="body_text"><b>Wednesday 2nd October</b></p><br />
<p class="body_text"> Performed miniprep and generated <a href="https://2013.igem.org/Team:UCL/Project/Protocols">glycerol stock</a> from incoluation on Oct 1st. </p><br />
<p class="body_text"> Proceeded with analytical digest and gel</p><br />
<p class="body_text"> Transformed CMV+MMP9 plasmid from Sinobiological into Top10 cells</p><br />
<br />
<p class="body_text"><b>Thursday 3nd October</b></p><br />
<p class="body_text"> Religated CMV with MMP9 and continued with transformation into Top10 cells. </p><br />
<p class="body_text"> Inoculated from plates of plasmid from Sinobiological</p><br />
<br />
<p class="body_text"><b>Friday 4th October</b></p><br />
<p class="body_text"> Performed <a href="https://2013.igem.org/Team:UCL/Project/Protocols">miniprep</a>, generation of glycerol stock on inoculation from Oct 3rd</p><br />
<p class="body_text"> Continued with analytical digest and <a href="https://2013.igem.org/Team:UCL/Project/Protocols">gel</a></p><br />
<br />
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<p class="major_title">October</p><br />
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<p class="body_text"><b>Monday 30th September</b></p><br />
<p class="body_text">Ligated CMV with MMP9 and transformed into Top10 cells. These were subsequently plated on <a href="https://2013.igem.org/Team:UCL/Project/Protocols">chloramphenicol plates</a>.<br />
<br />
<p class="body_text"><b>Tuesday 1st October</b></p><br />
<p class="body_text">Inoculated colonies from plates from September 30th.</p><br />
<br />
<p class="body_text"><b>Wednesday 2nd October</b></p><br />
<p class="body_text"> Performed miniprep and generated <a href="https://2013.igem.org/Team:UCL/Project/Protocols">glycerol stock</a> from incoluation on Oct 1st. </p><br />
<p class="body_text"> Proceeded with analytical digest and gel</p><br />
<p class="body_text"> Transformed CMV+MMP9 plasmid from Sinobiological into Top10 cells</p><br />
<br />
<p class="body_text"><b>Thursday 3nd October</b></p><br />
<p class="body_text"> Religated CMV with MMP9 and continued with transformation into Top10 cells. </p><br />
<p class="body_text"> Inoculated from plates of plasmid from Sinobiological</p><br />
<br />
<p class="body_text"><b>Friday 4th October</b></p><br />
<p class="body_text"> Performed <a href="https://2013.igem.org/Team:UCL/Project/Protocols">miniprep</a>, generation of glycerol stock on inoculation from Oct 3rd</p><br />
<p class="body_text"> Continued with analytical digest and <a href="https://2013.igem.org/Team:UCL/Project/Protocols">gel</a></p><br />
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<p class="body_text"> <a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a> <br />
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<p class="body_text"><b>Bacterial Labs<b></p><br />
<p class="body_text"><b>Monday 30th September</b></p><br />
</div><br />
<br />
<p class="major_title">October</p><br />
<br />
<div class="full_row"> <br />
<div class="gap"><br />
</div><br />
<p class="body_text"><b>Monday 30th September</b></p><br />
<p class="body_text">Ligated CMV with MMP9 and transformed into Top10 cells. These were subsequently plated on <a href="https://2013.igem.org/Team:UCL/Project/Protocols">chloramphenicol plates</a>.<br />
<br />
<p class="body_text"><b>Tuesday 1st October</b></p><br />
<p class="body_text">Inoculated colonies from plates from September 30th.</p><br />
<br />
<p class="body_text"><b>Wednesday 2nd October</b></p><br />
<p class="body_text"> Performed miniprep and generated <a href="https://2013.igem.org/Team:UCL/Project/Protocols">glycerol stock</a> from incoluation on Oct 1st. </p><br />
<p class="body_text"> Proceeded with analytical digest and gel</p><br />
<p class="body_text"> Transformed CMV+MMP9 plasmid from Sinobiological into Top10 cells</p><br />
<br />
<p class="body_text"><b>Thursday 3nd October</b></p><br />
<p class="body_text"> Religated CMV with MMP9 and continued with transformation into Top10 cells. </p><br />
<p class="body_text"> Inoculated from plates of plasmid from Sinobiological</p><br />
<br />
<p class="body_text"><b>Friday 4th October</b></p><br />
<p class="body_text"> Performed <a href="https://2013.igem.org/Team:UCL/Project/Protocols">miniprep</a>, generation of glycerol stock on inoculation from Oct 3rd</p><br />
<p class="body_text"> Continued with analytical digest and <a href="https://2013.igem.org/Team:UCL/Project/Protocols">gel</a></p><br />
<br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/AchievementsTeam:UCL/Achievements2013-10-05T03:49:28Z<p>Naruto: </p>
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<br />
<p class="major_title">MEDAL CRITERIA</p><br />
<br />
<p class="minor_title">Bronze Medal</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have registered our <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">team</a> of eleven undergraduates. <br />
</p><br />
<p class="body_text"><br />
• We have completed the judging form.<br />
</p><br />
<p class="body_text"><br />
• We have created a beautiful team wiki in keeping with the iGEM guidelines.<br />
</p><br />
<p class="body_text"><br />
• We have a poster and presentation ready for the regional jamboree in Lyon.<br />
</p><br />
<p class="body_text"><br />
• We have developed two new <a href="https://2013.igem.org/Team:UCL/Project/Parts" target="_blank">BioBricks</a> and have submitted them to the iGEM Registry<br />
</div><br />
<br />
<p class="minor_title">Silver Medal</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have proved our zeocin resistance BioBrick <a href="https://2013.igem.org/Team:UCL/Project/Parts" target="_blank">worked as expected</a> by characterising it in HeLa cells. We did this by by creating a zeocin kill curve with and without our <a href="https://2013.igem.org/Team:UCL/Project/Marker" target="_blank">zeocin resistance</a> BioBrick. <br />
</p><br />
<p class="body_text"><br />
• Due to the gravity of Alzheimer's disease and the perceived sovereignty of the brain, we have taken the ethics of using synthetic biological treatments very seriously. We produced a <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">neuroethics</a> and a <a href="https://2013.igem.org/Team:UCL/Practice/Report" target="_blank">feasibility</a> report, consulted numerous experts and provided a concise but detailed <a href="https://2013.igem.org/Team:UCL/Background" target="_blank">background</a> to our project, which shows how our proposed <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">genetic circuit</a> is advised by multiple theories for the causation of Alzheimer's pathology.<br />
</p><br />
</div><br />
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<p class="minor_title"><br />
Gold Medal<br />
</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
• We collaborated with iGEM Westminster, by modelling how well their bed-bug killing device will operate in a bedroom as well advising them on how to run a speed debate using our format. One of our team members, Alex Bates, also attended as guest speaker at their speed debate event.<br />
</p><br />
<p class="body_text"><br />
Full details on collaboration with iGEM Westminster can be found below.<br />
</p><br />
<p class="body_text"><br />
• Our <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">Human Practices</a> deal with an entirely new area for iGEM and, indeed, almost a completely new avenue of research for synthetic biology as a field - the <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">fusion</a> of neuroscience and synthetic biology. We use a variety of strong methods for social and ethical analysis, and outreach. Please see the section below for a summary of what we achieved.<br />
</p><br />
<p class="body_text"><br />
• Outside of this theme, we also engaged in outreach by training and advising the UCL Academy iGEM high school team. This was the first time a British iGEM team has helped run an iGEM HS team.<br />
</p><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<p class="major_title">ABOVE AND BEYOND</p><br />
<br />
<p class="minor_title">Project</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• Developed and experimentally validated the first selectable marker (Zeocin) in a mammalian system (HeLa cells). <br />
</p><br />
<p class="body_text"><br />
• Plan to use genetically engineered microglia cells; the first human brain cells to be used in iGEM, when they arrive so results will be available post jamboree. <br />
</p><br />
</div><br />
<br />
<p class="minor_title">Human Practices</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have looked at an entirely new ethical area for iGEM that has also essentially not been covered in academia; the neuroethics of genetic engineering. We have dubbed this <a href="https://2013.igem.org/Team:UCL/Practice" target="_blank">'Neuro-genethics'</a>. <br />
</p><br />
<p class="body_text"><br />
• We have produced an extensive 20 page <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">report</a> that looks into neuro-genethics and what synthetic biology could achieve in neuroscience.<br />
</p><br />
<p class="body_text"><br />
• We have engaged the public on this topic by getting their opinions at the <a href="http://www.artscatalyst.org/" target="_blank">Arts Catalyst</a>, running a <a href="https://2013.igem.org/Team:UCL/Practice/Debate" target="_blank">speed debate</a> and a <a href="https://2013.igem.org/Team:UCL/Practice/TED" target="_blank">TED debate</a>, conducting an <a href="https://2013.igem.org/Team:UCL/Practice/Survey" target="_blank">online survey</a> and producing a <a href="https://2013.igem.org/Team:UCL/Practice/Documentary" target="_blank">documentary </a> on synthetic neurobiology.<br />
</p><br />
<p class="body_text"><br />
• We ran one of iGEM's first <a href="https://2013.igem.org/Team:UCL/Practice/Creative" target="_blank">creative writing competitions</a>, to gauge public opinion on brain modification and highlight the impact of fiction on society's views.<br />
</p><br />
<p class="body_text"><br />
• Petcha Kutcha style presentation to enguage prospective students of the UCL Engineering Department about iGEM.<br />
</p><br />
<p class="body_text"><br />
• We created an original memory bank, <a href="https://2013.igem.org/Team:UCL/Memories" target="_blank">Eternal Sunshine</a>, which highlights how precious memories are, indicating the desperate need to cure Alzheimer's disease. <br />
</p><br />
<p class="body_text"><br />
• We created a <a href="https://2013.igem.org/Team:UCL/Practice/Report" target="_blank">feasibility report</a> on implementing our treatment.<br />
</p><br />
<p class="body_text"><br />
• Attended and aided in the running of YSB 1.0 (held at UCL) along with other UK iGEM teams to discuss projects and potential collaborations. <br />
</p><br />
<p class="body_text"><br />
• Through Eternal Sunshine and the creative writing competition we have successfully reached out to a diverse range of people from every corner of the globe, from the USA , to China, to Saudi Arabia and so on. <br />
</p><br />
</div><br />
<br />
<p class="minor_title">Modelling</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We are the first iGEM team to use a <a href="https://2013.igem.org/Team:UCL/Modeling" target="_blank">protein network analysis approach</a>. Network based bioinformatics can feedback into synthetic biology by informing the choice of parts in therapeutic genetic circuits.<br />
</p><br />
<p class="body_text"><br />
• We have developed <a href="https://2013.igem.org/Team:UCL/Modeling/Overview" target="_blank">an advanced piece of software</a> in C# with both numerical and graphical output. The model takes into account all major elements of the proposed mechanism.<br />
</p><br />
<p class="body_text"><br />
• We have used the model to qualitatively demonstrate the feasibility of our proposed mechanism – setting all variables with their real values, microglia cells effectively clear plaques from a region of the brain.<br />
</p><br />
<p class="body_text"><br />
• We have also used our model to <a href="https://2013.igem.org/Team:UCL/Modeling/Two" target="_blank">investigate the effect of altering various parameters</a>. Although the model is artificial, since it accurately represents the real world, the results of these investigations could be used to better design our plaque clearing mechanism. Additionally, we used the model to produce an accelerated-time animation of microglia cells clearing plaques, and 3D images of signal distribution once simulation is complete.<br />
</p><br />
<p class="body_text"><br />
• We have made the entire source code available <span class="plainlinks"><a href="https://github.com/21robin12/ucligemmodel" target="_blank">via GitHub</a></span>.<br />
</p><br />
<p class="body_text"><br />
• Furthermore, we have developed <a href="https://2013.igem.org/Team:Westminster/Modelling" target="_blank">an entirely separate model for the Westminster iGEM team</a>. The entire source code for this has been sent to them for further development, in addition to a published version of the software which members of their team, even those without any coding experience, can use.<br />
</p<br />
<br />
<br />
<br />
<br />
</p><br />
</div><br />
<br />
<p class="minor_title">Wiki</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have developed an, original attractive wiki using art work by our artists in residence, <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Fong Yi Khoo</a> and <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Oran Maguire</a>.<br />
</p><br />
<p class="body_text"><br />
• We have included an extensive neuroscience background section, which explains and compares multiple theories for the causation of Alzheimer's disease, so that readers can fully understand the pros and cons of our genetic circuit. <br />
</p><br />
<p class="body_text"><br />
• We have included a full complement of citations that link to PubMed pages so that it is easy to see from where our ideas and explanations have been drawn, and which papers have inspired us.<br />
</p><br />
</div><br />
<br />
<p class="minor_title">Collaboration with Westminster iGEM</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
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<b>Model</b><br />
</p><br />
<p class="body_text"><br />
We have modelled the movement of bed-bugs towards blood meal, which can be found on our <a href="https://2013.igem.org/Team:UCL/Modeling/Westminster" target="_blank">modelling section</a>.<br />
<br />
</div><br />
<br />
<p class="minor_title">Mentoring iGEM HS</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Two members of the UCL iGEM team volunteered as advisers to the UCL Academy iGEM team – <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Ruxi</a> and <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Khaicheng</a>, under the guidance of Aurelija Grigonyte, a member of the UCL iGEM 2012 team. During the high school team’s brainstorming process, we provided them with guidance and resources for their research. We also supervised their lab work in the UCL Biochemical Engineering department.<br />
</p><br />
<p class="body_text"><br />
UCL is the first university in the UK to be the sole sponsor of an academy – a non-selective mixed state school in our home borough of Camden. UCL Academy represents a unique opportunity to blur the boundaries between secondary and higher education.<br />
</p><br />
<p class="body_text"><br />
The academy is one of the first UK high schools to participate in iGEM this year, and is the only UK team so far to have attended the <a href="https://2013hs.igem.org/Main_Page" target="_blank">High School iGEM Jamboree</a> at MIT, Boston. The team aimed to revolutionise the recycling industry by proposing a home system that converts cellulose into glucose, allowing the up-cycling of paper into a commercial product of bioplastic - polyhydroxybutyrate (PHB) <br />
</p><br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/b0/IMG_4769-1024x68211.jpg');height:280px;width:650px;"><br />
</div><br />
</p><br />
<p class="body_text"><br />
For information about their iGEM project, check out their wiki <a href="https://2013hs.igem.org/Team:UCL_Academy" target="_blank">here</a>. <br />
</p><br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/AchievementsTeam:UCL/Achievements2013-10-05T03:49:07Z<p>Naruto: </p>
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<br />
<p class="major_title">MEDAL CRITERIA</p><br />
<br />
<p class="minor_title">Bronze Medal</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have registered our <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">team</a> of eleven undergraduates. <br />
</p><br />
<p class="body_text"><br />
• We have completed the judging form.<br />
</p><br />
<p class="body_text"><br />
• We have created a beautiful team wiki in keeping with the iGEM guidelines.<br />
</p><br />
<p class="body_text"><br />
• We have a poster and presentation ready for the regional jamboree in Lyon.<br />
</p><br />
<p class="body_text"><br />
• We have developed two new <a href="https://2013.igem.org/Team:UCL/Project/Parts" target="_blank">BioBricks</a> and have submitted them to the iGEM Registry<br />
</div><br />
<br />
<p class="minor_title">Silver Medal</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have proved our zeocin resistance BioBrick <a href="https://2013.igem.org/Team:UCL/Project/Parts" target="_blank">worked as expected</a> by characterising it in HeLa cells. We did this by by creating a zeocin kill curve with and without our <a href="https://2013.igem.org/Team:UCL/Project/Marker" target="_blank">zeocin resistance</a> BioBrick. <br />
</p><br />
<p class="body_text"><br />
• Due to the gravity of Alzheimer's disease and the perceived sovereignty of the brain, we have taken the ethics of using synthetic biological treatments very seriously. We produced a <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">neuroethics</a> and a <a href="https://2013.igem.org/Team:UCL/Practice/Report" target="_blank">feasibility</a> report, consulted numerous experts and provided a concise but detailed <a href="https://2013.igem.org/Team:UCL/Background" target="_blank">background</a> to our project, which shows how our proposed <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">genetic circuit</a> is advised by multiple theories for the causation of Alzheimer's pathology.<br />
</p><br />
</div><br />
<br />
<p class="minor_title"><br />
Gold Medal<br />
</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
• We collaborated with iGEM Westminster, by modelling how well their bed-bug killing device will operate in a bedroom as well advising them on how to run a speed debate using our format. One of our team members, Alex Bates, also attended as guest speaker at their speed debate event.<br />
</p><br />
<p class="body_text"><br />
Full details on collaboration with iGEM Westminster can be found below.<br />
</p><br />
<p class="body_text"><br />
• Our <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">Human Practices</a> deal with an entirely new area for iGEM and, indeed, almost a completely new avenue of research for synthetic biology as a field - the <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">fusion</a> of neuroscience and synthetic biology. We use a variety of strong methods for social and ethical analysis, and outreach. Please see the section below for a summary of what we achieved.<br />
</p><br />
<p class="body_text"><br />
• Outside of this theme, we also engaged in outreach by training and advising the UCL Academy iGEM high school team. This was the first time a British iGEM team has helped run an iGEM HS team.<br />
</p><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<p class="major_title">ABOVE AND BEYOND</p><br />
<br />
<p class="minor_title">Project</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• Developed and experimentally validated the first selectable marker (Zeocin) in a mammalian system (HeLa cells). <br />
</p><br />
<p class="body_text"><br />
• Plan to use genetically engineered microglia cells; the first human brain cells to be used in iGEM, when they arrive so results will be available post jamboree. <br />
</p><br />
</div><br />
<br />
<p class="minor_title">Human Practices</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have looked at an entirely new ethical area for iGEM that has also essentially not been covered in academia; the neuroethics of genetic engineering. We have dubbed this <a href="https://2013.igem.org/Team:UCL/Practice" target="_blank">'Neuro-genethics'</a>. <br />
</p><br />
<p class="body_text"><br />
• We have produced an extensive 20 page <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">report</a> that looks into neuro-genethics and what synthetic biology could achieve in neuroscience.<br />
</p><br />
<p class="body_text"><br />
• We have engaged the public on this topic by getting their opinions at the <a href="http://www.artscatalyst.org/" target="_blank">Arts Catalyst</a>, running a <a href="https://2013.igem.org/Team:UCL/Practice/Debate" target="_blank">speed debate</a> and a <a href="https://2013.igem.org/Team:UCL/Practice/TED" target="_blank">TED debate</a>, conducting an <a href="https://2013.igem.org/Team:UCL/Practice/Survey" target="_blank">online survey</a> and producing a <a href="https://2013.igem.org/Team:UCL/Practice/Documentary" target="_blank">documentary </a> on synthetic neurobiology.<br />
</p><br />
<p class="body_text"><br />
• We ran one of iGEM's first <a href="https://2013.igem.org/Team:UCL/Practice/Creative" target="_blank">creative writing competitions</a>, to gauge public opinion on brain modification and highlight the impact of fiction on society's views.<br />
</p><br />
<p class="body_text"><br />
• Petcha Kutcha style presentation to enguage prospective students of the UCL Engineering Department about iGEM.<br />
</p><br />
<p class="body_text"><br />
• We created an original memory bank, <a href="https://2013.igem.org/Team:UCL/Memories" target="_blank">Eternal Sunshine</a>, which highlights how precious memories are, indicating the desperate need to cure Alzheimer's disease. <br />
</p><br />
<p class="body_text"><br />
• We created a <a href="https://2013.igem.org/Team:UCL/Practice/Report" target="_blank">feasibility report</a> on implementing our treatment.<br />
</p><br />
<p class="body_text"><br />
• Attended and aided in the running of YSB 1.0 (held at UCL) along with other UK iGEM teams to discuss projects and potential collaborations. <br />
</p><br />
<p class="body_text"><br />
• Through Eternal Sunshine and the creative writing competition we have successfully reached out to a diverse range of people from every corner of the globe, from the USA , to China, to Saudi Arabia and so on. <br />
</p><br />
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<p class="minor_title">Modelling</p><br />
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• We are the first iGEM team to use a <a href="https://2013.igem.org/Team:UCL/Modeling" target="_blank">protein network analysis approach</a>. Network based bioinformatics can feedback into synthetic biology by informing the choice of parts in therapeutic genetic circuits.<br />
</p><br />
<p class="body_text"><br />
• We have developed <a href="https://2013.igem.org/Team:UCL/Modeling/Overview" target="_blank">an advanced piece of software</a> in C# with both numerical and graphical output. The model takes into account all major elements of the proposed mechanism.<br />
</p><br />
<p class="body_text"><br />
• We have used the model to qualitatively demonstrate the feasibility of our proposed mechanism – setting all variables with their real values, microglia cells effectively clear plaques from a region of the brain.<br />
</p><br />
<p class="body_text"><br />
• We have also used our model to <a href="https://2013.igem.org/Team:UCL/Modeling/Two" target="_blank">investigate the effect of altering various parameters</a>. Although the model is artificial, since it accurately represents the real world, the results of these investigations could be used to better design our plaque clearing mechanism. Additionally, we used the model to produce an accelerated-time animation of microglia cells clearing plaques, and 3D images of signal distribution once simulation is complete.<br />
</p><br />
<p class="body_text"><br />
• We have made the entire source code available <span class="plainlinks"><a href="https://github.com/21robin12/ucligemmodel" target="_blank">via GitHub</a></span>.<br />
</p><br />
<p class="body_text"><br />
• Furthermore, we have developed <a href="https://2013.igem.org/Team:Westminster/Modelling" target="_blank">an entirely separate model for the Westminster iGEM team</a>. The entire source code for this has been sent to them for further development, in addition to a published version of the software which members of their team, even those without any coding experience, can use.<br />
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<p class="minor_title">Wiki</p><br />
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• We have developed an, original attractive wiki using art work by our artists in residence, <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Fong Yi Khoo</a> and <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Oran Maguire</a>.<br />
</p><br />
<p class="body_text"><br />
• We have included an extensive neuroscience background section, which explains and compares multiple theories for the causation of Alzheimer's disease, so that readers can fully understand the pros and cons of our genetic circuit. <br />
</p><br />
<p class="body_text"><br />
• We have included a full complement of citations that link to PubMed pages so that it is easy to see from where our ideas and explanations have been drawn, and which papers have inspired us.<br />
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<p class="minor_title">Collaboration with Westminster iGEM</p><br />
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<b>Model</b><br />
</p><br />
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We have modelled the movement of bed-bugs towards blood meal, which can be found on our <a href="https://2013.igem.org/Team:UCL/Modeling/Westminster" target="_blank">Modelling section</a>.<br />
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<p class="minor_title">Mentoring iGEM HS</p><br />
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Two members of the UCL iGEM team volunteered as advisers to the UCL Academy iGEM team – <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Ruxi</a> and <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Khaicheng</a>, under the guidance of Aurelija Grigonyte, a member of the UCL iGEM 2012 team. During the high school team’s brainstorming process, we provided them with guidance and resources for their research. We also supervised their lab work in the UCL Biochemical Engineering department.<br />
</p><br />
<p class="body_text"><br />
UCL is the first university in the UK to be the sole sponsor of an academy – a non-selective mixed state school in our home borough of Camden. UCL Academy represents a unique opportunity to blur the boundaries between secondary and higher education.<br />
</p><br />
<p class="body_text"><br />
The academy is one of the first UK high schools to participate in iGEM this year, and is the only UK team so far to have attended the <a href="https://2013hs.igem.org/Main_Page" target="_blank">High School iGEM Jamboree</a> at MIT, Boston. The team aimed to revolutionise the recycling industry by proposing a home system that converts cellulose into glucose, allowing the up-cycling of paper into a commercial product of bioplastic - polyhydroxybutyrate (PHB) <br />
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For information about their iGEM project, check out their wiki <a href="https://2013hs.igem.org/Team:UCL_Academy" target="_blank">here</a>. <br />
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
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<p class="major_title">October</p><br />
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<p class="minor_title">1st October</p><br />
<p class="body_text"><br />
Darren visited us in the Mammalian Lab and gave us the CMV-MMP9 control plasmid.<br />
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<p class="minor_title">2nd October</p><br />
<p class="body_text"><br />
The entire team met Darren to rehearse the presentation for the Jamboree in Lyon.<br />
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<br />
<p class="minor_title">3rd October</p><br />
<p class="body_text"><br />
Robin took charge of the collaboration on Modelling for Westminster iGEM team.<br />
</p><br />
<p class="body_text"><br />
FYi finalised the circuit drawing which was mounted on the Wiki.<br />
The digestion of cmv+MMP9 recombinant plasmid showed promising results.<br />
</p><br />
<br />
<p class="minor_title">4th October</p><br />
<p class="body_text"><br />
The official UCL team shirts were collected. <br />
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<p class="body_text"><br />
The team reunited at Robin's in order to upload the remaining wiki content before the wiki freeze at 4:59 am. <br />
</p><br />
<p class="body_text"> <br />
We also took advantage of this event and had our group photo taken with all of us wearing our brand new Spotless mind T-shirts! We also included Stjohn's photo who wasn't able to be with us tonight but with whom we'll be finally reunited in Lyon! Yey!<br />
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<p class="major_title">A BIOINFORMATICS APPROACH</p><br />
<p class="minor_title">Finding New Parts</p><br />
<p class="body_text"><br />
Bioinformatics creates and enhances methods for storing, retrieving, organising and analysing biological data. We decided to take a completely new approach in our dry lab work and look into bioinformatic approaches to studying <a href="https://2013.igem.org/Team:UCL/Background/Alzheimers" target="_blank">Alzheimer’s disease (AD)</a>. <br />
</p> <br />
<p class="body_text"><br />
The rationale behind this is simple. In order to make a genetic circuit in a synthetic biological construct as effective as possible in a medical application, we may need to target key dysfunctional genes within the problematic biological entity. There are many risk factors for AD and so predicting the key, ‘driver genes’, and the group of proteins with which they interact is invaluable in knowing what we want our construct to produce, in order to mitigate AD. The idea is that bioinformatics work can feed back into synthetic biology, and though we did not have the time to demonstrate this full circle, we feel bioinformatics can have a place in iGEM, helping teams to decide which dysfunctional genes to target in medical projects.</p><br />
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<a href="https://static.igem.org/mediawiki/2013/0/03/Human_interactome.jpg" data-lightbox="image-1" title="The Human Interactome"><br />
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<p class="minor_title">Bioinformatics and Alzheimer’s Disease</p> <br />
<p class="body_text"><br />
Recent progress in characterising AD has lead to the identification of dozens of highly interconnected genetic risk factors, yet it is likely that many more remain undiscovered <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3044851/" target="_blank">(Soler-Lopez et al. 2011)</a> and the elucidation of their roles in AD could prove pivotal in beating the condition. AD is genetically complex, linked with many defects both mutational or of susceptibility. These defects produce alterations in the molecular interactions of cellular pathways, the collective effect of which may be gauged through the structure of the protein network <a href="http://www.sciencedirect.com/science/article/pii/S0092867413003875" target="_blank">(Zhang et al. 2013)</a>. In other words, there is a strong link between protein connectivity and the disease phenotype. AD arises from the downstream interplay between genetic and non-genetic alterations in the human protein interaction network <a href="http://www.sciencedirect.com/science/article/pii/S0092867413003875" target="_blank">(Zhang et al. 2013)</a>. <br />
</p><p class="body_text"><br />
Recent progress in characterising AD has lead to the identification of dozens of highly interconnected genetic risk factors, yet it is likely that many more remain undiscovered <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3044851/" target="_blank">(Soler-Lopez et al. 2011)</a> and the elucidation of their roles in AD could prove pivotal in beating the condition. AD is genetically complex, linked with many defects both mutational or of susceptibility. These defects produce alterations in the molecular interactions of cellular pathways, the collective effect of which may be gauged through the structure of the protein network <a href="http://www.sciencedirect.com/science/article/pii/S0092867413003875" target="_blank">(Zhang et al. 2013)</a>. In other words, there is a strong link between protein connectivity and the disease phenotype. AD arises from the downstream interplay between genetic and non-genetic alterations in the human protein interaction network <a href="http://www.sciencedirect.com/science/article/pii/S0092867413003875" target="_blank">(Zhang et al. 2013)</a>.<br />
</p><p class="body_text"><br />
In all pathologies, the most common way to predict driver genes is to target commonly recurrent genes. However, this approach misses misses rare altered genes which comprise the majority of genetic defects leading to, for example, carcinogenesis and arguably AD. This is partly because alterations in a single protein module can lead to the same disease phenotype. Thus, identification may best be attempted on a modular level. Yet it is also important to note correlation events between modules. Simply put, many rare gene alterations that influence the module they belong to and co-altered modules can collectively generate the disease pathology (Gu et al. 2013).<br />
<div class="gap"><br />
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<p class="minor_title">Our Programme</p> <br />
<p class="body_text"><br />
Under the guidance and tutelage of <a href="http://bmm.cancerresearchuk.org/~cheng03/" target="_blank">Dr Tammy Cheng</a> from the <a href="http://bmm.cancerresearchuk.org/" target="_blank">Biomolecular Modelling (BMM) lab</a> at Cancer Research UK, team member <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Alexander Bates</a> coded in python a network analysis programme based on a method devised by Gu et al. and originally applied to the study of glioblastoma (brain cancer). The programme tries to reveal driver genes and co-altered functional modules for AD. The analysis procedure involves mapping altered genes (mutations, amplifications, repressions, etc.) in patient microRNA data to the protein interaction network (PIT), which currently accounts for 48,480 interactions between 10,982 human genes. This is termed the ‘AD altered network’, and is searched with the algorithm suggested by Gu et al. (which has been re-coded from scratch).<br />
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<p class="body_text"><br />
The programme builds up gene sets, two at a time, starting from two seed genes. These sets are termed 'modules'. Pairs of modules (‘G1’ and ‘G2’ in equation) are assumed to be co-altered if any gene within each module is altered in a proportion of AD sufferers, and genes between the modules are often altered together. For two modules, G1 and G2, we must calculate the probability, P, of observing than the number of the samples in the patient gene expression data that by chance simultaneously carry alterations in both gene sets. The gene expression data originates from post-mortem brain samples.<br />
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‘n’ is the total number of patient samples, ‘a’ is the number of patients with alterations in both G1 and G2, ‘b’ is the number of patients with alteration in just G1, ‘c’ is the number of patients with alterations in only G2, and ‘d’ is the number of patients with alterations in neither set. The co-altered score’ S, is defined below. A high score indicates that the two modules tend to be altered together in AD.<br />
</p><p class="body_text"><br />
Fig.1 depicts the searching algorithm. It searches and builds co-altered module pairs for the gene combinations within them that have the greatest co-alteration scores. In step 1, it methodically choose two seed genes from the AD altered network. The ellipsoids in the diagram denote direct interaction partners for these genes. These are added to the seeds to make temporary module pairs. The dashed line represents co-alteration. In step 2, the co-alteration score for each temporary module pair is calculated. Only the pair with the maximal S score is retained for subsequent searching. This maximal group becomes the new seeds group in step 3. In step 4, temporary modules are again derived, this time from step 3, and the maximum score is kept. In step 5, it must determine whether or not this group of genes is going to continue to expand. Each new addition save for the original two starting seeds is removed and S is recalculated. If in one of these configurations S becomes smaller, we loop through steps 3 to 5 again. Otherwise, if all combinations equate to the S value of the gene groups chosen from step 4, the process stops, having assumed that we have reached maximal module size for the two starting seeds.<br />
</p><br />
<p class="body_text"><br />
In other words, we try to build up gene sets within a module as large was we can, whilst with each new addition increasing the co-alteration score.<br />
</p><br />
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We should end up with modules that frequently exhibit significant co-alteration in AD patients, and their gene products are therefore likely to be biochemically significant in the disease state.<br />
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<p class="minor_title">Results</p> <br />
<p class="body_text"><br />
Originally we planned, as previously suggested, to use the entirety of the human interactome to create an AD interactome and then run our programme in such a way as to build modules from this interactome. However, the estimated run time of the programme over-shot the iGEM 'wiki freeze' deadline. Therefore, we used the expression data for 311 hub genes, whose proteins are points of high connectivity in the human interactome, across 62 modules defined by Zhang et al., and searched for the hub genes combinations that produced the greatest co-alteration scores. The 62 modules are named after colours. <br />
</p><br />
<p class="body_text"><br />
<b>Module groups: </b> <a href="https://static.igem.org/mediawiki/2013/e/ec/AlzModules.txt" target="_blank">AlzModules.py</a><br />
<p class="body_text"><br />
<b>Hub expression data:</b> <a href="https://static.igem.org/mediawiki/2013/7/7a/ALzData2.txt" target="_blank">AlzData.py</a><br />
</p><br />
<p class="body_text"><br />
<b>Module matrix:</b> <a href="https://static.igem.org/mediawiki/2013/5/5f/AlzList.txt" target="_blank">AlzMatrix.py</a><br />
</p><br />
<p class="body_text"><br />
The code for our network analysis programme can be found <a href="https://static.igem.org/mediawiki/2013/4/40/Alex4.txt" target="_blank">here</a>. It needs to be converted to a .py file to be used. Please note that the output is given as a set of numbers that as assigned to genes. For example, the final output for the data we ran can be found <a href="https://static.igem.org/mediawiki/2013/0/0f/AlzFinal.txt" target="_blank">here</a>.<br />
</p><br />
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<th><p class="citation_text">Fig.1 Histogram showing the frequency of gene sets by co-alteration score.</p></th><br />
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<p class="body_text"><br />
We used the output of our programme to produce a histogram, which shows that the frequency of gene combinations falls exponentially with increasing co-alteration score This suggests that a significant few combinations are regularly co-altered in Alzheimer's disease, in modules that may help drive the disease state. Because we are only looking at which hub genes within modules, we are most interested in what modules are co-altered in the high score end of the histogram, and not the hub genes specifically.</p><br />
<p class="body_text"><br />
Below, Fig.2 shows the twenty gene set pairs between two modules, which yielded the greatest co-alteration score. The module pair with the highest score, and that recurs most frequently in the top twenty, are the 'Khaki' and 'Honey Dew' modules. The most enriched functional category of the khaki module is the biosynthesis of a neurotransmitter called GABA. GABA is responsible for neuronal excitability and muscle tone. The Honey Dew module is primarily involved in muscle contraction, though the hub genes AHCYL1 and C9orf61 are thought to be involved in inositol signaling and are possibly associated with another brain condition, bi-polar disorder. However, since the gene expression data is from generally older patients, given the profile of AD, these muscle associated modules may be altered together because of changing muscle usage with age (there is no muscle in the brain but this may represent brain cell structural integrity). Both of these modules have almost 100% of their total brain gene expression in the prefrontal cortex, and area known to be heavily impacted in AD, causing cognitive and intellectual damage. This suggests that our genetic circuit could be adapted to target signaling mechanisms in this area.</p><br />
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<table><br />
<th><p class="citation_text">Fig.2 Table of the top 20 gene combinations and their modules by co-alteration score.</p></th><br />
</table><br />
<table><br />
<tr><br />
<th>Module Name and Gene Set</th><br />
<th>Module Name and Gene Set</th><br />
<th>Co-alteration Score</th><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>20.39 </td><br />
<tr><br />
<td>SLC15A2, FXYD1</td><br />
<td>AHCYL1, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>19.73 </td><br />
<tr><br />
<td>GJA1, FXYD1</td><br />
<td>RFX4, AHCYL1, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>19.37 </td><br />
<tr><br />
<td>GJA1, FXYD1, ATP13A4</td><br />
<td>C20orf141, RFX4, AHCYL1, DGCR6</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Cyan</b></td><br />
<td>18.99 </td><br />
<tr><br />
<td>DYNC2LI1, CIRBP, ACRC, RBM4</td><br />
<td>Contig47252_RC, IFITM2, CDK2</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Cyan</b></td><br />
<td>18.81 </td><br />
<tr><br />
<td>DYNC2LI1, CIRBP, ACRC, RBM4</td><br />
<td>ENST00000289005, Contig47252_RC, IFITM2, CDK2</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>17.69 </td><br />
<tr><br />
<td>GJA1, FXYD1, SLC15A2</td><br />
<td>RFX4, AHCYL1, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Green 4</b></td><br />
<td><b>Yellow 3</b></td><br />
<td>17.57 </td><br />
<tr><br />
<td>RRM2, NM_022346, FAM64A</td><br />
<td>OR4F5, GRAP, XM_166973</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Wheat</b></td><br />
<td>17.49 </td><br />
<tr><br />
<td>DYNC2LI1, RBM4</td><br />
<td>AF087999</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Green 4</b></td><br />
<td><b>Yellow 3</b></td><br />
<td>16.95 </td><br />
<tr><br />
<td>HMMR</td><br />
<td>OR4F5, GRAP</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Green 4</b></td><br />
<td><b>Yellow 3</b></td><br />
<td>16.95 </td><br />
<tr><br />
<td>HMMR</td><br />
<td>OR4F5, GRAP, CRYBA2</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Wheat</b></td><br />
<td>16.78 </td><br />
<tr><br />
<td>CIRBP, RBM4</td><br />
<td>AF087999</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Green 4</b></td><br />
<td><b>Yellow 3</b></td><br />
<td>16.64 </td><br />
<tr><br />
<td>RRM2, NMMR, FAM64A</td><br />
<td>KRTHB4, GRAP, XM_166973</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Cyan</b></td><br />
<td>16.47 </td><br />
<tr><br />
<td>DYNC2LI1, CIRBP, ACRC, RCC1, RBM4</td><br />
<td>Contig47252_RC, IFITM2</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Cyan</b></td><br />
<td>16.46 </td><br />
<tr><br />
<td>DYNC2LI1, CIRBP, ACRC, RCC1, RBM4</td><br />
<td>Contig47252_RC, IFITM2, CDK2</td><br />
</tr> <br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Forestgreen</b></td><br />
<td><b>Cyan</b></td><br />
<td>16.43 </td><br />
<tr><br />
<td>IFITM3, CSDA</td><br />
<td>CSDA</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Cyan</b></td><br />
<td>16.38 </td><br />
<tr><br />
<td>DYNC2LI1, CIRBP, ACRC, RCC1, RBM4</td><br />
<td>ENST00000289005, Contig47252_RC, IFITM2</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>16.27 </td><br />
<tr><br />
<td>FXYD1, ATP13A4, SLC15A2</td><br />
<td>AHCYL1, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>16.25 </td><br />
<tr><br />
<td>FXYD1, ATP13A4</td><br />
<td>DGCR6, AHCYL1, C20orf141, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Gold 2</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>16.21 </td><br />
<tr><br />
<td>TUBB2B, NM_178525</td><br />
<td>AHCYL1, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>16.04 </td><br />
<tr><br />
<td>SPON1, FXYD1, SLC15A2</td><br />
<td>AHCYL1, C9orf61</td><br />
</tr><br />
</table> <br />
<br />
</p><br />
<p class="minor_title">Analysis and Feedback into Circuit</p><br />
<p class="body_text"><br />
The second highest scoring module pair, and the second most frequent in the top twenty, are 'Turquoise' and 'Cyan'. The former is primarily involved with NAD(P) homeostasis, and so is significant in cells' metabolism, while the genes in the later mainly play a role in vasculature development. This suggests that co-alteration in genes involved within these two modules could impact cell vitality and trophic support and help cause AD. This suggests that our circuit could be improved by being adapted to help maintain general cell health and energy supply in the brain. </p><br />
<p class="body_text"><br />
The third highest scoring module pair, and the third most frequent in the top twenty, are 'Green 4' and 'Yellow 3'. Green 4 is involved in cell cycle regulation, and area that has already been targeted by our circuit, which produces <b>BDNF</b> to help avoid chromosomal division in the neurons of AD patients. Yellow 3 is associated with the peripheral nervous system. Co-alteration here may again be indicative of gene expression changes with age, and its link with Green 4 may suggest that this is to do with a deficiency in cell division, regeneration and growth, but this is not directly related to AD, although hub genes like GRAP do play a role in cytoplasmic signaling in cells including neurons and glia, This suggests that our circuit could be improved by being adapted to help maintain general cell health and energy supply in the brain. </p><br />
<p class="body_text"><br />
Other module pairs that feature in the top twenty include 'Wheat' and 'Turqouise', 'Forestgreen' and 'Cyan' and 'Gold 2' and 'Honey Dew'. Wheat is involved in protein folding and responses to unfolded and mis-folded protein. This is significant because incorrectly formed and folded amyloid is strongly associated with the progression of AD. This is something out circuit already seeks to address, but by targeting elements of the 'Wheat' module and similar modules it could aim to avoid mis-creation in the first place, and the nucleation of other mis-folded proteins. Forestgreen is involved in immune functions, which implicates microglia and the cellular response to inflammation in neurons - factors our circuit already tries to help address by acting to prevent neuroinflammation. Its association with Cyan could imply that negative inflammatory effects may be inked with brain vasculature in AD. Gold 2 is associated with the cytoskeleton and axonal cytoskeletal control.In AD, the formation of plaques and protein tangles disrupts the cytoskeleton and perturb axonal connections, engendering cell death. Our circuit tries to target this already by removing the plaques, but perhaps a future improvement should to be to create an element capable to supporting a healthy cytoskeleton or able to remove cytoskeletal protein tangles. Its association with Honey Dew, however, could point to unusual gene expression in this module being due to the lessened use of muscle in old age.</p><br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T03:25:03Z<p>Naruto: </p>
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<div class="gap"></div><br />
<p class="major_title">Bacterial Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="full_page"><br />
<p class="body_text"><br />
<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
</p><br />
</div><div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">1.4% Agar</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Agar</td><br />
<td>7g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500mL</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">5X M9 Salts</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Na2HPO4</td><br />
<td>32g</td><br />
</tr><br />
<tr><br />
<td>KH2PO4</td><br />
<td>7.5g</td><br />
</tr><br />
<tr><br />
<td>NaCl</td><br />
<td>1.25g</td><br />
</tr><br />
<tr><br />
<td>NH4Cl</td><br />
<td>2.5g</td><br />
</tr><br />
<tr><br />
<td>RO HCL</td><br />
<td>500ml</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
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<div class="gap"><br />
</div><br />
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<div class="protocol"><br />
<p class="minor_title"> 0.1M CaCl2/15% glycerol</p><br />
<p class="body_text"><br />
In a 50mL Falcon insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>100% Glycerol</td><br />
<td>7.5</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>37.5</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Minimal Agar</p><br />
<p class="body_text"><br />
Mix:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>5x M9 Salts</td><br />
<td>10mL</td><br />
</tr><br />
<tr><br />
<td>2 mg/ml Thiamine</td><br />
<td>50µl</td><br />
</tr><br />
<tr><br />
<td>20% D Glucose</td><br />
<td>1 mL</td><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5µl</td><br />
</tr><br />
<tr><br />
<td>1M MgSO4</td><br />
<td>100µl</td><br />
</tr><br />
<tr><br />
<td>1.4% Agar</td><br />
<td>39 mL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">LB Media</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Broth</td><br />
<td>10g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500 mL</td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Generating Competence Cells</p><br />
<p class="body_text"><br />
Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.<br />
</p><br />
<p class="body_text"><br />
Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.<br />
</p><br />
<p class="body_text"><br />
After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).<br />
</p><br />
</div><br />
</div><br />
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<div class="gap"></div><br />
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<div class="row_small"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4b/Streakingplatesigemucl2013.png');"></div><br />
<div class="description"><br />
<p class="minor_title">Streaking Plates</p><br />
<p class="body_text"><br />
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight. <br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Plate generation (AMP, CMP & NoDrug)</p><br />
<p class="body_text"><br />
Heat up 50 mL of agar until molten (usually ~300 seconds using a 800W microwave). Douse in cold water to lower temperature. When still warm, but able to handle, it is possible to add an antibiotic drug for selection purposes (~50 uL). Once this complete, pour ~10mL into a petri dish and ensure that the whole surface is covered. Leave lid off for 30 minutes. Place lid on dish and then use, or store at ~5C.<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Glycerol Stock Generation</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Media</td><br />
<td>3mL</td><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>3 µl</td><br />
</tr><br />
<tr><br />
<td>Relevant Anti-bioitc</td><br />
<td>3 µl</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
12-16 hour 37C incubation. Insert into 1.5mL microcentrifuge tubes. Note absorbance. Add below, then store (-20C).<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µl)</th><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>500</td><br />
</tr><br />
<tr><br />
<td>Glycerol Stock</td><br />
<td>166</td><br />
</tr><br />
</table><br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">50X to 1X Dilution</p><br />
<p class="body_text"><br />
To a 1L Duran bottle, insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>50X TAE Buffer</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>980</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Transformation</p><br />
<p class="body_text"><br />
<b>1)</b> Remove one of your aliquots of competent cells from the -80C freezer and place onto ice.<br />
<b>2)</b> Add ~2uL of DNA to the competent cells. Leave in ice for ~45 minutes.<br />
<b>3)</b> Place tubes into 37C water bath for 10 minutes (heat shock).<br />
<b>4)</b> Place tubes into ice for 2 minutes.<br />
<b>5)</b> Add 1.3mL of Lb to the tubes and transfer all of the contents to new tubes. Incubate for 1 hour at 37C.<br />
<b>6)</b> Centrifuge at high RPM for 2 minutes. Discard the supernatant.<br />
<b>7)</b> Resuspend cell pellet into 100uL of LB.<br />
<b>8)</b> Spread contents onto petri dishes containing LB agar (may also contain antibiotic resistance for better selectivity.<br />
<b>9)</b> Incubate for 16 hours at 37C and then pick colonies if growth has occurred.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Maxi/mini Preparation</p><br />
<p class="body_text"><br />
See <a href="http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDsQFjAA&url=http%3A%2F%2Fwww.qiagen.com%2Fresources%2FDownload.aspx%3Fid%3D%257B46205595-0440-459E-9D93-50EB02E5707E%257D%26lang%3Den%26ver%3D2&ei=-YpFUpvcF-yd0wWIw4HACg&usg=AFQjCNFGR5hl0QYv64lnVZDZWaw26BKA0A&sig2=JjaWz8EP2dxWJAMCpnLxCA&bvm=bv.53217764,d.d2k<br />
" target="_blank">protocol</a> for mini/maxi prep from Qiagen:<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Analytical Digest</p><br />
<p class="body_text"><br />
Add the following items to a 1.5mL microcentrifuge tube and briefly (<10s) centrifuge to ensure all contents are mixed:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µL)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>1.5g</td><br />
</tr><br />
<tr><br />
<td>Enzyme 1</td><br />
<td>150mL</td><br />
</tr><br />
<tr><br />
<td>Enzyme 2</td><br />
<td>3mL</td><br />
</tr><br />
</table><br />
</p><br />
<p class="body_text"><br />
Heat up the solution in a conical flask, until agarose has dissolved completely and the solution becomes clear. To the clear solution, add 2 ul of Ethidium Bromide and shake. Pour solution onto the the gel plate with the comb. Wait until gel has solidified after 20 minutes, the gel may now be ready for digest. <br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Gel Electrophoreisis</p><br />
<p class="body_text"><br />
Add loading buffer to all samples (including laddder), then remove the comb from the solidified agarose gel, place the solidified agarose gel onto gel box and cover the gel box with 1x TAE buffer. Carefully load samples into the gel wells. Then cover the gel box with the lid. Run the gel on 120 Volts, 60 minutes condition. <br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/bd/Dongchanchoi.png');height:200px;width:321px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Nanodrop</p><br />
<p class="body_text"><br />
Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present. Open the Nanodrop program and the appropriate module (e.g., DNA). Wipe off the top and bottom sensors of the instrument with a Kimwipe. Pipette 1 μL of RO water onto the sensor. Bring down the lever arm. Follow the onscreen prompts to calibrate. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer EB or whatever solution your prep is in). Bring down the lever arm. Follow the onscreen prompts to blank. Wipe the sensors and pipette on 2 μL of your sample. Bring down the lever arm. Click Measure and record the concentration measured. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280 ratio should be between 1.8 and 2.0. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary. Clean the sample surfaces once more after you are finished.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">PCR</p><br />
<p class="body_text"><br />
in a small 100uL reaction tube, add the following reagents:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Component</b></th><br />
<th>50ul Reaction</th><br />
<th>Final Concentration </th><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>to 50uL</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>5X NED Phusion buffer</td><br />
<td>10uL</td><br />
<td>1X</td><br />
</tr><br />
<tr><br />
<td>10mM dNTP</td><br />
<td>1ul</td><br />
<td>200uM</td><br />
<tr><br />
<td>10mM Forward Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>10mM Reverse Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>Template DNA</td><br />
<td>2uL</td><br />
<td><250ng</td><br />
</tr><br />
<tr><br />
<td>DMSO (optional)</td><br />
<td>1.5uL</td><br />
<td>3%</td><br />
</tr><br />
<tr><br />
<td>Phusion DNA Polymerase</td><br />
<td>0.5uL</td><br />
<td>1.0 units</td><br />
</tr><br />
</tr><br />
</table><br />
<br />
<p class="body_text"><br />
Thermocycling conditions:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Step</b></th><br />
<th>Temperature</th><br />
<th>Time </th><br />
</tr><br />
<tr><br />
<td>Initial Denaturation</td><br />
<td>98C</td><br />
<td>30s</td><br />
</tr><br />
<tr><br />
<td>25-35 cycles</td><br />
<td>98C<br />
<br />
45-72C<br />
<br />
72C</td><br />
<td>5-10s<br />
<br />
10-30s<br />
<br />
15-30s</td><br />
</tr><br />
<tr><br />
<td>Final Extension</td><br />
<td>72C</td><br />
<td>5-10min</td><br />
<tr><br />
<td>Hold</td><br />
<td>4-10C</td><br />
<td></td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</p><br />
<p class="body_text"><br />
In a 1.5mL eppendorf tube, add the following reagents, adds up to 10uL reaction volume<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Volume</th><br />
</tr><br />
<tr><br />
<td>T4 Ligase</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>Insert</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>Backbone</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>4uL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Mammalian Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4f/Weiling_Labs.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Passaging Adherent Cells</p><br />
<p class="body_text"><br />
In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood. <br />
<p class="body_text"><br />
<b>1)</b> Pipette spent medium and discard to waste.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C. <br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>NOTE:</b> Care should be taken to<br />
avoid leaving cells exposed to the trypsin<br />
longerthan necessary. Care should also be<br />
taken that the cells not be forced to detach<br />
prematurely, as this may result in clumping.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b>Add serum-containing medium(10mL) and pipette the cells up and<br />
down until the cells are dispersed into<br />
a single cells suspension. <br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Add the appropriate volume of cell<br />
suspension (dependent on confluence/cell count - generally for 100% confluence split 1:4) to a new flask/dish containing medium (end volume 10mL).<br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Place dish(es) in incubator at 37C. Leave for 3-4 days before next passage. <br />
</p><br />
</div><br />
</div><br />
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<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a5/KC_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Stable Transfection Of Adherent Cells</p><br />
<p class="body_text"><br />
For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.<br />
<p class="body_text"><br />
<b>1)</b> The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Incubate at room temperature for 5-10mins to allow for complex formation.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml. <br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well. <br />
</p><br />
<p class="body_text"><br />
<b>8)</b> Change medium and wash with PBS. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Incubate for 24-48 hours. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Assay for gene expression. <br />
</p><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/1/16/ALex_Bates_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Amyloid Degradation Assay</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. <br />
</p><br />
<p class="body_text"><br />
b. For fAβ digestions, 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.<br />
</p><br />
<p class="body_text"><br />
c. After digestion, analyse the reaction by Congo red assay.<br />
</p><br />
<p class="body_text"><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"></div><br />
<div class="description"><br />
<p class="minor_title">Congo Red Spectrophotometric assay </p><br />
<p class="body_text"><br />
<b>1)</b> Make up a 7 mg/mL solution of Congo Red in a buffer solution of 5mM potassium phosphate, 150mM NaCl (pH7.4). Filter through a 0.2µm syringe immediately before using.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> At room temperature zero a UV–Vis spectrophotometer between 400 and700 nm with a disposable cuvette containing 1mL phosphate buffer.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> To the the phosphate buffer, add 5µL of the Congo Red solution. Scan between 400 and 700 nm and take a record of the spectrum.<br />
</p><br />
</p><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
<b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> For a microscopic analysis, transfer the protein/lysate-Congo Red solution to a centrifuge tube. Centrifuge (12,000–14,000 rpm) to pellet the fibrils. Wash the fibrils with water, resuspend the fibrils in a small amount of water, and place on a microscope slide. Let the sample dry in air and analyse under polarized light. If transfection with MMP-9 has been successful, the assay should not be strongly indicative of fibrils.<br />
</p><br />
</div><br />
</div><br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/Project/ExperimentsTeam:UCL/Project/Experiments2013-10-05T03:21:25Z<p>Naruto: </p>
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<p class="body_text"><br />
<b> Here we explain some of the experiments and procedures we undertook. The details of our procedures are presented in our <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank">protocols page</a>. </b><br />
</p><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<p class="major_title">Bacterial Lab Experiments And Procedures</p><br />
<div class="gap"></div><br />
<br />
<p class="minor_title">Creating Competent Bacteria</p><br />
<div class="full_row"><br />
<p class="body_text"><br />
E. coli are not naturally transformable, which means they lack the ability to take up plasmids (competency). Competency is induced by divalent cations such as calcium. These alter the permeability of the membranes enveloping the bacterium to plasmids. Normally macromolecules on the outer surface of bacteria are negatively charged which means the negative charges of incoming DNA would be repelled. The addition of calcium chloride facilitates the movement of DNA into the cell.<br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<p class="minor_title">Transformation</p><br />
<div class="full_row"><br />
<p class="body_text"><br />
To insert foreign DNA into our competent cells we used the heat shock treatment. Our competent bacteria are stored in -80C. To transform, DNA with a selectable marker and competent bacteria are mixed together and kept on ice for thirty minutes to allow interactions between calcium ions and the negative charges on the bacterial envelope. The mixture is exposed to a brief period of 38C (heat shock). The rapid shift in temperature alters the fluidity of the membrane therefore allowing DNA to enter the cell.<br />
Afterwards, the bacteria containing the foreign DNA are streaked on selective plates. Bacteria containing the foreign DNA with the selectable marker, such as ampicillin resistance, would be the only bacteria growing on the selective plates.<br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<p class="minor_title">Minipreparation</p><br />
<div class="full_row"><br />
<p class="body_text"><br />
This step involves purifying plasmid DNA from bacteria. This allows us to prepare a stock of DNA ready for digest, ligation or further transformation.<br />
This is done by first lysing the cells then applying centrifugation on the sample in spin filters. Eventually after the process DNA would be purified as it elutes through the filter along with elution buffer.<br />
</p><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<p class="minor_title">Analytical Digest</p><br />
<div class="full_row"><br />
<p class="body_text"><br />
Gel electrophoresis exploits the fact that the negatively charged DNA fragments would move in a electric field. Furthermore, in the gel matrix, smaller fragments of DNA would move with more ease. This allows separation of shorter fragments of DNA from longer fragments. <br />
Our DNA sample is first digested into fragments with restriction enzymes. Finally the digested DNA sample is added to the gel. After exposure to an electric field for an hour, separated fragments can be observed under UV.<br />
</p><br />
</div><br />
<br />
<p class="minor_title">Nanodrop</p><br />
<div class="full_row"><br />
<p class="body_text"><br />
The nanodrop, or spectrophotometer, quantitatively measures the purity of DNA in a sample. It uses the fact that nucleic acids absorb UV light in a specific pattern. The Beer-Lambert Law is used to determine the concentration of DNA as a function of absorbance.<br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<p class="minor_title">Preparative Digest</p><br />
<div class="full_row"><br />
<p class="body_text"><br />
This experiment is used to build up a stock of a specific fragment of DNA. Afterwards, the digested sample is run on gel electrophoresis. Compared to the analytical digest, this is done in larger quantities of DNA sample and enzymes. To isolate the wanted DNA fragment we extracted the band with the correct length on the gel.<br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<p class="minor_title">Gel Extraction</p><br />
<div class="full_row"><br />
<p class="body_text"><br />
This procedure is used to extract the DNA fragment, with the expected band, on the gel. With the use of UV light, DNA bound to ethidium bromide illuminates and can be carefully extracted from the agarose gel. The removed fragment of gel would contain the desired DNA fragment. Furthermore, DNA would have to be purified from the gel fragment using a gel purification procedure. This DNA could be utilized for ligation, transformation and so on.<br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<p class="minor_title">Polymerase Chain Reaction</p><br />
<div class="full_row"><br />
<p class="body_text"><br />
The polymerase chain reaction uses enzymes and primers to generate a large quantity of copies of a DNA sequence. This allows us to amplify a gene or sample to a more workable quantity. In addition, with the correct use of primers, we would be able to modify a gene so that illegal sites could be mutagenised and addition of the iGEM standardized prefixes and suffixes. <br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<p class="minor_title">Ligation</p><br />
<div class="full_row"><br />
<p class="body_text"><br />
If an insert needs to be attached to a backbone (vector) containing a selectable marker, a ligation is used. Insert and backbone will need to have compatible sticky ends prepared from the preparative digest. Ligase, Ligase buffer, correct conditions are used in the process to catalyse this reaction. <br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<p class="minor_title">Maxipreparation</p><br />
<div class="full_row"><br />
<p class="body_text"><br />
Similar to a minipreparation, the maximpreparation is used to purify DNA. The maxipreparation, however, produces more pure and higher concentrations of DNA.<br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Mammalian Lab Experiments And Procedures</p><br />
<div class="gap"></div><br />
<br />
<p class="minor_title">Zeocin Kill Curve</p><br />
<div class="full_row"><br />
<p class="body_text"><br />
Before doing a transfection experiment, it is important to determine the concentration of selection reagent required for efficient selection. For this, we growed HeLa cells in various concentrations of Zeocin - 0 nM, 50 nM, 100 nM, 250 nM, 500 nM and 1000 nM.<br />
<br />
We conducted the kill curve in a 6-well plate. A T25 flask of HeLa is split in 10 ml medium. Each well is filled with 1 ml of cells. Incubation is carried out at 37°C overnight to allow cells to attach before adding inhibitor (Zeocin). Medium is removed and replaced with 2 ml of DMEM + Glu + FBS + Zeocin. Confluency of cells is observed in each well every day for 5 days. Viability can be assessed using a Vicell machine, which stains dead cells blue.<br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<p class="minor_title">HeLa Growth Curve</p><br />
<div class="full_row"><br />
<p class="body_text"><br />
We conducted a growth curve for HeLa cells in a 6-well plate. A T25 flask of HeLa is split in 10 ml medium. Each well is filled with 1 ml of cells. Incubation is carried out at 37°C overnight. Medium is removed and replaced with 2 ml of DMEM + Glu + FBS. Confluency of cells is observed in each well every day for 5 days. <br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<p class="minor_title">Transfection</p><br />
<div class="full_row"><br />
<p class="body_text"><br />
<br />
Hela cell culture (0.1x106 cells/ml) was seeded in four 6-well plates 24 hours prior to transfection to attain 70% confluency. When cells reached 70% confluency they were transfected. Control flasks are transfected with only plasmid backbone pSB1C3. <br />
</p><br />
<p class="body_text"><br />
5ug of BBa_K1018001 plasmid DNA (12.5ul of a 401 ng/ul stock) was added to 5ml of DMEM supplemented with 2mM L-glutamine only in a 15ml falcon tube. The solution was vortexed briefly and incubated at room temperature for 5 minutes. 20ml of a 1mg/ml branched 25KDa polyethylenimine (or SuperFect) was added to the solution before vortexing briefly and a further 5-minute incubation at room temperature.<br />
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The media in the cell culture flasks was aspirated and replaced with the transfection solution and cells were returned to incubation conditions. Control flasks were treated with 5ml DMEM supplemented with 2mM L-glutamine and 20ml of branched PEI (or SuperFect) as above. <br />
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After 24 hours the media was replaced with DMEM supplemented with 10% FCS, 1% PenStrep antibiotic, 2mM L-Glutamine and 150mg/ml Zeocin. <br />
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Medium is removed and replaced with 2 ml of DMEM + Glu + FCS + Zeocin. Confluency of cells is observed in each well every day for 5 days. <br />
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<p class="minor_title">Amyloid Degradation Assay</p><br />
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The 42-amino acid peptide (Aβ1-42), the predominant peptide length found in senile plaques, has a remarkable propensity to aggregate at high concentrations to form a β-pleated sheet structure. While plaques and amyloid fibrils have been viewed by some as resistant to proteolytic degradation, it is possible that certain proteases, such as MMP-9 may contribute to endogenous mechanisms leading to plaque clearance. Our assay, inspired by Yan et al., demonstrates our BioBrick's capability to do this.<br />
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<p class="minor_title">Congo Red Assay</p><br />
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The birefringence version of this assay was originally developed to study amyloid <i>in vivo</i> but has since been expanded into frequent <i> in vitro </i> use. It depends on the birefringence of amyloid; an optical property of a material that has a refractive index dependent on the polarisation and propagation of light (i.e. orientation differences within the molecule in question), meaning that there is a double refraction of light. Many materials can be birefringent, including phosphate buffer, and this assay is subjective, so a known fibrillar material needs to be used as a control. The spectroscopic version of this assay is less subjective and less prone to misinterpretation, and involves recording absorbance spectrums. <br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/GalleryTeam:UCL/Gallery2013-10-05T03:19:27Z<p>Naruto: </p>
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<p class="major_title">The Creative Angle</p><br />
<p class="minor_title">Visual Art</p><br />
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How does the public interpret visually the invasion of synthetic biology into neuroscience? <br />
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We have heavily integrated visual art into our project, especially the website, to help engage the public and stage our ethical ideas. Our team includes two artists-in-residence who have helped design thought-provoking images. <br />
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This Gallery page exhibits primarily poster art pieces that have been created by team member Fong Yi Khoo. We have incorporated these art pieces into our project by using them as promotional posters, speed debate event posters, presentation and so on. You can find other art pieces created by our artists on every corner of our wiki page.<br />
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<p class="body_text">We used this main poster on our T-shirts and on our promotional material. It was imortant to show graphically the damage wrought on a brain by the onset of Alzheimer's Disease, and at the same time, display the vibrancy of the solution that we propose. The watercoulour blots are the figures of gial cells and their processes spreading through the brain. The dark mug from the brain mirrors its stem. It is round, like the figure of a closed circuit of our treatment (we do not propose that the transfected microglia should perpetuate themselves into the second generation). It symbolises the plasmid, and the marks of coffee mugs through the late nights we spent on the project.<br />
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<p class="body_text">The Brain - A Memory Bank?</p><br />
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<p class="body_text">How much do you know about how memories are stored in the brain? No-one really knows for sure, and the theories shared among Psychologists and Neuroscientists are far from common knowledge. At present, there are just under 500,000 people in the UK. We can't show with sores, or with injured limbs: something subtle, yet devastating is happening inside the skull. All that's clear is that the things which make us who we are steadily come apart. <br />
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<p class="body_text">Against the backdrop of damage shifting like heavy cloud through the tissues, what about the brain loss outside?</p><br />
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<p class="body_text">Homepage Piece</p><br />
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<p class="body_text">Brain Plaques</p><br />
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<p class="body_text">Synthetic Biology On Brain</p><br />
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<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/f/f4/Ecoli.png');height:660px;width:470px"></div><br />
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The Biology that we engineer forces us to think of the machinery in very clear cut terms, but what of the consequences, how will it react?<br />
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<p class="body_text">Brain Maze</p><br />
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<p class="body_text">Neuroethics is confusing isn't it?</p><br />
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<p class="body_text">Error: Unable to Save</p><br />
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<p class="body_text">Delete</p><br />
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<p class="minor_title">Four detailed drawings of the Brain</p><br />
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<p class="body_text">Coronal section of a brain afflicted with Alzheimers (in pencil)</p><br />
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<p class="body_text">Coronal section of a healthy brain (in pencil)</p><br />
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<p class="body_text">Aspect of the left Cortex (in charcoal)</p><br />
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<p class="body_text">Saggital section of a healthy brain (in charcoal)</p><br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/Notebook/AugustTeam:UCL/Notebook/August2013-10-05T03:15:31Z<p>Naruto: </p>
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
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<p class="major_title">August</p><br />
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<p class="minor_title">1st August</p><br />
<p class="body_text"><br />
Bacterial lab had good results today in the preparation of a new stock of competent cells.<br />
In the evening we celebrated the success of the speed debate.<br />
</p><br />
<p class="minor_title">2nd August</p><br />
<p class="body_text"><br />
Stjohn designed the linkers for the Mammalian Oxidative Stress Inducible Promoter.<br />
The team met to discuss fundraising ideas such as Kickstarter. Starting idea: brain-with-plaques-for-sale.<br />
</p><br />
<p class="body_text"><br />
The concept of a Memory Lane, where people could upload their memories onto a page within the wiki, was discussed. <br />
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<p class="body_text"><br />
Alex suggested a collaboration with Westminster iGEM team regarding the speed debate idea.<br />
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<p class="minor_title">5th August</p><br />
<p class="body_text"><br />
Snapshots of the team members were taken!<br />
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<p class="body_text"><br />
Team abstracts were written and uploaded onto the wiki. <br />
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<p class="body_text"><br />
Alex contacted the Imperial iGEM team regarding an eventual collaboration. <br />
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<p class="minor_title">6th August</p><br />
<p class="body_text"><br />
Rob invited the team at 12 noon in the Anatomy Hub to discuss about the wiki design in order to make sure that all the ideas about this matter are taken into account.<br />
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<p class="minor_title">7th August</p><br />
<p class="body_text"><br />
Barbeque evening, venue Wilkins Roof Garden!<br />
</p><br />
<p class="body_text"><br />
Prof. Eli Keshavarz-Moore was our guest and at 3 pm we also had the chance to present our project. (venue: Malet Place Engineering LT 1.03)<br />
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<p class="minor_title">8th August</p><br />
<p class="body_text"><br />
The team discussed about the work on zeocin,pA-f1-Zec biobrick, which will indeed be an improvement of BBa_J176124 because:<br />
</p><br />
<p class="body_text"><br />
i) it gives most of the functionality of BBa_J176124 but is compatible with standard assembly<br />
</p><br />
<p class="body_text"><br />
ii) it allows people to simply insert a PROMOTER-ORF fragment upstream of a pA to give an expression cassette for the ORF of interest, and a ZEC to select stable transfectants. <br />
</p><br />
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<p class="minor_title">9th August</p><br />
<p class="body_text"><br />
Project description is up on Wiki!<br />
</p><br />
<p class="body_text"><br />
Dr Darren Nesbeth visted the lab on numerous occasions to check project progress. <br />
The requested batch of biobricks arrived as glycerol stocks.<br />
</p><br />
<p class="body_text"><br />
The team further discussed crowdfunding via Kickstarter. Beautiful pieces of art are to be generated by FYi for the Memory Palace. <br />
</p><br />
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<p class="minor_title">12th August</p><br />
<p class="body_text"><br />
A strategy meeting was held in the lab with Dr Darren Nesbeth. <br />
</p><br />
<p class="body_text"><br />
FYi drawn the wiki background for the diary section. She also made the illustrations for the T-shirts.<br />
The team also debated on the wiki design and a consensus was reached regarding the site map, default banner and logo.<br />
</p><br />
<p class="body_text"><br />
In 'Memory Lane', we are going to ask people to 'leave one strong memory' on one page whether in text or pictures. These will be done anonymously but they will leave their emails with us so they will be notified when the 'compilation' is up. <br />
</p><br />
<p class="body_text"><br />
The website came to life today!<br />
</p><br />
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<p class="minor_title">13th August</p><br />
<p class="body_text"><br />
Alex and Oran came up with the idea of a Creative writing competition. <br />
</p><br />
<p class="body_text"><br />
FYi, Robin, Alex and Stjohn and Oran focused on wiki building for the weeks to come while the rest of the team worked in the Bacterial Labs.<br />
</p><br />
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<p class="minor_title">14th August</p><br />
<p class="body_text"><br />
The advertisement for the competition was written and the competition was launched. More details about the outcome can be found on the ‘Competition’ subsection.<br />
</p><br />
<p class="body_text"><br />
Met the Westminister team to discuss about the potential modelling collaboration. It was a nice gathering.<br />
</p><br />
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<p class="minor_title">15th August</p><br />
<p class="body_text"><br />
Continued intensively planning and brainstorming for the design of our wiki, especially on the front page design. <br />
</p><br />
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<p class="minor_title">16th August</p><br />
<p class="body_text"><br />
Alex finished the essay on Neuroethics on which he has dedicated around 2 weeks of research.<br />
</p><br />
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<p class="minor_title">19th August</p><br />
<p class="body_text"><br />
Alex advertised the writing competition on prizemagic.co.uk.<br />
</p><br />
<p class="body_text"><br />
Stjohn released a new set of rules for managing wiki content in order to make work easier before the wiki freeze.<br />
</p><br />
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<p class="minor_title">20th August</p><br />
<p class="body_text"><br />
The actual work on the main poster on the frontal page started. FYi produced the first sketch and the team gave feedback.<br />
</p><br />
<p class="body_text"><br />
The members’ Profiles are ready to be uploaded on wiki!<br />
</p><br />
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<p class="minor_title">21th August</p><br />
<p class="body_text"><br />
The lab was closed in the morning, however in the afternoon the Bacteria Team prepared selective plates and selective media in order to culture the last arrived biobricks from the HQ. Darren assisted us.<br />
</p><br />
<p class="body_text"><br />
The linkers designed by Stjohn: IGM Ox L1, L2, L3, L4 as well primers for cmv promoter were ordered.<br />
</p><br />
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<p class="minor_title">22th August</p><br />
<p class="body_text"><br />
The first Creative Competition Entry! Yey! Thank you!<br />
</p><br />
<p class="body_text"><br />
The atmosphere in the Bacterial Lab became slightly more cheerful. The amplification of zeocin from the 2 types of ordered primers was successful as well as the digestion of K812014 and pSB1C3 and pSB1A3. We decided to use the zec bb F,R primers for the further amplification of zeocin. <br />
</p><br />
<p class="body_text"><br />
The Zeocin kill curve was derived, a concentration of 150 ug/ml was used.<br />
</p><br />
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<p class="minor_title">23th August</p><br />
<p class="body_text"><br />
The main poster for the front page was finalised. Well done FYi!<br />
</p><br />
<p class="body_text"><br />
New submissions for the Creative writing! <br />
Lonza confirmed a sponsorship of £1, 207. Happy Happy Joy Joy! Well done Weiling!<br />
</p><br />
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<br />
<p class="minor_title">26th August</p><br />
<p class="body_text"><br />
The lab was closed today hence we all focused on the wiki content.<br />
</p><br />
<p class="body_text"><br />
The front page poster background - wasteland was completed.<br />
</p><br />
<br />
<br />
<p class="minor_title">27th August</p><br />
<p class="body_text"><br />
Weiling emailed Geneious and Eppendorf with regards to Sponsorship.<br />
</p><br />
<br />
<p class="minor_title">28th August</p><br />
<p class="body_text"><br />
The Biosafety forms were filled in as necessary. These must be signed by Darren before the 30th.<br />
</p><br />
<p class="body_text"><br />
We met Darren at 4 pm in the lab to discuss about the biobrick processing.<br />
</p><br />
<br />
<p class="minor_title">29th August</p><br />
<p class="body_text"><br />
We considered the strategy to deal with the linker region. First step is to achieve the annealing of the oligonucleotides making up this linker. We're still waiting for these sequences.<br />
</p><br />
<p class="body_text"><br />
Agreed on the final design of the T-shirts. We're aiming to order them as soon as possible.<br />
</p><br />
<br />
<p class="minor_title">30th August</p><br />
<p class="body_text"><br />
We uploaded the first samples of memories on the Memory Lane page.<br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/Notebook/JuneTeam:UCL/Notebook/June2013-10-05T03:02:48Z<p>Naruto: </p>
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
</p> <br />
</div><br />
<br />
<p class="major_title">June</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">5th June</p><br />
<p class="body_text"><br />
Group discussion concerning the project idea to be carried forward - favouring the 'Anti cancer project'. Roles were then assigned to team members present for intial research roles for the week:<br />
</p><br />
<p class="body_text"><br />
Cancer research roles:<br />
</p><br />
<p class="body_text"><br />
1. Ruxi Comisel - Proteins upregulated in cancer of the intestines. Specifically in the outer epithelial cell (enterocytes) – in microvilli. Also, what actually is... gut cancer? A general overview would be useful…<br />
</p><br />
<p class="body_text"><br />
2. Khaicheng Kiew - Our chassis (bearing in mind that we will also build it in E. coli as a backup). We need to think what would make a good chassis in our case (ie. naturally found in the gut in an obvious one), and how well does the chassis fit.<br />
</p><br />
<p class="body_text"><br />
3. Alex Bates - What will the killing mechanism be? A broad overview of cancer treatments is required, specifically detailing how a bacterium can administer the treatment.<br />
</p><br />
<p class="body_text"><br />
Considerations:<br />
</p><br />
<p class="body_text"><br />
a. The bacteria may secrete a toxin etc – how will we ensure that it doesn’t simply diffuse through the gut? <br />
b. If it is a toxin, what sort of biosynthetic pathway is required?<br />
c. Does the bacteria trigger apoptosis in the cancer cells (ie. an intracellular killing mechanism)? How can this be done from an extracellular bacterium? Perhaps beta-arrestin?<br />
d. Are there any treatments which we can take advantage of specifically because we are using bacteria? <br />
e. For example, a protein which creates holes in the cancer cells? Does using a bacterium open up the possibility of using a different cure that currently isn’t in use because we cannot target it to cancer cells – could the use of bacteria allow this?<br />
</p><br />
<p class="body_text"><br />
4. Weiling Yuan - Targeting – do we use antibodies? What previous projects have used bacteria expressing antibodies? Are there any other ways of doing this? Perhaps the latching and initiation mechanisms can be incorporated into one protein?<br />
</p><br />
<p class="body_text"><br />
5. StJohn Townsend - Initiation – mechanoreceptor activated upon latching? What other ways are there of doing this?<br />
</p><br />
<p class="body_text"><br />
6. Tom Johnson - Past iGEM projects which we could incorporate into our own: Cancer projects, Gut projects, Protein engineering, Antibodies expressed in bacteria etc.<br />
</p><br />
<br />
<p class="minor_title">7th June</p><br />
<p class="body_text"><br />
The team discusses findings from the initial research - further agreement that the 'Anti Cancer' project seemed to be the best idea, preparation of 'project sheets' to be sent to Dr. Darren Nesbeth for review and subsequent meetings.<br />
</p><br />
<p class="minor_title">11th June</p><br />
<p class="body_text"><br />
looked a bit at the possible chassis species: salmonella, clostridium, helicobacter, E. coli. according to the tissue type/cancer type we shall decide which works with which. We start with E. coli in the lab.<br />
</p><br />
<p class="body_text"><br />
We considered a pro-drug approach - bacterially directed enzyme pro-drug therapy which suggests that we may establish a transformed bacterial population with an enzyme capable to activate an ingested prodrug. This pro-drug would be connected to an antibody (possibly part of the tail) and would also have linking consensus sequence targeted by the enzyme produced locally by our bacteria.<br />
</p><br />
<p class="body_text"><br />
From this above point Alex distinguished 2 scenarios built on the circuit sketch that he and Laia posted a while ago. These would be:<br />
</p><br />
<p class="body_text"><br />
1) Kill unit produces tailed protein pro-drug (possibly tailed perforin) and signaling molecule, A. When A reaches a threshold amount, perforin and a protease to remove the confounding tail is produced, bacteria lyses and activated pro-drug acts on surrounding cells.<br />
</p><br />
<p class="body_text"><br />
2) No protease is produced, because the tail can be cleaved off by matrix metalloproteases.<br />
</p><br />
<p class="body_text"><br />
Goals for the end of this week: <br />
</p><br />
<p class="body_text"><br />
- Alex, Andy and Weiling continue investigating possible candidates to fill in the parts for the scenarios<br />
</p><br />
<p class="body_text"><br />
-Tom, KC and Ruxi make sure we have everything set up to start the work in the lab: protocol, parts etc.<br />
</p><br />
<p class="minor_title">12th June</p><br />
<p class="body_text"><br />
Ruxi and Tom went through a general cloning protocol but then realised that the best way to prepare for the lab is to get familiarised with the iGEM distribution kits. We discovered that we are given almost everything we need in order to get it right.<br />
</p><br />
<p class="body_text"><br />
Alex filled in the form with our proposal requested by Darren - we have the sequences and details of potential new biobricks. <br />
</p><br />
<p class="body_text"><br />
We formulated a new proposal regarding the Alzheimer’s disease amyloid plaque degradation.<br />
</p><br />
<p class="body_text"><br />
Andy searched potential cancer killer molecules:<br />
</p><br />
<p class="body_text"><br />
- CD95 - <a href="http://www.nature.com/cdd/journal/v14/n4/full/4402051a.html" target="_blank">Fas agonist</a><br />
</p><br />
<p class="body_text"><br />
- Tumor Necrosis Factor, Histamine - induces inflammation<br />
</p><br />
<p class="body_text"><br />
- HAMLET (human a-lactalbumin) - induces apoptosis <br />
</p><br />
<p class="body_text"><br />
- endostatin, thrombospondin - reduce cancer growth<br />
</p><br />
<p class="body_text"><br />
Weiling looked at potential promotors: <br />
</p><br />
<p class="body_text"><br />
- RacA (based on increased DNA damaged due to radiation) to start the killing cascade and CD95 as a potential killer molecule<br />
</p><br />
<p class="body_text"><br />
- Lux pR promotor<br />
</p><br />
<p class="body_text"><br />
- Lld promoter<br />
</p><br />
<p class="body_text"><br />
- Vgb promotor <br />
</p><br />
<p class="body_text"><br />
- HIP-1<br />
</p><br />
<p class="body_text"><br />
<a href="http://www.biomedcentral.com/1471-2180/11/96" target="_blank">gastric Oxygen levels</a> <br />
</p><br />
<p class="body_text"><br />
For promoter 1 (switches on the pro-drug and signaling molecule transcription), a very <br />
good candidate is HIP 1 promoter - hypoxia-inducible promoter which drives reporter gene expression under both acute and chronic hypoxia. It was <a href=" http://www.landesbioscience.com/journals/cbt/article/2951/mengesha5-9.pdf" target="_blank">developed in attenuated Salmonella species</a>. <br />
</p><br />
<p class="body_text"><br />
We need to register this part!<br />
</p><br />
<br />
<p class="minor_title">13th June</p><br />
<p class="body_text"><br />
Alex sent the 3 main project proposals to Dr. Darren Nesbeth for review.<br />
</p><br />
<p class="body_text"><br />
Tom and Andy edited the wiki page adding various sections and elaborating on previously created pages.<br />
</p><br />
<p class="body_text"><br />
Weiling researched on killing mechanisms being able to target hypoxic regions of solid tumors and promoters in hypoxia environments.<br />
</p><br />
<p class="body_text"><br />
Catrin - General project research<br />
</p><br />
<p class="body_text"><br />
Ruxi - Further researched the potential promoters esp HIP 1 and the Fas regulated programmed apoptosis.<br />
</p><br />
<p class="body_text"><br />
We attended a Synthetic Biology talk by Neil Dixon, University of Manchester (Tom and Andy).<br />
</p><br />
<p class="body_text"><br />
Had a general meeting for discussion of what has been accomplished so far, and the subsequent actions, which are to be undertaken by team members. Further documents were also submitted to Dr. Darren Nesbeth concerning 'team roles'. The team then began to do individual research or other activity:<br />
</p><br />
<p class="body_text"><br />
Tom and Robin - Edited the iGEM wiki, added team information and removed the unnecessary tutorial information, replacing it with more useful information and streamlining the whole interface.<br />
</p><br />
<p class="body_text"><br />
Weiling and Alex - Further development of circuit ideas, taking inspiration from previous iGEM ideas as well as further research into the CD95L molecule.<br />
</p><br />
<p class="body_text"><br />
Ruxi and Catrin - Research into latching molecules for a bacteria to tumour interface to increase target specificity. Idea encounted from Hong Kong 2012 where Colon Cancer was targeted.<br />
</p><br />
<br />
<p class="minor_title">14th June</p><br />
<p class="body_text"><br />
Tom - Website design for: Main Page, UCL information, Team based pages and Notebook pages<br />
</p><br />
<p class="body_text"><br />
Robin - Coding in HTML for website<br />
</p><br />
<p class="body_text"><br />
Ruxi, Catrin, Weiling - Further investigation of Hong Kong 2010 to see what parts may be improved or of use to the project, these were: a blue light activated promoter, how can the quorum sensing and CagA be exploited, a negative regulatory system for drug secretion.<br />
</p><br />
<p class="body_text"><br />
Alex - searched for potential bacterial receptor to be modified in order to be a good target for something else in the environment/cancer cell surface.<br />
</p><br />
<p class="minor_title">17th June</p><br />
<p class="body_text"><br />
The group had a meeting to discuss what had been achieved so far and what needed to be done today. <br />
</p><br />
<p class="body_text"><br />
Tom - Continued on website design and wrote several pieces concerning UCL to be used on the website when it goes live.<br />
</p><br />
<p class="body_text"><br />
Robin - Continued on website coding.<br />
</p><br />
<p class="body_text"><br />
Weiling & Catrin - Researched for project sponsors and potential contacts.<br />
</p><br />
<p class="body_text"><br />
Alex, Ruxi, StJohn & Andy - Continued research into the project ideas.<br />
</p><br />
<p class="minor_title">18th June</p><br />
<p class="body_text"><br />
The group met with advisors Darren Nesbeth and Philipp Boeing to discuss the three project suggestions. The 'Neural Network' proposal was effectively ruled out due to the high risk and low probablility of project success in terms of medals.<br />
</p><br />
<p class="body_text"><br />
The anti-cancer project was previously the favoured idea, but after extensive review ,the Alzheimers project gained favour due to being relatively new (and hence exciting) to iGEM compared to a cancer project, which has been done several times already at iGEM. No final decision has been made however, work has continued on researching both projects. The wiki is also still being worked on.<br />
</p><br />
<p class="body_text"><br />
The team also had a social gathering: pizza for lunch.<br />
</p><br />
<p class="minor_title">19th June</p><br />
<p class="body_text"><br />
The group continued work on all three projects in order to send improved proposals to Darren Nesbeth by the end of the day. Many professors and experts were also emailed to seek guidance, in particular for the Alzheimer's project which seems to be particularly difficult.<br />
</p><br />
<p class="minor_title">20th June</p><br />
<p class="body_text"><br />
Tom - Prepared a presentation to be given next week about iGEM to prospective UCL students to raise interest in the engineering faculty and also the iGEM competition. After this was complete, joined the rest of the group in research. Also performed wiki coding for the team page and notebook page.<br />
</p><br />
<p class="body_text"><br />
The group continued what was started yesterday: Rectifying the proposals, with both sent off at the end of the day once they were complete. A group meeting was held at the end of the day to gauge interest and vote for the most popular idea, followed by a social gathering.<br />
</p><br />
<p class="minor_title">21st June</p><br />
<p class="body_text"><br />
Tom - Continued wiki design, coding and content uploads.<br />
Alex - Continued to redraft the proposal for Alzheimer's<br />
StJohn - Continued to redraft the proposal for Cancer<br />
</p><br />
<p class="body_text"><br />
KC - Researched into other iGEM teams to colloborate with and initiated correspondence via email<br />
</p><br />
<p class="body_text"><br />
The team then discusses which project was favoured. It was fairly even but Alzheimer's was slightly more popular.<br />
</p><br />
<p class="minor_title">24th June</p><br />
<p class="body_text"><br />
Tom continued wiki design whilst the rest of the group performed research.<br />
</p><br />
<p class="body_text"><br />
Once this was complete, the group had a meeting with Yanika Borg and Philipp Boeing concerning the two project ideas. Philipp favoured the Alzheimer's project whilst Yanika was somewhat undecided. <br />
</p><br />
<p class="body_text"><br />
A vote was taken with Alzheimer's being the prefered project by the group as a whole once more, although consensus was not fully reached. The group agreed to decide on the project on Wednesday proceeding a meeting with Prof. Lazaros Lukas.<br />
</p><br />
<br />
<p class="minor_title">25th June</p><br />
<p class="body_text"><br />
The group continued with general research, and also went to the Wellcome trust to seek any extra information, although this was unfruitful.<br />
</p><br />
<br />
<p class="minor_title">27th June</p><br />
<p class="body_text"><br />
The group voted 29 -11 in favour of Alzheimer's after a meeting with Prof. Lazaro Lukas, who was helpful and seemed excited about the project. The group also met advisor Yanika Borg and she agreed with the choice. The group also scheduled lab safety training for next thursday.<br />
</p><br />
<br />
<p class="minor_title">28th June</p><br />
<p class="body_text"><br />
Tom presented to prospective students about the iGEM project for the day.<br />
</p><br />
<p class="body_text"><br />
Weiling, Alex, Andy & Catrin began to produce a 'stop motion' explanation of the Alzheimer's project.<br />
</p><br />
<p class="body_text"><br />
KC, Robin and StJohn discussed lab protocols and also modelling ideas.<br />
</p><br />
<p class="minor_title">29th June</p><br />
<p class="body_text"><br />
Tom, Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi & StJohn – Continued work on the proposals for the meeting with Dr. Nesbeth on Thursday.<br />
</p><br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/Notebook/JuneTeam:UCL/Notebook/June2013-10-05T03:00:50Z<p>Naruto: </p>
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
</p> <br />
</div><br />
<br />
<p class="major_title">June</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">5th June</p><br />
<p class="body_text"><br />
Group discussion concerning the project idea to be carried forward - favouring the 'Anti cancer project'. Roles were then assigned to team members present for intial research roles for the week:<br />
</p><br />
<p class="body_text"><br />
Cancer research roles:<br />
</p><br />
<p class="body_text"><br />
1. Ruxi Comisel - Proteins upregulated in cancer of the intestines. Specifically in the outer epithelial cell (enterocytes) – in microvilli. Also, what actually is... gut cancer? A general overview would be useful…<br />
</p><br />
<p class="body_text"><br />
2. Khaicheng Kiew - Our chassis (bearing in mind that we will also build it in E. coli as a backup). We need to think what would make a good chassis in our case (ie. naturally found in the gut in an obvious one), and how well does the chassis fit.<br />
</p><br />
<p class="body_text"><br />
3. Alex Bates - What will the killing mechanism be? A broad overview of cancer treatments is required, specifically detailing how a bacterium can administer the treatment.<br />
</p><br />
<p class="body_text"><br />
Considerations:<br />
</p><br />
<p class="body_text"><br />
a. The bacteria may secrete a toxin etc – how will we ensure that it doesn’t simply diffuse through the gut? <br />
b. If it is a toxin, what sort of biosynthetic pathway is required?<br />
c. Does the bacteria trigger apoptosis in the cancer cells (ie. an intracellular killing mechanism)? How can this be done from an extracellular bacterium? Perhaps beta-arrestin?<br />
d. Are there any treatments which we can take advantage of specifically because we are using bacteria? <br />
e. For example, a protein which creates holes in the cancer cells? Does using a bacterium open up the possibility of using a different cure that currently isn’t in use because we cannot target it to cancer cells – could the use of bacteria allow this?<br />
</p><br />
<p class="body_text"><br />
4. Weiling Yuan - Targeting – do we use antibodies? What previous projects have used bacteria expressing antibodies? Are there any other ways of doing this? Perhaps the latching and initiation mechanisms can be incorporated into one protein?<br />
</p><br />
<p class="body_text"><br />
5. StJohn Townsend - Initiation – mechanoreceptor activated upon latching? What other ways are there of doing this?<br />
</p><br />
<p class="body_text"><br />
6. Tom Johnson - Past iGEM projects which we could incorporate into our own: Cancer projects, Gut projects, Protein engineering, Antibodies expressed in bacteria etc.<br />
</p><br />
<br />
<p class="minor_title">7th June</p><br />
<p class="body_text"><br />
The team discusses findings from the initial research - further agreement that the 'Anti Cancer' project seemed to be the best idea, preparation of 'project sheets' to be sent to Dr. Darren Nesbeth for review and subsequent meetings.<br />
</p><br />
<p class="minor_title">11th June</p><br />
<p class="body_text"><br />
looked a bit at the possible chassis species: salmonella, clostridium, helicobacter, E. coli. according to the tissue type/cancer type we shall decide which works with which. We start with E. coli in the lab.<br />
</p><br />
<p class="body_text"><br />
We considered a pro-drug approach - bacterially directed enzyme pro-drug therapy which suggests that we may establish a transformed bacterial population with an enzyme capable to activate an ingested prodrug. This pro-drug would be connected to an antibody (possibly part of the tail) and would also have linking consensus sequence targeted by the enzyme produced locally by our bacteria.<br />
</p><br />
<p class="body_text"><br />
From this above point Alex distinguished 2 scenarios built on the circuit sketch that he and Laia posted a while ago. These would be:<br />
</p><br />
<p class="body_text"><br />
1) Kill unit produces tailed protein pro-drug (possibly tailed perforin) and signaling molecule, A. When A reaches a threshold amount, perforin and a protease to remove the confounding tail is produced, bacteria lyses and activated pro-drug acts on surrounding cells.<br />
</p><br />
<p class="body_text"><br />
2) No protease is produced, because the tail can be cleaved off by matrix metalloproteases.<br />
</p><br />
<p class="body_text"><br />
Goals for the end of this week: <br />
</p><br />
<p class="body_text"><br />
- Alex, Andy and Weiling continue investigating possible candidates to fill in the parts for the scenarios<br />
</p><br />
<p class="body_text"><br />
-Tom, KC and Ruxi make sure we have everything set up to start the work in the lab: protocol, parts etc.<br />
</p><br />
<p class="minor_title">12th June</p><br />
<p class="body_text"><br />
Ruxi and Tom went through a general cloning protocol but then realised that the best way to prepare for the lab is to get familiarised with the iGEM distribution kits. We discovered that we are given almost everything we need in order to get it right.<br />
</p><br />
<p class="body_text"><br />
Alex filled in the form with our proposal requested by Darren - we have the sequences and details of potential new biobricks. <br />
</p><br />
<p class="body_text"><br />
We formulated a new proposal regarding the Alzheimer’s disease amyloid plaque degradation.<br />
</p><br />
<p class="body_text"><br />
Andy searched potential cancer killer molecules:<br />
</p><br />
<p class="body_text"><br />
- CD95 - <a href="http://www.nature.com/cdd/journal/v14/n4/full/4402051a.html" target="_blank">Fas agonist</a><br />
- Tumor Necrosis Factor, Histamine - induces inflammation<br />
- HAMLET (human a-lactalbumin) - induces apoptosis <br />
- endostatin, thrombospondin - reduce cancer growth<br />
</p><br />
<p class="body_text"><br />
Weiling looked at potential promotors: <br />
</p><br />
<p class="body_text"><br />
- RacA (based on increased DNA damaged due to radiation) to start the killing cascade and CD95 as a potential killer molecule<br />
- Lux pR promotor<br />
- Lld promoter<br />
- Vgb promotor <br />
- HIP-1<br />
</p><br />
<p class="body_text"><br />
(about gastric Oxygen levels: http://www.biomedcentral.com/1471-2180/11/96) <br />
</p><br />
<p class="body_text"><br />
For promoter 1 (switches on the pro-drug and signaling molecule transcription), a very <br />
good candidate is HIP 1 promoter - hypoxia-inducible promoter which drives reporter gene expression under both acute and chronic hypoxia. It was <a href=" http://www.landesbioscience.com/journals/cbt/article/2951/mengesha5-9.pdf" target="_blank">developed in attenuated Salmonella species</a>. <br />
</p><br />
<p class="body_text"><br />
We need to register this part!<br />
</p><br />
<br />
<p class="minor_title">13th June</p><br />
<p class="body_text"><br />
Alex sent the 3 main project proposals to Dr. Darren Nesbeth for review.<br />
</p><br />
<p class="body_text"><br />
Tom and Andy edited the wiki page adding various sections and elaborating on previously created pages.<br />
</p><br />
<p class="body_text"><br />
Weiling researched on killing mechanisms being able to target hypoxic regions of solid tumors and promoters in hypoxia environments.<br />
</p><br />
<p class="body_text"><br />
Catrin - General project research<br />
</p><br />
<p class="body_text"><br />
Ruxi - Further researched the potential promoters esp HIP 1 and the Fas regulated programmed apoptosis.<br />
</p><br />
<p class="body_text"><br />
We attended a Synthetic Biology talk by Neil Dixon, University of Manchester (Tom and Andy).<br />
</p><br />
<p class="body_text"><br />
Had a general meeting for discussion of what has been accomplished so far, and the subsequent actions, which are to be undertaken by team members. Further documents were also submitted to Dr. Darren Nesbeth concerning 'team roles'. The team then began to do individual research or other activity:<br />
</p><br />
<p class="body_text"><br />
Tom and Robin - Edited the iGEM wiki, added team information and removed the unnecessary tutorial information, replacing it with more useful information and streamlining the whole interface.<br />
</p><br />
<p class="body_text"><br />
Weiling and Alex - Further development of circuit ideas, taking inspiration from previous iGEM ideas as well as further research into the CD95L molecule.<br />
</p><br />
<p class="body_text"><br />
Ruxi and Catrin - Research into latching molecules for a bacteria to tumour interface to increase target specificity. Idea encounted from Hong Kong 2012 where Colon Cancer was targeted.<br />
</p><br />
<br />
<p class="minor_title">14th June</p><br />
<p class="body_text"><br />
Tom - Website design for: Main Page, UCL information, Team based pages and Notebook pages<br />
</p><br />
<p class="body_text"><br />
Robin - Coding in HTML for website<br />
</p><br />
<p class="body_text"><br />
Ruxi, Catrin, Weiling - Further investigation of Hong Kong 2010 to see what parts may be improved or of use to the project, these were: a blue light activated promoter, how can the quorum sensing and CagA be exploited, a negative regulatory system for drug secretion.<br />
</p><br />
<p class="body_text"><br />
Alex - searched for potential bacterial receptor to be modified in order to be a good target for something else in the environment/cancer cell surface.<br />
</p><br />
<p class="minor_title">17th June</p><br />
<p class="body_text"><br />
The group had a meeting to discuss what had been achieved so far and what needed to be done today. <br />
</p><br />
<p class="body_text"><br />
Tom - Continued on website design and wrote several pieces concerning UCL to be used on the website when it goes live.<br />
</p><br />
<p class="body_text"><br />
Robin - Continued on website coding.<br />
</p><br />
<p class="body_text"><br />
Weiling & Catrin - Researched for project sponsors and potential contacts.<br />
</p><br />
<p class="body_text"><br />
Alex, Ruxi, StJohn & Andy - Continued research into the project ideas.<br />
</p><br />
<p class="minor_title">18th June</p><br />
<p class="body_text"><br />
The group met with advisors Darren Nesbeth and Philipp Boeing to discuss the three project suggestions. The 'Neural Network' proposal was effectively ruled out due to the high risk and low probablility of project success in terms of medals.<br />
</p><br />
<p class="body_text"><br />
The anti-cancer project was previously the favoured idea, but after extensive review ,the Alzheimers project gained favour due to being relatively new (and hence exciting) to iGEM compared to a cancer project, which has been done several times already at iGEM. No final decision has been made however, work has continued on researching both projects. The wiki is also still being worked on.<br />
</p><br />
<p class="body_text"><br />
The team also had a social gathering: pizza for lunch.<br />
</p><br />
<p class="minor_title">19th June</p><br />
<p class="body_text"><br />
The group continued work on all three projects in order to send improved proposals to Darren Nesbeth by the end of the day. Many professors and experts were also emailed to seek guidance, in particular for the Alzheimer's project which seems to be particularly difficult.<br />
</p><br />
<p class="minor_title">20th June</p><br />
<p class="body_text"><br />
Tom - Prepared a presentation to be given next week about iGEM to prospective UCL students to raise interest in the engineering faculty and also the iGEM competition. After this was complete, joined the rest of the group in research. Also performed wiki coding for the team page and notebook page.<br />
</p><br />
<p class="body_text"><br />
The group continued what was started yesterday: Rectifying the proposals, with both sent off at the end of the day once they were complete. A group meeting was held at the end of the day to gauge interest and vote for the most popular idea, followed by a social gathering.<br />
</p><br />
<p class="minor_title">21st June</p><br />
<p class="body_text"><br />
Tom - Continued wiki design, coding and content uploads.<br />
Alex - Continued to redraft the proposal for Alzheimer's<br />
StJohn - Continued to redraft the proposal for Cancer<br />
</p><br />
<p class="body_text"><br />
KC - Researched into other iGEM teams to colloborate with and initiated correspondence via email<br />
</p><br />
<p class="body_text"><br />
The team then discusses which project was favoured. It was fairly even but Alzheimer's was slightly more popular.<br />
</p><br />
<p class="minor_title">24th June</p><br />
<p class="body_text"><br />
Tom continued wiki design whilst the rest of the group performed research.<br />
</p><br />
<p class="body_text"><br />
Once this was complete, the group had a meeting with Yanika Borg and Philipp Boeing concerning the two project ideas. Philipp favoured the Alzheimer's project whilst Yanika was somewhat undecided. <br />
</p><br />
<p class="body_text"><br />
A vote was taken with Alzheimer's being the prefered project by the group as a whole once more, although consensus was not fully reached. The group agreed to decide on the project on Wednesday proceeding a meeting with Prof. Lazaros Lukas.<br />
</p><br />
<br />
<p class="minor_title">25th June</p><br />
<p class="body_text"><br />
The group continued with general research, and also went to the Wellcome trust to seek any extra information, although this was unfruitful.<br />
</p><br />
<br />
<p class="minor_title">27th June</p><br />
<p class="body_text"><br />
The group voted 29 -11 in favour of Alzheimer's after a meeting with Prof. Lazaro Lukas, who was helpful and seemed excited about the project. The group also met advisor Yanika Borg and she agreed with the choice. The group also scheduled lab safety training for next thursday.<br />
</p><br />
<br />
<p class="minor_title">28th June</p><br />
<p class="body_text"><br />
Tom presented to prospective students about the iGEM project for the day.<br />
</p><br />
<p class="body_text"><br />
Weiling, Alex, Andy & Catrin began to produce a 'stop motion' explanation of the Alzheimer's project.<br />
</p><br />
<p class="body_text"><br />
KC, Robin and StJohn discussed lab protocols and also modelling ideas.<br />
</p><br />
<p class="minor_title">29th June</p><br />
<p class="body_text"><br />
Tom, Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi & StJohn – Continued work on the proposals for the meeting with Dr. Nesbeth on Thursday.<br />
</p><br />
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
</p> <br />
</div><br />
<br />
<p class="major_title">June</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">5th June</p><br />
<p class="body_text"><br />
Group discussion concerning the project idea to be carried forward - favouring the 'Anti cancer project'. Roles were then assigned to team members present for intial research roles for the week:<br />
</p><br />
<p class="body_text"><br />
Cancer research roles:<br />
</p><br />
<p class="body_text"><br />
1. Ruxi Comisel - Proteins upregulated in cancer of the intestines. Specifically in the outer epithelial cell (enterocytes) – in microvilli. Also, what actually is... gut cancer? A general overview would be useful…<br />
</p><br />
<p class="body_text"><br />
2. Khaicheng Kiew - Our chassis (bearing in mind that we will also build it in E. coli as a backup). We need to think what would make a good chassis in our case (ie. naturally found in the gut in an obvious one), and how well does the chassis fit.<br />
</p><br />
<p class="body_text"><br />
3. Alex Bates - What will the killing mechanism be? A broad overview of cancer treatments is required, specifically detailing how a bacterium can administer the treatment.<br />
</p><br />
<p class="body_text"><br />
Considerations:<br />
</p><br />
<p class="body_text"><br />
a. The bacteria may secrete a toxin etc – how will we ensure that it doesn’t simply diffuse through the gut? <br />
b. If it is a toxin, what sort of biosynthetic pathway is required?<br />
c. Does the bacteria trigger apoptosis in the cancer cells (ie. an intracellular killing mechanism)? How can this be done from an extracellular bacterium? Perhaps beta-arrestin?<br />
d. Are there any treatments which we can take advantage of specifically because we are using bacteria? <br />
e. For example, a protein which creates holes in the cancer cells? Does using a bacterium open up the possibility of using a different cure that currently isn’t in use because we cannot target it to cancer cells – could the use of bacteria allow this?<br />
</p><br />
<p class="body_text"><br />
4. Weiling Yuan - Targeting – do we use antibodies? What previous projects have used bacteria expressing antibodies? Are there any other ways of doing this? Perhaps the latching and initiation mechanisms can be incorporated into one protein?<br />
</p><br />
<p class="body_text"><br />
5. StJohn Townsend - Initiation – mechanoreceptor activated upon latching? What other ways are there of doing this?<br />
</p><br />
<p class="body_text"><br />
6. Tom Johnson - Past iGEM projects which we could incorporate into our own: Cancer projects, Gut projects, Protein engineering, Antibodies expressed in bacteria etc.<br />
</p><br />
<br />
<p class="minor_title">7th June</p><br />
<p class="body_text"><br />
The team discusses findings from the initial research - further agreement that the 'Anti Cancer' project seemed to be the best idea, preparation of 'project sheets' to be sent to Dr. Darren Nesbeth for review and subsequent meetings.<br />
</p><br />
<p class="minor_title">11th June</p><br />
<p class="body_text"><br />
looked a bit at the possible chassis species: salmonella, clostridium, helicobacter, E. coli. according to the tissue type/cancer type we shall decide which works with which. We start with E. coli in the lab.<br />
</p><br />
<p class="body_text"><br />
We considered a pro-drug approach - bacterially directed enzyme pro-drug therapy which suggests that we may establish a transformed bacterial population with an enzyme capable to activate an ingested prodrug. This pro-drug would be connected to an antibody (possibly part of the tail) and would also have linking consensus sequence targeted by the enzyme produced locally by our bacteria.<br />
</p><br />
<p class="body_text"><br />
From this above point Alex distinguished 2 scenarios built on the circuit sketch that he and Laia posted a while ago. These would be:<br />
</p><br />
<p class="body_text"><br />
1) Kill unit produces tailed protein pro-drug (possibly tailed perforin) and signaling molecule, A. When A reaches a threshold amount, perforin and a protease to remove the confounding tail is produced, bacteria lyses and activated pro-drug acts on surrounding cells.<br />
</p><br />
<p class="body_text"><br />
2) No protease is produced, because the tail can be cleaved off by matrix metalloproteases.<br />
</p><br />
<p class="body_text"><br />
Goals for the end of this week: <br />
</p><br />
<p class="body_text"><br />
- Alex, Andy and Weiling continue investigating possible candidates to fill in the parts for the scenarios<br />
</p><br />
<p class="body_text"><br />
-Tom, KC and Ruxi make sure we have everything set up to start the work in the lab: protocol, parts etc.<br />
</p><br />
<p class="minor_title">12th June</p><br />
<p class="body_text"><br />
Ruxi and Tom went through a general cloning protocol but then realised that the best way to prepare for the lab is to get familiarised with the iGEM distribution kits. We discovered that we are given almost everything we need in order to get it right.<br />
</p><br />
<p class="body_text"><br />
Alex filled in the form with our proposal requested by Darren - we have the sequences and details of potential new biobricks. <br />
</p><br />
<p class="body_text"><br />
We formulated a new proposal regarding the Alzheimer’s disease amyloid plaque degradation.<br />
</p><br />
<p class="body_text"><br />
Andy searched potential cancer killer molecules:<br />
</p><br />
<p class="body_text"><br />
- CD95 - Fas agonist (http://www.nature.com/cdd/journal/v14/n4/full/4402051a.html)<br />
- Tumor Necrosis Factor, Histamine - induces inflammation<br />
- HAMLET (human a-lactalbumin) - induces apoptosis <br />
- endostatin, thrombospondin - reduce cancer growth<br />
</p><br />
<p class="body_text"><br />
Weiling looked at potential promotors: <br />
</p><br />
<p class="body_text"><br />
- RacA (based on increased DNA damaged due to radiation) to start the killing cascade and CD95 as a potential killer molecule<br />
- Lux pR promotor<br />
- Lld promoter<br />
- Vgb promotor <br />
- HIP-1<br />
</p><br />
<p class="body_text"><br />
(about gastric Oxygen levels: http://www.biomedcentral.com/1471-2180/11/96) <br />
</p><br />
<p class="body_text"><br />
For promoter 1 (switches on the pro-drug and signaling molecule transcription), a very <br />
good candidate is HIP 1 promoter - hypoxia-inducible promoter which drives reporter gene expression under both acute and chronic hypoxia. It was <a href=" http://www.landesbioscience.com/journals/cbt/article/2951/mengesha5-9.pdf" target="_blank">developed in attenuated Salmonella species</a>. <br />
</p><br />
<p class="body_text"><br />
We need to register this part!<br />
</p><br />
<br />
<p class="minor_title">13th June</p><br />
<p class="body_text"><br />
Alex sent the 3 main project proposals to Dr. Darren Nesbeth for review.<br />
</p><br />
<p class="body_text"><br />
Tom and Andy edited the wiki page adding various sections and elaborating on previously created pages.<br />
</p><br />
<p class="body_text"><br />
Weiling researched on killing mechanisms being able to target hypoxic regions of solid tumors and promoters in hypoxia environments.<br />
</p><br />
<p class="body_text"><br />
Catrin - General project research<br />
</p><br />
<p class="body_text"><br />
Ruxi - Further researched the potential promoters esp HIP 1 and the Fas regulated programmed apoptosis.<br />
</p><br />
<p class="body_text"><br />
We attended a Synthetic Biology talk by Neil Dixon, University of Manchester (Tom and Andy).<br />
</p><br />
<p class="body_text"><br />
Had a general meeting for discussion of what has been accomplished so far, and the subsequent actions, which are to be undertaken by team members. Further documents were also submitted to Dr. Darren Nesbeth concerning 'team roles'. The team then began to do individual research or other activity:<br />
</p><br />
<p class="body_text"><br />
Tom and Robin - Edited the iGEM wiki, added team information and removed the unnecessary tutorial information, replacing it with more useful information and streamlining the whole interface.<br />
</p><br />
<p class="body_text"><br />
Weiling and Alex - Further development of circuit ideas, taking inspiration from previous iGEM ideas as well as further research into the CD95L molecule.<br />
</p><br />
<p class="body_text"><br />
Ruxi and Catrin - Research into latching molecules for a bacteria to tumour interface to increase target specificity. Idea encounted from Hong Kong 2012 where Colon Cancer was targeted.<br />
</p><br />
<br />
<p class="minor_title">14th June</p><br />
<p class="body_text"><br />
Tom - Website design for: Main Page, UCL information, Team based pages and Notebook pages<br />
</p><br />
<p class="body_text"><br />
Robin - Coding in HTML for website<br />
</p><br />
<p class="body_text"><br />
Ruxi, Catrin, Weiling - Further investigation of Hong Kong 2010 to see what parts may be improved or of use to the project, these were: a blue light activated promoter, how can the quorum sensing and CagA be exploited, a negative regulatory system for drug secretion.<br />
</p><br />
<p class="body_text"><br />
Alex - searched for potential bacterial receptor to be modified in order to be a good target for something else in the environment/cancer cell surface.<br />
</p><br />
<p class="minor_title">17th June</p><br />
<p class="body_text"><br />
The group had a meeting to discuss what had been achieved so far and what needed to be done today. <br />
</p><br />
<p class="body_text"><br />
Tom - Continued on website design and wrote several pieces concerning UCL to be used on the website when it goes live.<br />
</p><br />
<p class="body_text"><br />
Robin - Continued on website coding.<br />
</p><br />
<p class="body_text"><br />
Weiling & Catrin - Researched for project sponsors and potential contacts.<br />
</p><br />
<p class="body_text"><br />
Alex, Ruxi, StJohn & Andy - Continued research into the project ideas.<br />
</p><br />
<p class="minor_title">18th June</p><br />
<p class="body_text"><br />
The group met with advisors Darren Nesbeth and Philipp Boeing to discuss the three project suggestions. The 'Neural Network' proposal was effectively ruled out due to the high risk and low probablility of project success in terms of medals.<br />
</p><br />
<p class="body_text"><br />
The anti-cancer project was previously the favoured idea, but after extensive review ,the Alzheimers project gained favour due to being relatively new (and hence exciting) to iGEM compared to a cancer project, which has been done several times already at iGEM. No final decision has been made however, work has continued on researching both projects. The wiki is also still being worked on.<br />
</p><br />
<p class="body_text"><br />
The team also had a social gathering: pizza for lunch.<br />
</p><br />
<p class="minor_title">19th June</p><br />
<p class="body_text"><br />
The group continued work on all three projects in order to send improved proposals to Darren Nesbeth by the end of the day. Many professors and experts were also emailed to seek guidance, in particular for the Alzheimer's project which seems to be particularly difficult.<br />
</p><br />
<p class="minor_title">20th June</p><br />
<p class="body_text"><br />
Tom - Prepared a presentation to be given next week about iGEM to prospective UCL students to raise interest in the engineering faculty and also the iGEM competition. After this was complete, joined the rest of the group in research. Also performed wiki coding for the team page and notebook page.<br />
</p><br />
<p class="body_text"><br />
The group continued what was started yesterday: Rectifying the proposals, with both sent off at the end of the day once they were complete. A group meeting was held at the end of the day to gauge interest and vote for the most popular idea, followed by a social gathering.<br />
</p><br />
<p class="minor_title">21st June</p><br />
<p class="body_text"><br />
Tom - Continued wiki design, coding and content uploads.<br />
Alex - Continued to redraft the proposal for Alzheimer's<br />
StJohn - Continued to redraft the proposal for Cancer<br />
</p><br />
<p class="body_text"><br />
KC - Researched into other iGEM teams to colloborate with and initiated correspondence via email<br />
</p><br />
<p class="body_text"><br />
The team then discusses which project was favoured. It was fairly even but Alzheimer's was slightly more popular.<br />
</p><br />
<p class="minor_title">24th June</p><br />
<p class="body_text"><br />
Tom continued wiki design whilst the rest of the group performed research.<br />
</p><br />
<p class="body_text"><br />
Once this was complete, the group had a meeting with Yanika Borg and Philipp Boeing concerning the two project ideas. Philipp favoured the Alzheimer's project whilst Yanika was somewhat undecided. <br />
</p><br />
<p class="body_text"><br />
A vote was taken with Alzheimer's being the prefered project by the group as a whole once more, although consensus was not fully reached. The group agreed to decide on the project on Wednesday proceeding a meeting with Prof. Lazaros Lukas.<br />
</p><br />
<br />
<p class="minor_title">25th June</p><br />
<p class="body_text"><br />
The group continued with general research, and also went to the Wellcome trust to seek any extra information, although this was unfruitful.<br />
</p><br />
<br />
<p class="minor_title">27th June</p><br />
<p class="body_text"><br />
The group voted 29 -11 in favour of Alzheimer's after a meeting with Prof. Lazaro Lukas, who was helpful and seemed excited about the project. The group also met advisor Yanika Borg and she agreed with the choice. The group also scheduled lab safety training for next thursday.<br />
</p><br />
<br />
<p class="minor_title">28th June</p><br />
<p class="body_text"><br />
Tom presented to prospective students about the iGEM project for the day.<br />
</p><br />
<p class="body_text"><br />
Weiling, Alex, Andy & Catrin began to produce a 'stop motion' explanation of the Alzheimer's project.<br />
</p><br />
<p class="body_text"><br />
KC, Robin and StJohn discussed lab protocols and also modelling ideas.<br />
</p><br />
<p class="minor_title">29th June</p><br />
<p class="body_text"><br />
Tom, Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi & StJohn – Continued work on the proposals for the meeting with Dr. Nesbeth on Thursday.<br />
</p><br />
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<p class="body_text"> <a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a> <br />
</p> <br />
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<p class="body_text"><b>Bacterial Labs<b></p><br />
<p class="body_text"><b>Monday 30th September</b></p><br />
</div><br />
<br />
<p class="major_title">October</p><br />
<br />
<div class="full_row"> <br />
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</div><br />
<p class="body_text"><b>Monday 30th September</b></p><br />
<p class="body_text">Ligated CMV with MMP9 and transformed into Top10 cells. These were subsequently plated on <a href="https://2013.igem.org/Team:UCL/Project/Protocols">chloramphenicol plates</a>.<br />
<br />
<p class="body_text"><b>Tuesday 1st October</b></p><br />
<p class="body_text">Inoculated colonies from plates from September 30th.</p><br />
<br />
<p class="body_text"><b>Wednesday 2nd October</b></p><br />
<p class="body_text"> Performed miniprep and generated <a href="https://2013.igem.org/Team:UCL/Project/Protocols">glycerol stock</a> from incoluation on Oct 1st. </p><br />
<p class="body_text"> Proceeded with analytical digest and gel</p><br />
<p class="body_text"> Transformed CMV+MMP9 plasmid from Sinobiological into Top10 cells</p><br />
<br />
<p class="body_text"><b>Thursday 3nd October</b></p><br />
<p class="body_text"> Religated CMV with MMP9 and continued with transformation into Top10 cells. </p><br />
<p class="body_text"> Inoculated from plates of plasmid from Sinobiological</p><br />
<br />
<p class="body_text"><b>Friday 4th October</b></p><br />
<p class="body_text"> Performed <a href="https://2013.igem.org/Team:UCL/Project/Protocols">miniprep</a>, generation of glycerol stock on inoculation from Oct 3rd</p><br />
<p class="body_text"> Continued with analytical digest and <a href="https://2013.igem.org/Team:UCL/Project/Protocols">gel</a></p><br />
<br />
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<p class="body_text"><br />
<b>Mammalian Labs</b><br />
</p><br />
<br />
<p class="body_text"><b>Monday 30th September<b/></p><br />
<br />
<p class="body_text"><b>Tuesday 1st October</b></p><br />
<br />
<p class="body_text"><b>Wednesday 2nd October</b></p><br />
<br />
<p class="body_text"><b>Thursday 3nd October</b></p><br />
<br />
<p class="body_text"><b>Friday 4th October</b></p><br />
<br />
</div><br />
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<!-- END CONTENT ------------------------------------------------------------------------------------------------------><br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/Project/DevelopmentsTeam:UCL/Project/Developments2013-10-05T02:48:46Z<p>Naruto: </p>
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<p class="major_title">OTHER CIRCUIT COMPONENTS</p><br />
<p class="minor_title">Avoiding Inflammation And Supporting Neurons</p><br />
<p class="body_text"><br />
Unfortunately, we did not have time to attempt to create all the parts envisioned in our original potential circuit. However, we believe that they are theoretically significant, and so here we explain what more could be done to improve this project of ours, as we work on these improvements after the ‘WikiFreeze’ for the Regional Jamboree of the iGEM competition.<br />
</p><br />
<p class="body_text"><br />
The strength of our <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">system</a> is that the <a href="https://2013.igem.org/Team:UCL/Background/Microglia" target="_blank">microglial chassis</a> already detect and engage other microglia.<br />
</p><br />
<p class="body_text"><br />
This means that our systems can create proteins in situ to improve the <a href="https://2013.igem.org/Team:UCL/Background/Alzheimers" target="_blank">Alzheimer’s disease (AD)</a> state. However, amyloid proteases such as <a href="https://2013.igem.org/Team:UCL/Project/Degradation" target="_blank">MMP-9</a> would only have a positive impact on the pathology if the <a href="https://2013.igem.org/Team:UCL/Background/Microglia" target="_blank">'Amyloid Hypothesis'</a> is correct, and there is some evidence to suggest that it may not be.<br />
</p><br />
<p class="body_text"><br />
It is thought that AD may be exacerbated into a neurodegenerative condition by the action of microglia themselves, the custodians of the brain. They can inflame the plaque area, and this damages neurons. Therefore, we propose producing a de-activating agent, such as vasoactive intestinal peptide (VIP), BioBrick with an <a href="https://2013.igem.org/Team:UCL/Project/Detection" target="_blank">oxidative stress promoter</a>. This means that our genetically engineered microglia (GEM) would activate when it detects a plaque and move towards that plaque. As a GEM approaches, oxidative stress increases so that once near the plaque the de-activating agent would return the GEM and wild-type microglia surrounding the plaque into their resting state, avoiding neuroinflammation. This would stop them from producing amyloid proteases such as neprilysin. However, our MMP-9 BioBrick can ensure that amyloid degradation continues (the positive action of microglia in AD) without inflammation (the negative action of microglia in AD). <br />
</p><br />
<p class="body_text"><br />
It is also thought that AD may initiate due to cell-cycle re-entry on account of a disbalance in <a href="https://2013.igem.org/Team:UCL/Background/Microglia" target="_blank">neurotrophin signalling</a>. Brain-derived neurotrophic factor (BDNF) is a signal that sustains neurons. If expressed by engineered microglia at plaque localities it could support dying neurons and stop other neurons progressing into an AD state.<br />
</p><br />
<br />
</div><br />
<br />
<div class="gap"></div><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="part" style="background-image:url('https://static.igem.org/mediawiki/2013/9/93/VIP_png2.png');"></div><br />
<div class="description"><br />
<p class="minor_title">Vasoactive Intestinal Peptide</p><br />
<p class="body_text"><br />
This is a 28 amino acid long secreted neuropeptide. It stimulates heart contractility, vasodilation and glycogenolysis, muscle relaxation in the gastrointestinal tract and lowers arterial blood pressure and relaxes the smooth muscle of trachea, stomach and gall bladder <a href="http://www.ncbi.nlm.nih.gov/pubmed/6129023" target="_blank">(Fahrenkrug and Emson 1982)</a>. In the brain, it plays a key role in circadian rhythm control in the hypothalamus. It has been suggested as a therapeutic as it is also known to have a neuroprotective role. Studies have shown that VIP prevents activated microglia inducing the neuroinflammatory conditions that engender and may drive neurodegeneration <a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Neuroprotective+effect+of+vasoactive+intestinal+peptide+(VIP)+in+a+mouse+model+of+Parkinson%E2%80%99s" target="_blank">(Delgado and Ganea March 2003)</a><a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Vasoactive+intestinal+peptide+prevents+activated+microglia+neurodegeneration+under+inflammatory+conditions%3Ainduced" target="_blank">(Delgado and Ganea Jan 2003)</a>.<br />
</p><br />
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<div class="row_small"><br />
<div class="part" style="background-image:url('https://static.igem.org/mediawiki/2013/8/8a/Coolstuffbdnf.png');"></div><div class="description"><br />
<p class="minor_title">Brain-Derived Neurotrophic Factor</p><br />
<p class="body_text"><br />
This is a secreted, 252 amino acid long neurotrophic protein. it is made in the endoplasmic reticulum and secreted from dense core vesicle. it’s assortment into these vesicles is aided by the enzyme carboxypeptidase E. Decreased levels of BDNF have been associated with Alzheimer’s, depression and epilepsy - and so this would be a very useful medical BioBrick. It mainly mediates it effects through membrane protein TrkB. BDNF promotes the neurons’ survival, growth, differentiation and maintenance. It is active at the communicative connections between neurons (synapses), where it helps sustain the synapse and facilitate changes in that synapse’s strength over time, in response to experience. This is called ‘synaptic plasticity’, and is believed to play a key role in learning and memory. A decrease in BDNF could engender cell cycle re-entry <a href="http://www.ncbi.nlm.nih.gov/pubmed/20436277" target="_blank">(Frade & Lopez-Sanchez 2010)</a> and the senile plaques in AD can disrupt synapses, meaning that they receive less BDNF.<br />
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</html></div>Narutohttp://2013.igem.org/File:Coolstuffbdnf.pngFile:Coolstuffbdnf.png2013-10-05T02:48:20Z<p>Naruto: </p>
<hr />
<div></div>Narutohttp://2013.igem.org/Team:UCL/Project/DevelopmentsTeam:UCL/Project/Developments2013-10-05T02:46:17Z<p>Naruto: </p>
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<p class="major_title">OTHER CIRCUIT COMPONENTS</p><br />
<p class="minor_title">Avoiding Inflammation And Supporting Neurons</p><br />
<p class="body_text"><br />
Unfortunately, we did not have time to attempt to create all the parts envisioned in our original potential circuit. However, we believe that they are theoretically significant, and so here we explain what more could be done to improve this project of ours, as we work on these improvements after the ‘WikiFreeze’ for the Regional Jamboree of the iGEM competition.<br />
</p><br />
<p class="body_text"><br />
The strength of our <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">system</a> is that the <a href="https://2013.igem.org/Team:UCL/Background/Microglia" target="_blank">microglial chassis</a> already detect and engage other microglia.<br />
</p><br />
<p class="body_text"><br />
This means that our systems can create proteins in situ to improve the <a href="https://2013.igem.org/Team:UCL/Background/Alzheimers" target="_blank">Alzheimer’s disease (AD)</a> state. However, amyloid proteases such as <a href="https://2013.igem.org/Team:UCL/Project/Degradation" target="_blank">MMP-9</a> would only have a positive impact on the pathology if the <a href="https://2013.igem.org/Team:UCL/Background/Microglia" target="_blank">'Amyloid Hypothesis'</a> is correct, and there is some evidence to suggest that it may not be.<br />
</p><br />
<p class="body_text"><br />
It is thought that AD may be exacerbated into a neurodegenerative condition by the action of microglia themselves, the custodians of the brain. They can inflame the plaque area, and this damages neurons. Therefore, we propose producing a de-activating agent, such as vasoactive intestinal peptide (VIP), BioBrick with an <a href="https://2013.igem.org/Team:UCL/Project/Detection" target="_blank">oxidative stress promoter</a>. This means that our genetically engineered microglia (GEM) would activate when it detects a plaque and move towards that plaque. As a GEM approaches, oxidative stress increases so that once near the plaque the de-activating agent would return the GEM and wild-type microglia surrounding the plaque into their resting state, avoiding neuroinflammation. This would stop them from producing amyloid proteases such as neprilysin. However, our MMP-9 BioBrick can ensure that amyloid degradation continues (the positive action of microglia in AD) without inflammation (the negative action of microglia in AD). <br />
</p><br />
<p class="body_text"><br />
It is also thought that AD may initiate due to cell-cycle re-entry on account of a disbalance in <a href="https://2013.igem.org/Team:UCL/Background/Microglia" target="_blank">neurotrophin signalling</a>. Brain-derived neurotrophic factor (BDNF) is a signal that sustains neurons. If expressed by engineered microglia at plaque localities it could support dying neurons and stop other neurons progressing into an AD state.<br />
</p><br />
<br />
</div><br />
<br />
<div class="gap"></div><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="part" style="background-image:url('https://static.igem.org/mediawiki/2013/9/93/VIP_png2.png');"></div><br />
<div class="description"><br />
<p class="minor_title">Vasoactive Intestinal Peptide</p><br />
<p class="body_text"><br />
This is a 28 amino acid long secreted neuropeptide. It stimulates heart contractility, vasodilation and glycogenolysis, muscle relaxation in the gastrointestinal tract and lowers arterial blood pressure and relaxes the smooth muscle of trachea, stomach and gall bladder <a href="http://www.ncbi.nlm.nih.gov/pubmed/6129023" target="_blank">(Fahrenkrug and Emson 1982)</a>. In the brain, it plays a key role in circadian rhythm control in the hypothalamus. It has been suggested as a therapeutic as it is also known to have a neuroprotective role. Studies have shown that VIP prevents activated microglia inducing the neuroinflammatory conditions that engender and may drive neurodegeneration <a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Neuroprotective+effect+of+vasoactive+intestinal+peptide+(VIP)+in+a+mouse+model+of+Parkinson%E2%80%99s" target="_blank">(Delgado and Ganea March 2003)</a><a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Vasoactive+intestinal+peptide+prevents+activated+microglia+neurodegeneration+under+inflammatory+conditions%3Ainduced" target="_blank">(Delgado and Ganea Jan 2003)</a>.<br />
</p><br />
</div><br />
<br />
</div><br />
<br />
<div class="gap"></div><br />
<div class="row_small"><br />
<div class="part" style="background-image:url('https://2013.igem.org/File:BDNF_pndfsdfg2.png');"></div><div class="description"><br />
<p class="minor_title">Brain-Derived Neurotrophic Factor</p><br />
<p class="body_text"><br />
This is a secreted, 252 amino acid long neurotrophic protein. it is made in the endoplasmic reticulum and secreted from dense core vesicle. it’s assortment into these vesicles is aided by the enzyme carboxypeptidase E. Decreased levels of BDNF have been associated with Alzheimer’s, depression and epilepsy - and so this would be a very useful medical BioBrick. It mainly mediates it effects through membrane protein TrkB. BDNF promotes the neurons’ survival, growth, differentiation and maintenance. It is active at the communicative connections between neurons (synapses), where it helps sustain the synapse and facilitate changes in that synapse’s strength over time, in response to experience. This is called ‘synaptic plasticity’, and is believed to play a key role in learning and memory. A decrease in BDNF could engender cell cycle re-entry <a href="http://www.ncbi.nlm.nih.gov/pubmed/20436277" target="_blank">(Frade & Lopez-Sanchez 2010)</a> and the senile plaques in AD can disrupt synapses, meaning that they receive less BDNF.<br />
</div><br />
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</html></div>Narutohttp://2013.igem.org/File:VIP_png2.pngFile:VIP png2.png2013-10-05T02:43:31Z<p>Naruto: uploaded a new version of &quot;File:VIP png2.png&quot;</p>
<hr />
<div></div>Narutohttp://2013.igem.org/Team:UCL/Project/SafetyTeam:UCL/Project/Safety2013-10-05T02:41:24Z<p>Naruto: </p>
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<br />
<p class="major_title">KEEPING SAFE</p><br />
<p class="minor_title">It's Just Good Lab Practice</p><br />
<p class="body_text"><br />
The health and safety considerations of the project are vital for the well-being of the project members and the laboratory environment. Some of the key considerations to be made involve handling bio-hazardous materials, process chemicals and mechanical operations; requiring planning and awareness in order prevent hazards from occurring, as well as appropriate plans of action in order to deal with any situation that may occur.<br />
</p><br />
<p class="body_text"><br />
The Escherichia coli strain used is not considered pathogenic, and thus not of considerable risk to the environment or team members. Despite this, there must still be a level of Good Laboratory Practice (GLP) to reduce risk associated with performing experiments on a daily basis. As a minimum requirement, all members will wear some form of protective clothing, which generally consists of a lab coat and goggles, as well as single use gloves.<br />
</p><br />
<p class="body_text"><br />
For disposal of spent materials, any item should be isolated and sealed in a container which prevents physical contact with any of the facility. The material should then be removed from the room through the waste corridor and disposed of accordingly (chemical or steam treatment for example).<br />
</p><br />
<p class="body_text"><br />
Continual monitoring and safety reviews of the facility is required to maintain GLP and the condition of the plant. By undertaking continual assessments of laboratory safety, the risk of a hazard occurring in the plant can be reduced. Training of team members in safe operation in general safety protocols is essential to reduce risk; so safety training and education in good laboratory practise was undertaken by all team members before experiments could begin.<br />
</p><br />
<p class="body_text"><br />
The main principle behind performing experiments in a safe environment is to minimise risk. Risk is considered as the amalgamation of the potential damage that a hazard could cause, combined with the likelihood of said hazard occurring. The damage a hazard could cause is normally set by the equipment present in the laboratory, whilst laboratory team members can often decrease risk by decreasing the likelihood of a hazard occurring. Below the three main categories (Microbiological, Chemical and Mechanical) are defined with tables showing which hazard fall into said category, and how the risk is minimised in each case.<br />
</p><br />
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<br />
<p class="minor_title">Microbiological Hazards</p><br />
<p class="body_text"><br />
The key focus of microbiological hazard control concerns the Escherichia coli expression system being used. The majority of Escherichia coli strains are categorised into Bio-safety levels 1 or 2, with non-pathogenic strains placed into level 1 under World Health Organisation classifications. Therefore the Escherichia coli strain used in the facility is considered to be Bio-safety level one, which often requires less stringent safety standards. The genetically engineered strains also may require more safety regulation requirements than wild type micro-organisms; however advances in synthetic biology are allowing more control over the expression system in terms of pathogenicity and also the ability to express suicide genes, which can prevent any live Escherichia coli from surviving outside of the facility environment. Regardless, the facility must ensure and validate that all of the Escherichia coli is killed and effectively disposed of. Pressure systems and directed ventilation are in place to prevent escape of any strains, although this may lead to possible contaminants entering the halls, which dictates that the live Escherichia coli must be sealed in containers or vessels at all times where possible in order to prevent infection. The use of mammalian systems in the project also present a degree of hazard, although the associated risk is lower when compared to using bacteria.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Escape of Escherichia coli from lab</td><br />
<td>The strain(s) exit the laboratory environment, thus allowing the possibility of contamination of exterior objects and/or persons.</td><br />
<td>Pressure of lab is lower than of corridor, thus causing airflow into the lab as opposed of out, which minimises the chance of airborne or aerosol escape of E. coli. Laboratory coats are worn over clothes which do not leave the lab, thus reducing possibility of a team member inadvertently acting as a carrier out of the lab.</td><br />
</tr><br />
<tr><br />
<td>Contamination of E coli/ HeLa/ Microglia</td><br />
<td>Contamination of the desired strain leads to competition between several strains of bacteria, resulting in inaccurate stocks and therefore unusable data. Note that bacterial and mammalian experiments are undertaken in separate parts of the facility.</td><br />
<td>Exposure of strain to open air is minimised, which is standard practise for any container that may be involved with the desired E. coli strain. Single use gloves are used to minimise possible contamination when experiments are performed. Ethanol is applied on work surfaces before and after experimental procedures in order to minimise contamination on work surfaces etc.</td><br />
</tr><br />
<tr><br />
<td>Contamination of team members</td><br />
<td>Illness could ensue from working in the laboratory environment.</td><br />
<td>Team members are not advised to work when feeling unwell. Gloves, lab coat and goggles are also worn to minimise contact with organisms. Personal hygiene standards are advised to be upheld in particular for laboratory workers.</td><br />
</tr><br />
<tr><br />
<td>HeLa Cells</td><br />
<td>Level 2 biosafety class cells</td><br />
<td>Cell experiments always performed in a sterile environment and a fume hood, with a supervisor providing assistance to the experiment when necessary</td><br />
</tr><br />
<tr><br />
<td>Microglia cells</td><br />
<td>Level 2 biosafety class cells</td><br />
<td>Dr. Darren Nesbeth will perform the neccesary assays in place of iGEm members due to the safety regulations</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Chemical Hazards</p><br />
<p class="body_text"><br />
When performing experiments, it may be necessary to use dangerous chemicals during certain processes. Whilst using hazardous chemicals is avoided where necessary, in some cases it is required, so in each case where this occurs the procedure must be executed in a fashion which is as safe as reasonably possible. Therefore standard protocols such as wearing protective clothing may be added to by performing experiments in fume hoods or other such devices to minimise any chance of contact via evaporation etc.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Ethidium Bromide (EtBr)</td><br />
<td>Mutagenic.<br />
Minor toxicity issue</td><br />
<td>All operations using EtBr are carried out in a fume cupboard - separate equipment (pipettes etc.) are used only in the fume cupboard specifically for EtBr.<br />
Gloves and long sleeved protective gowns MUST be worn, disposal of items in contact with EtBr are disposed of separately to other wastes.<br />
Wash hands after gloves are removed.</td><br />
</tr><br />
<tr><br />
<td>High concentration Ethanol (EtOH) and other alcohols</td><br />
<td>Flammable.</td><br />
<td>Securely stored (glass container) in a separate cupboard in volumes less than 2 Litres.</td><br />
</tr><br />
<tr><br />
<td>Dimethyl sulfoxide & Zeocin</td><br />
<td>Irritant to operator if contact with skin is made</td><br />
<td>Chemical handled with extreme caution, with particular attention to no skin exposed when performing an experiment, particularly goggles must be worn</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Mechanical Hazards</p><br />
<p class="body_text"><br />
For this project, mechanical hazards pose the lowest probability of occurring out of the three and thus may be considered as the lowest risk group. However, there are hazards present which must be minimised. The predominant issue here is the use of the desktop centrifuge system, which poses considerable danger if a blowout occurs.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Centrifuge</td><br />
<td>Centrifuge mechanical failure (eg. of rotor) can cause severe damage to the machine, and possibly personnel in the nearby vicinity.</td><br />
<td><br />
Regular inspections and maintenance of the machine is required (performed by external engineers). <br />
Machine not to be used if in non optimal condition.<br />
Centrifuge must be balanced to minimise chance of rotor failure.<br />
Centrifuge must be properly closed before use (required for the machine to work.)<br />
Attention must be paid to ensure that nothing is spilled in the centrifuge bowl, or a build up of dust or aerosol should also be avoided.</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Concluding Comments</p><br />
<p class="body_text"><br />
The safety of the team members, produced material and environment are key aspects to the smooth running of the project, and it must be ensured that adequate measures are in place to provide protection and ensuring that guidelines can be followed proceeding mandatory safety training. Current Good Laboratory Practice must also be adhered to at all times. By following guidelines and acting in an appropriate manner whilst in the laboratory, the general risk in terms of micro-biological, chemical and mechanical aspects will be reduced to a level which is as low as reasonably possible for all project members involved.<br />
</p><br />
<p class="body_text"><br />
Due to the relatively small scale of the experiments performed, the quantities used of material is not large, but still must be handled and respected properly at all times. Proper containment and labelling of materials in sealed containers is necessary, especially when of particular hazard such as live material or Ethidium Bromide. Flammability concerns are also present with alcohol and other flammable materials stored in the lab, although the volumes (generally less than 2L) are not considerably large enough, although should still be used and handled properly when used, with fire extinguishers available and also emergency exits present if required. Protective clothing also should be worn where appropriate to minimise the possibility of skin contact, with wash and first aid station available in key areas where allowable due to the area classification.<br />
</p><br />
<p class="body_text"><br />
The mechanical safety predominantly concerns the proper maintenance of the centrifuge system, which can cause a considerable risk if a failure occurs. Regular inspections should be made, with the centrifuge often restricted access when it was considered unfit for use. This room is also placed away from mammalian and bacterial processing centres. Prevention methods should also be in place such as emergency shut-off procedures.<br />
</p><br />
<p class="body_text"><br />
By following all guidelines set out in safety training and following supervisor instructions, the project may be performed in a safe and effective manner. Continual training and inspection of the facility will aid in ensuring safety levels are maintained, with review protocols in place to investigate any faults. Team members have been trained in general safety, with supervisors having first aid experience and training. These considerations combined will ensure the safety risk to the personnel; product and environment is as low as reasonably possible. For further reading, the Institute of Chemical Engineering (IChemE) has relevant information, as well as other safety guidance documents relating to the relevant country where the university is located.<br />
</p><br />
<br />
<p class="minor_title">Safety Forms</p><br />
<br />
<p class="body_text"><br />
<a href="https://static.igem.org/mediawiki/2013/2/20/IGEM_2013_Basic_Safety_Form.pdf" target="_blank">Basic safety form</a> & <a href="https://static.igem.org/mediawiki/2013/7/71/IGEM_Biosafety_Form_Part_2.pdf" target="_blank">Biosafety form</a><br />
</p><br />
<br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/Project/SafetyTeam:UCL/Project/Safety2013-10-05T02:40:46Z<p>Naruto: </p>
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<br />
<p class="major_title">KEEPING SAFE</p><br />
<p class="minor_title">It's Just Good Lab Practice</p><br />
<p class="body_text"><br />
The health and safety considerations of the project are vital for the well-being of the project members and the laboratory environment. Some of the key considerations to be made involve handling bio-hazardous materials, process chemicals and mechanical operations; requiring planning and awareness in order prevent hazards from occurring, as well as appropriate plans of action in order to deal with any situation that may occur.<br />
</p><br />
<p class="body_text"><br />
The Escherichia coli strain used is not considered pathogenic, and thus not of considerable risk to the environment or team members. Despite this, there must still be a level of Good Laboratory Practice (GLP) to reduce risk associated with performing experiments on a daily basis. As a minimum requirement, all members will wear some form of protective clothing, which generally consists of a lab coat and goggles, as well as single use gloves.<br />
</p><br />
<p class="body_text"><br />
For disposal of spent materials, any item should be isolated and sealed in a container which prevents physical contact with any of the facility. The material should then be removed from the room through the waste corridor and disposed of accordingly (chemical or steam treatment for example).<br />
</p><br />
<p class="body_text"><br />
Continual monitoring and safety reviews of the facility is required to maintain GLP and the condition of the plant. By undertaking continual assessments of laboratory safety, the risk of a hazard occurring in the plant can be reduced. Training of team members in safe operation in general safety protocols is essential to reduce risk; so safety training and education in good laboratory practise was undertaken by all team members before experiments could begin.<br />
</p><br />
<p class="body_text"><br />
The main principle behind performing experiments in a safe environment is to minimise risk. Risk is considered as the amalgamation of the potential damage that a hazard could cause, combined with the likelihood of said hazard occurring. The damage a hazard could cause is normally set by the equipment present in the laboratory, whilst laboratory team members can often decrease risk by decreasing the likelihood of a hazard occurring. Below the three main categories (Microbiological, Chemical and Mechanical) are defined with tables showing which hazard fall into said category, and how the risk is minimised in each case.<br />
</p><br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">Microbiological Hazards</p><br />
<p class="body_text"><br />
The key focus of microbiological hazard control concerns the Escherichia coli expression system being used. The majority of Escherichia coli strains are categorised into Bio-safety levels 1 or 2, with non-pathogenic strains placed into level 1 under World Health Organisation classifications. Therefore the Escherichia coli strain used in the facility is considered to be Bio-safety level one, which often requires less stringent safety standards. The genetically engineered strains also may require more safety regulation requirements than wild type micro-organisms; however advances in synthetic biology are allowing more control over the expression system in terms of pathogenicity and also the ability to express suicide genes, which can prevent any live Escherichia coli from surviving outside of the facility environment. Regardless, the facility must ensure and validate that all of the Escherichia coli is killed and effectively disposed of. Pressure systems and directed ventilation are in place to prevent escape of any strains, although this may lead to possible contaminants entering the halls, which dictates that the live Escherichia coli must be sealed in containers or vessels at all times where possible in order to prevent infection. The use of mammalian systems in the project also present a degree of hazard, although the associated risk is lower when compared to using bacteria.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Escape of Escherichia coli from lab</td><br />
<td>The strain(s) exit the laboratory environment, thus allowing the possibility of contamination of exterior objects and/or persons.</td><br />
<td>Pressure of lab is lower than of corridor, thus causing airflow into the lab as opposed of out, which minimises the chance of airborne or aerosol escape of E. coli. Laboratory coats are worn over clothes which do not leave the lab, thus reducing possibility of a team member inadvertently acting as a carrier out of the lab.</td><br />
</tr><br />
<tr><br />
<td>Contamination of E coli/ HeLa/ Microglia</td><br />
<td>Contamination of the desired strain leads to competition between several strains of bacteria, resulting in inaccurate stocks and therefore unusable data. Note that bacterial and mammalian experiments are undertaken in separate parts of the facility.</td><br />
<td>Exposure of strain to open air is minimised, which is standard practise for any container that may be involved with the desired E. coli strain. Single use gloves are used to minimise possible contamination when experiments are performed. Ethanol is applied on work surfaces before and after experimental procedures in order to minimise contamination on work surfaces etc.</td><br />
</tr><br />
<tr><br />
<td>Contamination of team members</td><br />
<td>Illness could ensue from working in the laboratory environment.</td><br />
<td>Team members are not advised to work when feeling unwell. Gloves, lab coat and goggles are also worn to minimise contact with organisms. Personal hygiene standards are advised to be upheld in particular for laboratory workers.</td><br />
</tr><br />
<tr><br />
<td>HeLa Cells</td><br />
<td>Level 2 biosafety class cells</td><br />
<td>Cell experiments always performed in a sterile environment and a fume hood, with a supervisor providing assistance to the experiment when necessary</td><br />
</tr><br />
<tr><br />
<td>Microglia cells</td><br />
<td>Level 2 biosafety class cells</td><br />
<td>Dr. Darren Nesbeth will perform the neccesary assays in place of iGEm members due to the safety regulations</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Chemical Hazards</p><br />
<p class="body_text"><br />
When performing experiments, it may be necessary to use dangerous chemicals during certain processes. Whilst using hazardous chemicals is avoided where necessary, in some cases it is required, so in each case where this occurs the procedure must be executed in a fashion which is as safe as reasonably possible. Therefore standard protocols such as wearing protective clothing may be added to by performing experiments in fume hoods or other such devices to minimise any chance of contact via evaporation etc.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Ethidium Bromide (EtBr)</td><br />
<td>Mutagenic.<br />
Minor toxicity issue</td><br />
<td>All operations using EtBr are carried out in a fume cupboard - separate equipment (pipettes etc.) are used only in the fume cupboard specifically for EtBr.<br />
Gloves and long sleeved protective gowns MUST be worn, disposal of items in contact with EtBr are disposed of separately to other wastes.<br />
Wash hands after gloves are removed.</td><br />
</tr><br />
<tr><br />
<td>High concentration Ethanol (EtOH) and other alcohols</td><br />
<td>Flammable.</td><br />
<td>Securely stored (glass container) in a separate cupboard in volumes less than 2 Litres.</td><br />
</tr><br />
<tr><br />
<td>Dimethyl sulfoxide & Zeocin</td><br />
<td>Irritant to operator if contact with skin is made</td><br />
<td>Chemical handled with extreme caution, with particular attention to no skin exposed when performing an experiment, particularly goggles must be worn</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Mechanical Hazards</p><br />
<p class="body_text"><br />
For this project, mechanical hazards pose the lowest probability of occurring out of the three and thus may be considered as the lowest risk group. However, there are hazards present which must be minimised. The predominant issue here is the use of the desktop centrifuge system, which poses considerable danger if a blowout occurs.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Centrifuge</td><br />
<td>Centrifuge mechanical failure (eg. of rotor) can cause severe damage to the machine, and possibly personnel in the nearby vicinity.</td><br />
<td><br />
Regular inspections and maintenance of the machine is required (performed by external engineers). <br />
Machine not to be used if in non optimal condition.<br />
Centrifuge must be balanced to minimise chance of rotor failure.<br />
Centrifuge must be properly closed before use (required for the machine to work.)<br />
Attention must be paid to ensure that nothing is spilled in the centrifuge bowl, or a build up of dust or aerosol should also be avoided.</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Concluding Comments</p><br />
<p class="body_text"><br />
The safety of the team members, produced material and environment are key aspects to the smooth running of the project, and it must be ensured that adequate measures are in place to provide protection and ensuring that guidelines can be followed proceeding mandatory safety training. Current Good Laboratory Practice must also be adhered to at all times. By following guidelines and acting in an appropriate manner whilst in the laboratory, the general risk in terms of micro-biological, chemical and mechanical aspects will be reduced to a level which is as low as reasonably possible for all project members involved.<br />
</p><br />
<p class="body_text"><br />
Due to the relatively small scale of the experiments performed, the quantities used of material is not large, but still must be handled and respected properly at all times. Proper containment and labelling of materials in sealed containers is necessary, especially when of particular hazard such as live material or Ethidium Bromide. Flammability concerns are also present with alcohol and other flammable materials stored in the lab, although the volumes (generally less than 2L) are not considerably large enough, although should still be used and handled properly when used, with fire extinguishers available and also emergency exits present if required. Protective clothing also should be worn where appropriate to minimise the possibility of skin contact, with wash and first aid station available in key areas where allowable due to the area classification.<br />
</p><br />
<p class="body_text"><br />
The mechanical safety predominantly concerns the proper maintenance of the centrifuge system, which can cause a considerable risk if a failure occurs. Regular inspections should be made, with the centrifuge often restricted access when it was considered unfit for use. This room is also placed away from mammalian and bacterial processing centres. Prevention methods should also be in place such as emergency shut-off procedures.<br />
</p><br />
<p class="body_text"><br />
By following all guidelines set out in safety training and following supervisor instructions, the project may be performed in a safe and effective manner. Continual training and inspection of the facility will aid in ensuring safety levels are maintained, with review protocols in place to investigate any faults. Team members have been trained in general safety, with supervisors having first aid experience and training. These considerations combined will ensure the safety risk to the personnel; product and environment is as low as reasonably possible. For further reading, the Institute of Chemical Engineering (IChemE) has relevant information, as well as other safety guidance documents relating to the relevant country where the university is located.<br />
</p><br />
<br />
<p class="body_text"><br />
<a href="https://static.igem.org/mediawiki/2013/2/20/IGEM_2013_Basic_Safety_Form.pdf" target="_blank">Basic safety form</a> & <a href="https://static.igem.org/mediawiki/2013/7/71/IGEM_Biosafety_Form_Part_2.pdf" target="_blank">Biosafety form</a><br />
</p><br />
<br />
</div><br />
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<br />
<p class="major_title">KEEPING SAFE</p><br />
<p class="minor_title">It's Just Good Lab Practice</p><br />
<p class="body_text"><br />
The health and safety considerations of the project are vital for the well-being of the project members and the laboratory environment. Some of the key considerations to be made involve handling bio-hazardous materials, process chemicals and mechanical operations; requiring planning and awareness in order prevent hazards from occurring, as well as appropriate plans of action in order to deal with any situation that may occur.<br />
</p><br />
<p class="body_text"><br />
The Escherichia coli strain used is not considered pathogenic, and thus not of considerable risk to the environment or team members. Despite this, there must still be a level of Good Laboratory Practice (GLP) to reduce risk associated with performing experiments on a daily basis. As a minimum requirement, all members will wear some form of protective clothing, which generally consists of a lab coat and goggles, as well as single use gloves.<br />
</p><br />
<p class="body_text"><br />
For disposal of spent materials, any item should be isolated and sealed in a container which prevents physical contact with any of the facility. The material should then be removed from the room through the waste corridor and disposed of accordingly (chemical or steam treatment for example).<br />
</p><br />
<p class="body_text"><br />
Continual monitoring and safety reviews of the facility is required to maintain GLP and the condition of the plant. By undertaking continual assessments of laboratory safety, the risk of a hazard occurring in the plant can be reduced. Training of team members in safe operation in general safety protocols is essential to reduce risk; so safety training and education in good laboratory practise was undertaken by all team members before experiments could begin.<br />
</p><br />
<p class="body_text"><br />
The main principle behind performing experiments in a safe environment is to minimise risk. Risk is considered as the amalgamation of the potential damage that a hazard could cause, combined with the likelihood of said hazard occurring. The damage a hazard could cause is normally set by the equipment present in the laboratory, whilst laboratory team members can often decrease risk by decreasing the likelihood of a hazard occurring. Below the three main categories (Microbiological, Chemical and Mechanical) are defined with tables showing which hazard fall into said category, and how the risk is minimised in each case.<br />
</p><br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">Microbiological Hazards</p><br />
<p class="body_text"><br />
The key focus of microbiological hazard control concerns the Escherichia coli expression system being used. The majority of Escherichia coli strains are categorised into Bio-safety levels 1 or 2, with non-pathogenic strains placed into level 1 under World Health Organisation classifications. Therefore the Escherichia coli strain used in the facility is considered to be Bio-safety level one, which often requires less stringent safety standards. The genetically engineered strains also may require more safety regulation requirements than wild type micro-organisms; however advances in synthetic biology are allowing more control over the expression system in terms of pathogenicity and also the ability to express suicide genes, which can prevent any live Escherichia coli from surviving outside of the facility environment. Regardless, the facility must ensure and validate that all of the Escherichia coli is killed and effectively disposed of. Pressure systems and directed ventilation are in place to prevent escape of any strains, although this may lead to possible contaminants entering the halls, which dictates that the live Escherichia coli must be sealed in containers or vessels at all times where possible in order to prevent infection. The use of mammalian systems in the project also present a degree of hazard, although the associated risk is lower when compared to using bacteria.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Escape of Escherichia coli from lab</td><br />
<td>The strain(s) exit the laboratory environment, thus allowing the possibility of contamination of exterior objects and/or persons.</td><br />
<td>Pressure of lab is lower than of corridor, thus causing airflow into the lab as opposed of out, which minimises the chance of airborne or aerosol escape of E. coli. Laboratory coats are worn over clothes which do not leave the lab, thus reducing possibility of a team member inadvertently acting as a carrier out of the lab.</td><br />
</tr><br />
<tr><br />
<td>Contamination of E coli/ HeLa/ Microglia</td><br />
<td>Contamination of the desired strain leads to competition between several strains of bacteria, resulting in inaccurate stocks and therefore unusable data. Note that bacterial and mammalian experiments are undertaken in separate parts of the facility.</td><br />
<td>Exposure of strain to open air is minimised, which is standard practise for any container that may be involved with the desired E. coli strain. Single use gloves are used to minimise possible contamination when experiments are performed. Ethanol is applied on work surfaces before and after experimental procedures in order to minimise contamination on work surfaces etc.</td><br />
</tr><br />
<tr><br />
<td>Contamination of team members</td><br />
<td>Illness could ensue from working in the laboratory environment.</td><br />
<td>Team members are not advised to work when feeling unwell. Gloves, lab coat and goggles are also worn to minimise contact with organisms. Personal hygiene standards are advised to be upheld in particular for laboratory workers.</td><br />
</tr><br />
<tr><br />
<td>HeLa Cells</td><br />
<td>Level 2 biosafety class cells</td><br />
<td>Cell experiments always performed in a sterile environment and a fume hood, with a supervisor providing assistance to the experiment when necessary</td><br />
</tr><br />
<tr><br />
<td>Microglia cells</td><br />
<td>Level 2 biosafety class cells</td><br />
<td>Dr. Darren Nesbeth will perform the neccesary assays in place of iGEm members due to the safety regulations</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Chemical Hazards</p><br />
<p class="body_text"><br />
When performing experiments, it may be necessary to use dangerous chemicals during certain processes. Whilst using hazardous chemicals is avoided where necessary, in some cases it is required, so in each case where this occurs the procedure must be executed in a fashion which is as safe as reasonably possible. Therefore standard protocols such as wearing protective clothing may be added to by performing experiments in fume hoods or other such devices to minimise any chance of contact via evaporation etc.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Ethidium Bromide (EtBr)</td><br />
<td>Mutagenic.<br />
Minor toxicity issue</td><br />
<td>All operations using EtBr are carried out in a fume cupboard - separate equipment (pipettes etc.) are used only in the fume cupboard specifically for EtBr.<br />
Gloves and long sleeved protective gowns MUST be worn, disposal of items in contact with EtBr are disposed of separately to other wastes.<br />
Wash hands after gloves are removed.</td><br />
</tr><br />
<tr><br />
<td>High concentration Ethanol (EtOH) and other alcohols</td><br />
<td>Flammable.</td><br />
<td>Securely stored (glass container) in a separate cupboard in volumes less than 2 Litres.</td><br />
</tr><br />
<tr><br />
<td>Dimethyl sulfoxide & Zeocin</td><br />
<td>Irritant to operator if contact with skin is made</td><br />
<td>Chemical handled with extreme caution, with particular attention to no skin exposed when performing an experiment, particularly goggles must be worn</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Mechanical Hazards</p><br />
<p class="body_text"><br />
For this project, mechanical hazards pose the lowest probability of occurring out of the three and thus may be considered as the lowest risk group. However, there are hazards present which must be minimised. The predominant issue here is the use of the desktop centrifuge system, which poses considerable danger if a blowout occurs.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Centrifuge</td><br />
<td>Centrifuge mechanical failure (eg. of rotor) can cause severe damage to the machine, and possibly personnel in the nearby vicinity.</td><br />
<td><br />
Regular inspections and maintenance of the machine is required (performed by external engineers). <br />
Machine not to be used if in non optimal condition.<br />
Centrifuge must be balanced to minimise chance of rotor failure.<br />
Centrifuge must be properly closed before use (required for the machine to work.)<br />
Attention must be paid to ensure that nothing is spilled in the centrifuge bowl, or a build up of dust or aerosol should also be avoided.</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Concluding Comments</p><br />
<p class="body_text"><br />
The safety of the team members, produced material and environment are key aspects to the smooth running of the project, and it must be ensured that adequate measures are in place to provide protection and ensuring that guidelines can be followed proceeding mandatory safety training. Current Good Laboratory Practice must also be adhered to at all times. By following guidelines and acting in an appropriate manner whilst in the laboratory, the general risk in terms of micro-biological, chemical and mechanical aspects will be reduced to a level which is as low as reasonably possible for all project members involved.<br />
</p><br />
<p class="body_text"><br />
Due to the relatively small scale of the experiments performed, the quantities used of material is not large, but still must be handled and respected properly at all times. Proper containment and labelling of materials in sealed containers is necessary, especially when of particular hazard such as live material or Ethidium Bromide. Flammability concerns are also present with alcohol and other flammable materials stored in the lab, although the volumes (generally less than 2L) are not considerably large enough, although should still be used and handled properly when used, with fire extinguishers available and also emergency exits present if required. Protective clothing also should be worn where appropriate to minimise the possibility of skin contact, with wash and first aid station available in key areas where allowable due to the area classification.<br />
</p><br />
<p class="body_text"><br />
The mechanical safety predominantly concerns the proper maintenance of the centrifuge system, which can cause a considerable risk if a failure occurs. Regular inspections should be made, with the centrifuge often restricted access when it was considered unfit for use. This room is also placed away from mammalian and bacterial processing centres. Prevention methods should also be in place such as emergency shut-off procedures.<br />
</p><br />
<p class="body_text"><br />
By following all guidelines set out in safety training and following supervisor instructions, the project may be performed in a safe and effective manner. Continual training and inspection of the facility will aid in ensuring safety levels are maintained, with review protocols in place to investigate any faults. Team members have been trained in general safety, with supervisors having first aid experience and training. These considerations combined will ensure the safety risk to the personnel; product and environment is as low as reasonably possible. For further reading, the Institute of Chemical Engineering (IChemE) has relevant information, as well as other safety guidance documents relating to the relevant country where the university is located.<br />
</p><br />
</div><br />
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<div></div>Narutohttp://2013.igem.org/File:IGEM_2013_Basic_Safety_Form.pdfFile:IGEM 2013 Basic Safety Form.pdf2013-10-05T02:35:32Z<p>Naruto: uploaded a new version of &quot;File:IGEM 2013 Basic Safety Form.pdf&quot;</p>
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<div>iGEM 2013 Basic Safety Form</div>Narutohttp://2013.igem.org/Team:UCL/Notebook/MarchTeam:UCL/Notebook/March2013-10-05T02:29:54Z<p>Naruto: </p>
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<div class="full_page"><br />
<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
</p> <br />
</div><br />
<br />
<p class="major_title">March</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Final few meetings were held before exams, both within UCL and at the Arts Catalyst. The most popular projects were brought forward and were narrowed down to the following: <br />
</p><br />
<p class="body_text"><br />
• Anti-cancer yoghurt<br />
</p><br />
<p class="body_text"><br />
• Zebrafish<br />
</p><br />
<p class="body_text"><br />
• Alzheimer's disease<br />
</p><br />
<p class="body_text"><br />
• Neural Network<br />
</p><br />
</p><br />
<p class="body_text"><br />
The team held a probiotic yoghurt workshop for the anti-cancer project. During this opinions regarding the projects were gathered as members of the public were allowed to make their very own yoghurt. Ethical concerns regarding the Alzheimer's project were frequently raised during the workshop. As a result, the team came to the conclusion that bioethics would play a large role within the project. <br />
</p><br />
</div><br />
<br />
<!-- END CONTENT ------------------------------------------------------------------------------------------------------><br />
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
</p> <br />
</div><br />
<br />
<p class="major_title">March</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Final few meetings were held before exams, both within UCL and at the Arts Catalyst. The most popular projects were brought forward and were narrowed down to the following: <br />
</p><br />
<p class="body_text"><br />
• Anti-cancer yoghurt<br />
</p><br />
<p class="body_text"><br />
• Zebrafish<br />
</p><br />
<p class="body_text"><br />
• Alzheimer's disease<br />
</p><br />
<p class="body_text"><br />
• Neural Network<br />
</p><br />
</p><br />
<p class="body_text"><br />
The team held a probiotic yoghurt workshop for the anti-cancer project. During this members of the public were allowed to make their very own yoghurt. Project opinions were gathered. Ethical concerns regarding the Alzheimer's project were frequently raised during the workshop. As a result, the team came to the conclusion that bioethics would play a large role within the project. <br />
</p><br />
</div><br />
<br />
<!-- END CONTENT ------------------------------------------------------------------------------------------------------><br />
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<p class="major_title">Lab Weeks</p><br />
<br />
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<div class="full_page"><br />
<p class="body_text"> <a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a> <br />
</p> <br />
</div><br />
<br />
<p class="minor_title">Week 17</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Bacterial Labs</b><br />
</p><br />
<p class="body_text"><br />
<b>Monday 23rd September</b><br />
</p><br />
<p class="body_text"><br />
Nanodrop readings<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Sample</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>MMP9</td><br />
<td>382.5</td><br />
<td>1.86</td><br />
</tr><br />
<tr><br />
<td>ZEC</td><br />
<td>401</td><br />
<td>1.49</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Nanodrops of the 6 colonies</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1x1</td><br />
<td>22.3</td><br />
<td>1.46</td><br />
</tr><br />
<tr><br />
<td>1x2</td><br />
<td>78.5</td><br />
<td>1.45</td><br />
</tr><br />
<tr><br />
<td>1x3</td><br />
<td>11.0</td><br />
<td>1.77</td><br />
</tr><br />
<tr><br />
<td>1x4</td><br />
<td>33.9</td><br />
<td>1.50</td><br />
</tr><br />
<tr><br />
<td>1x5</td><br />
<td>35.2</td><br />
<td>1.55</td><br />
</tr><br />
<tr><br />
<td>5x1</td><br />
<td>28.7</td><br />
<td>1.53</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Tuesday 24th September</b><br />
</p><br />
<p class="body_text"><br />
CMV PCR with Taq polymerase<br />
</p><br />
<p class="body_text"><br />
-10 reactions and 1 control<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Reaction components</th><br />
<th>Tubes 1-10</th><br />
<th>Control (11)</th><br />
</tr><br />
<tr><br />
<td>Taq Buffer</td><br />
<td>5</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>dNTP</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>F Primer</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>R Primer</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Template (pSecTag2A, 1ng/ul)</td><br />
<td>1.5</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Taq polymerase</td><br />
<td>0.25</td><br />
<td>0.25</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>40.25</td><br />
<td>41.75</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Tubes were labelled from 1 to 11.<br />
This was unsuccessful.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Wednesday 25th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Thursday 26th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Friday 27th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Saturday 28th September</b><br />
</p><br />
<p class="body_text"><br />
Made new inoculations from 5X CMP and 1X CMP plates for Ligated CMV-MMP9.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Sunday 29th September</b><br />
</p><br />
<p class="body_text"><br />
There were not enough colonies for B3&4. The rest of the samples were <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> minipreped</a><br />
and digested and run on a gel cut.<br />
</p><br />
<p class="body_text"><br />
4 more inoculations in 10 ml LB broth of A2, A5, B2 and C2. <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> Nanodrop</a> readings of these inoculations were poor.<br />
</p><br />
<br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Mammalian Labs</b><br />
</p><br />
<p class="body_text"><br />
<b>Monday 23rd September</b><br />
</p><br />
<p class="body_text"><br />
Seeded 2x105 cells (1x105 cells/ml) per well into 16 wells set in three 6-well plates. Stock Hela passaged (P21).<br />
</p><br />
<br />
<br />
<p class="body_text"><br />
24th September<br />
</p><br />
<p class="body_text"><br />
CMV PCR with Taq polymerase<br />
</p><br />
<p class="body_text"><br />
-10 reactions and 1 control<br />
<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Reaction Components</th><br />
<th>Tubes 1-10</th><br />
<th>Control 11</th><br />
</tr><br />
<tr><br />
<td>Taq buffer</td><br />
<td>5</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>dNTP</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>F primer</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>R primer</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Template (pSecTag2A, 1 ng/ul)</td><br />
<td>1.5</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Taq polymerase</td><br />
<td>0.25</td><br />
<td>0.25</td><br />
</tr><br />
<tr><br />
<td>Water</td><br />
<td>40.25</td><br />
<td>41.25</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>50</td><br />
<td>50</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
This was unsuccessful.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>25th September</b><br />
</p><br />
<p class="body_text"><br />
<b>Mammalian Lab</b><br />
</p><br />
<p class="body_text"><br />
Aim: To stably transfect Hela cells with ’’Str Ble’’.<br />
</p><br />
<p class="body_text"><br />
Confluency of dishes: 30-50%<br />
<br />
</p><br />
<p class="body_text"><br />
Per well of the 6-well plates: 13 well total.<br />
</p><br />
<p class="body_text"><br />
1. Mass of DNA: 2.0µg<br />
</p><br />
<p class="body_text"><br />
2. Volume of DNA dissolved in TE Buffer: 5.0 µl<br />
</p><br />
<p class="body_text"><br />
3. Final volume of DNA diluted in serum free media: 100 µl<br />
</p><br />
<p class="body_text"><br />
4. Volume of superfect (SF) reagent:10.0 µl<br />
</p><br />
<p class="body_text"><br />
5. Volume of serum-containing media: 600 µl<br />
</p><br />
<p class="body_text"><br />
2x ”mock transfected” wells (No DNA, No SE)<br />
</p><br />
<p class="body_text"><br />
Procedure:<br />
</p><br />
<p class="body_text"><br />
Made a ”mask mix” solution for the 13 well-plates to be <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> transfected</a>, with the following composition :<br />
</p><br />
<p class="body_text"><br />
1. 34 ng<br />
</p><br />
<p class="body_text"><br />
2. 93.5 ul<br />
</p><br />
<p class="body_text"><br />
3. 1870 ul<br />
</p><br />
<p class="body_text"><br />
4. 187 ul<br />
</p><br />
<p class="body_text"><br />
5. 11220 ul<br />
<br />
</p><br />
<p class="body_text"><br />
Transfer DNA-Buffer solution to 15 ml falcon tube. Add non-serum media (1776.5µl), Filter, Add SF, Vortex (10.6), Incubate for 5-10 min at room temperature. Meanwhile aspirate and wash cells with 4ml PBS. Add media with serum (600ml). Pipette up and down x2. Apply to cells(≈700µl). Incubate for 2-3 hours. Change media. Incubate (24 hours)<br />
</p><br />
<p class="body_text"><br />
Note : for characterisation , add 1µl of 100µg/ml zeocin to one plate, 2µl to another, and 3µl to the last to make up the concentrations to those over the page.<br />
</p><br />
<p class="body_text"><br />
<b>26th September</b><br />
</p><br />
<p class="body_text"><br />
<b>Mammalian Lab</b><br />
</p><br />
<p class="body_text"><br />
Characterisation:<br />
</p><br />
<p class="body_text"><br />
24 hours after exposure:<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Viability (%)</th><br />
<th>50 µg/ml Zec</th><br />
<th>100 µg/ml Zec</th><br />
<th>150 µg/ml Zec</th><br />
</tr><br />
<tr><br />
<td>Control</td><br />
<td>80</td><br />
<td>80</td><br />
<td>70</td><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>80</td><br />
<td>80</td><br />
<td>80</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>75</td><br />
<td>80</td><br />
<td>75</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>75</td><br />
<td>80</td><br />
<td>75</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>70</td><br />
<td>80</td><br />
<td>75</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Sunday 29th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>24 Hours</th><br />
<th>A (%)</th><br />
<th>F (%)</th><br />
<th>48 Hours</th><br />
<th>A (%)</th><br />
<th>F (%)</th><br />
<th>72 Hours</th><br />
<th>A (%)</th><br />
<th>F (%)</th><br />
</tr><br />
<tr><br />
<td>C1</td><br />
<td>0</td><br />
<td>1</td><br />
<td>C1</td><br />
<td>0</td><br />
<td>4</td><br />
<td>C1</td><br />
<td>0</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>C2</td><br />
<td>0</td><br />
<td>1</td><br />
<td>C1</td><br />
<td>0</td><br />
<td>5</td><br />
<td>C1</td><br />
<td>5</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>C3</td><br />
<td>0</td><br />
<td>2</td><br />
<td>C3</td><br />
<td>0</td><br />
<td>4</td><br />
<td>C3</td><br />
<td>1</td><br />
<td>40</td><br />
</tr><br />
<tr><br />
<td>P1</td><br />
<td>0</td><br />
<td>3</td><br />
<td>P1</td><br />
<td>0</td><br />
<td>5</td><br />
<td>P1</td><br />
<td>5</td><br />
<td>50</td><br />
</tr><br />
<tr><br />
<td>P2</td><br />
<td>0</td><br />
<td>2</td><br />
<td>P2</td><br />
<td>0</td><br />
<td>10</td><br />
<td>P2</td><br />
<td>1</td><br />
<td>15</td><br />
</tr><br />
<tr><br />
<td>P3</td><br />
<td>0</td><br />
<td>1</td><br />
<td>P3</td><br />
<td>0</td><br />
<td>10</td><br />
<td>P3</td><br />
<td>1</td><br />
<td>50</td><br />
</tr><br />
</table><br />
<br />
</div><br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/Labbook/Week17Team:UCL/Labbook/Week172013-10-05T01:43:57Z<p>Naruto: </p>
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<div class="gap"><br />
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<br />
<p class="major_title">Lab Weeks</p><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="full_page"><br />
<p class="body_text"> <a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a> <br />
</p> <br />
</div><br />
<br />
<p class="minor_title">Week 17</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Bacterial Labs</b><br />
</p><br />
<p class="body_text"><br />
<b>Monday 23rd September</b><br />
</p><br />
<p class="body_text"><br />
Nanodrop readings<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Sample</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>MMP9</td><br />
<td>382.5</td><br />
<td>1.86</td><br />
</tr><br />
<tr><br />
<td>ZEC</td><br />
<td>401</td><br />
<td>1.49</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Nanodrops of the 6 colonies</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1x1</td><br />
<td>22.3</td><br />
<td>1.46</td><br />
</tr><br />
<tr><br />
<td>1x2</td><br />
<td>78.5</td><br />
<td>1.45</td><br />
</tr><br />
<tr><br />
<td>1x3</td><br />
<td>11.0</td><br />
<td>1.77</td><br />
</tr><br />
<tr><br />
<td>1x4</td><br />
<td>33.9</td><br />
<td>1.50</td><br />
</tr><br />
<tr><br />
<td>1x5</td><br />
<td>35.2</td><br />
<td>1.55</td><br />
</tr><br />
<tr><br />
<td>5x1</td><br />
<td>28.7</td><br />
<td>1.53</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Tuesday 24th September</b><br />
</p><br />
<p class="body_text"><br />
CMV PCR with Taq polymerase<br />
</p><br />
<p class="body_text"><br />
-10 reactions and 1 control<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Reaction components</th><br />
<th>Tubes 1-10</th><br />
<th>Control (11)</th><br />
</tr><br />
<tr><br />
<td>Taq Buffer</td><br />
<td>5</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>dNTP</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>F Primer</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>R Primer</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Template (pSecTag2A, 1ng/ul)</td><br />
<td>1.5</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Taq polymerase</td><br />
<td>0.25</td><br />
<td>0.25</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>40.25</td><br />
<td>41.75</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Tubes were labelled from 1 to 11.<br />
This was unsuccessful.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Wednesday 25th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Thursday 26th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Friday 27th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Saturday 28th September</b><br />
</p><br />
<p class="body_text"><br />
Made new inoculations from 5X CMP and 1X CMP plates for Ligated CMV-MMP9.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Sunday 29th September</b><br />
</p><br />
<p class="body_text"><br />
There were not enough colonies for B3&4. The rest of the samples were <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> minipreped</a><br />
and digested and run on a gel cut.<br />
</p><br />
<p class="body_text"><br />
4 more inoculations in 10 ml LB broth of A2, A5, B2 and C2. <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> Nanodrop</a> readings of these inoculations were poor.<br />
</p><br />
<br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Mammalian Labs</b><br />
</p><br />
<p class="body_text"><br />
<b>Monday 23rd September</b><br />
</p><br />
<p class="body_text"><br />
Seeded 2x105 cells (1x105 cells/ml) per well into 16 wells set in three 6-well plates. Stock Hela passaged (P21).<br />
</p><br />
<br />
<br />
<p class="body_text"><br />
24th September<br />
</p><br />
<p class="body_text"><br />
CMV PCR with Taq polymerase<br />
</p><br />
<p class="body_text"><br />
-10 reactions and 1 control<br />
<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Reaction Components</th><br />
<th>Tubes 1-10</th><br />
<th>Control 11</th><br />
</tr><br />
<tr><br />
<td>Taq buffer</td><br />
<td>5</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>dNTP</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>F primer</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>R primer</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Template (pSecTag2A, 1 ng/ul)</td><br />
<td>1.5</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Taq polymerase</td><br />
<td>0.25</td><br />
<td>0.25</td><br />
</tr><br />
<tr><br />
<td>Water</td><br />
<td>40.25</td><br />
<td>41.25</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>50</td><br />
<td>50</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
This was unsuccessful.<br />
</p><br />
<br />
<p class="body_text"><br />
25th September<br />
</p><br />
<p class="body_text"><br />
<b>Mammalian Lab</b><br />
</p><br />
<p class="body_text"><br />
Aim: To stably transfect Hela cells with ’’Str Ble’’.<br />
</p><br />
<p class="body_text"><br />
Confluency of dishes: 30-50%<br />
<br />
</p><br />
<p class="body_text"><br />
Per well of the 6-well plates: 13 well total.<br />
</p><br />
<p class="body_text"><br />
1. Mass of DNA: 2.0µg<br />
</p><br />
<p class="body_text"><br />
2. Volume of DNA dissolved in TE Buffer: 5.0 µl<br />
</p><br />
<p class="body_text"><br />
3. Final volume of DNA diluted in serum free media: 100 µl<br />
</p><br />
<p class="body_text"><br />
4. Volume of superfect (SF) reagent:10.0 µl<br />
</p><br />
<p class="body_text"><br />
5. Volume of serum-containing media: 600 µl<br />
</p><br />
<p class="body_text"><br />
2x ”mock transfected” wells (No DNA, No SE)<br />
</p><br />
<p class="body_text"><br />
Procedure:<br />
</p><br />
<p class="body_text"><br />
Made a ”mask mix” solution for the 13 well-plates to be <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> transfected</a>, with the following composition :<br />
</p><br />
<p class="body_text"><br />
1. 34 ng<br />
</p><br />
<p class="body_text"><br />
2. 93.5 ul<br />
</p><br />
<p class="body_text"><br />
3. 1870 ul<br />
</p><br />
<p class="body_text"><br />
4. 187 ul<br />
</p><br />
<p class="body_text"><br />
5. 11220 ul<br />
<br />
</p><br />
<p class="body_text"><br />
Transfer DNA-Buffer solution to 15 ml falcon tube. Add non-serum media (1776.5µl), Filter, Add SF, Vortex (10.6), Incubate for 5-10 min at room temperature. Meanwhile aspirate and wash cells with 4ml PBS. Add media with serum (600ml). Pipette up and down x2. Apply to cells(≈700µl). Incubate for 2-3 hours. Change media. Incubate (24 hours)<br />
</p><br />
<p class="body_text"><br />
Note : for characterisation , add 1µl of 100µg/ml zeocin to one plate, 2µl to another, and 3µl to the last to make up the concentrations to those over the page.<br />
</p><br />
<p class="body_text"><br />
<b>26th September</b><br />
</p><br />
<p class="body_text"><br />
<b>Mammalian Lab</b><br />
</p><br />
<p class="body_text"><br />
Characterisation:<br />
</p><br />
<p class="body_text"><br />
24 hours after exposure:<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Viability (%)</th><br />
<th>50 µg/ml Zec</th><br />
<th>100 µg/ml Zec</th><br />
<th>150 µg/ml Zec</th><br />
</tr><br />
<tr><br />
<td>Control</td><br />
<td>80</td><br />
<td>80</td><br />
<td>70</td><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>80</td><br />
<td>80</td><br />
<td>80</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>75</td><br />
<td>80</td><br />
<td>75</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>75</td><br />
<td>80</td><br />
<td>75</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>70</td><br />
<td>80</td><br />
<td>75</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Sunday 29th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>24 Hours</th><br />
<th>A (%)</th><br />
<th>F (%)</th><br />
<th>48 Hours</th><br />
<th>A (%)</th><br />
<th>F (%)</th><br />
<th>72 Hours</th><br />
<th>A (%)</th><br />
<th>F (%)</th><br />
</tr><br />
<tr><br />
<td>C1</td><br />
<td>0</td><br />
<td>1</td><br />
<td>C1</td><br />
<td>0</td><br />
<td>4</td><br />
<td>C1</td><br />
<td>0</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>C2</td><br />
<td>0</td><br />
<td>1</td><br />
<td>C1</td><br />
<td>0</td><br />
<td>5</td><br />
<td>C1</td><br />
<td>5</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>C3</td><br />
<td>0</td><br />
<td>2</td><br />
<td>C3</td><br />
<td>0</td><br />
<td>4</td><br />
<td>C3</td><br />
<td>1</td><br />
<td>40</td><br />
</tr><br />
<tr><br />
<td>P1</td><br />
<td>0</td><br />
<td>3</td><br />
<td>P1</td><br />
<td>0</td><br />
<td>5</td><br />
<td>P1</td><br />
<td>5</td><br />
<td>50</td><br />
</tr><br />
<tr><br />
<td>P2</td><br />
<td>0</td><br />
<td>2</td><br />
<td>P2</td><br />
<td>0</td><br />
<td>10</td><br />
<td>P2</td><br />
<td>1</td><br />
<td>15</td><br />
</tr><br />
<tr><br />
<td>P3</td><br />
<td>0</td><br />
<td>1</td><br />
<td>P3</td><br />
<td>0</td><br />
<td>10</td><br />
<td>P3</td><br />
<td>1</td><br />
<td>50</td><br />
</tr><br />
</table><br />
<br />
</div><br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/Labbook/Week16Team:UCL/Labbook/Week162013-10-05T01:40:31Z<p>Naruto: </p>
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<div class="gap"><br />
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<br />
<p class="major_title">Lab Weeks</p><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="full_page"><br />
<p class="body_text"> <a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a> <br />
</p> <br />
</div><br />
<br />
<p class="minor_title">Week 16</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Bacterial Labs</b><br />
</p><br />
<p class="body_text"><br />
<b>Monday 16th September</b><br />
</p><br />
<p class="body_text"><br />
Results of the inoculations of transformations of 13/09 in 4xcmp: all showed growth apart from falcons 5.3, 5.2, 1.14 and 1.2. Glycerol stocks of the rest of 19 inoculations were made.<br />
</p><br />
<p class="body_text"><br />
Minipreps of the above inoculations were made only for 1.3, 1.7, 1.9, 1.10, 1.11, 1.15, 1.20 and 5.1 due to lack of chromatographic columns.<br />
</p><br />
<p class="body_text"><br />
Nanodrop result of the above minipreps showed concentration values below 16.0 ng/ul.<br />
Analytical digest of miniprep samples 1-9 (prepared the day before) as well as of 5.1 and 1.15 (which showed concentrations of about 15 ng/ul) with E and P<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Components</th><br />
<th>Volume (ul)<br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>EcoR1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>1.5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>10</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/9/9c/Andymon_was_here.png');height:515px;width:650px"></div><br />
</p><br />
<p class="body_text"><br />
Prep digest of miniprep of J632014 with E & P in order to keep up the pSB1C3 stocks<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Components</th><br />
<th>Cut</th><br />
<th>Uncut</th><br />
<th>Control - pSecTag2A</th><br />
</tr><br />
<tr><br />
<td>J632014 DNA</td><br />
<td>25</td><br />
<td>5</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>EocR1</td><br />
<td>2</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>2</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>4</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>6.5</td><br />
<td>5</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>40</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These were incubated for 2 hours at 37O C.<br />
Casey ran a gel of the following samples in the next order:<br />
Zeo BB: 26D, 26u; 28D, 28u; AuxD, AuxU <br />
</p><br />
<p class="body_text"><br />
Gel1: E+P double digest and P, single digest (Auxin bb): 3, 12, 13, 15.<br />
Gel2: D digest 26, 28 and Auxin bb.<br />
Minipreps: 26, 28, 3, 12, 13, 15, 21 (sample of which DNA is waiting to be eluted)<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Tuesday 17th September</b><br />
</p><br />
<p class="body_text"><br />
<a href="https://2013.igem.org/Team:UCL/Project/Protocols"> Miniprep</a> of the 16 tubes of inoculations of MMP9 as well as tube 21 of which DNA had to be eluted.<br />
</p><br />
<p class="body_text"><br />
Nanodrop results of the minipreps<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Tube no.</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>20.6</td><br />
<td>1.9</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>26.9</td><br />
<td>1.64</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>22.0</td><br />
<td>3.2</td><br />
</tr><br />
<tr><br />
<td>5</td><br />
<td>4.8</td><br />
<td>2.16</td><br />
</tr><br />
<tr><br />
<td>6</td><br />
<td>22.7</td><br />
<td>2.99</td><br />
</tr><br />
<tr><br />
<td>7</td><br />
<td>16.4</td><br />
<td>2.41</td><br />
</tr><br />
<tr><br />
<td>8</td><br />
<td>16.9</td><br />
<td>2.01</td><br />
</tr><br />
<tr><br />
<td>9</td><br />
<td>7.1</td><br />
<td>1.49</td><br />
</tr><br />
<tr><br />
<td>10</td><br />
<td>27.3</td><br />
<td>1.79</td><br />
</tr><br />
<tr><br />
<td>11</td><br />
<td>16.0</td><br />
<td>1.72</td><br />
</tr><br />
<tr><br />
<td>12</td><br />
<td>22.2</td><br />
<td>1.41</td><br />
</tr><br />
<tr><br />
<td>13</td><br />
<td>11.0</td><br />
<td>1.73</td><br />
</tr><br />
<tr><br />
<td>14</td><br />
<td>12.0</td><br />
<td>1.6</td><br />
</tr><br />
<tr><br />
<td>15</td><br />
<td>18.4</td><br />
<td>1.57</td><br />
</tr><br />
<tr><br />
<td>17</td><br />
<td>45.1</td><br />
<td>1.62</td><br />
</tr><br />
<tr><br />
<td>18</td><br />
<td>9.2</td><br />
<td>1.72</td><br />
</tr><br />
<tr><br />
<td>21 (kc)</td><br />
<td>21.3</td><br />
<td>1.81</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Nanodrop readings (Tom's)<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Sample</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>J63A (glyc stock)</td><br />
<td>62.3</td><br />
<td>1.74</td><br />
</tr><br />
<tr><br />
<td>CCB4 (4xcmp comp cells)</td><br />
<td>46.8</td><br />
<td>1.94</td><br />
</tr><br />
<tr><br />
<td>CCB2 (2xcmp comp cells)</td><br />
<td>57.6</td><br />
<td>1.87</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Analytical digest of zeo+pSB1C3 (from AA1 ligation) potential clones with xba1<br />
Eight clones (AA1 col x miniprep 15/09 RC) + AA1 5xcmp mini prep were digested with xba1 following the recipe:<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Components</th><br />
<th>Volume (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Xba1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 2</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>2.5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>10</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Samples were briefly centrifuged and incubated at 37O C for circa 3 hours. After that, samples were supplemented with 3 ul dye and run on a gel.<br />
</p><br />
<p class="body_text"><br />
<u>Purification of 8 PCR reactions to amplify Zeo and BB hangers</u><br />
</p><br />
<p class="body_text"><br />
These reactions (total volume of 400 ul) were left in the thermocycler at 4O C overnight. The samples were run on a gel together with 70 ul dye and the correct bands (1.8 kb) were gel extracted. This gel was purified and eluted in 40 ul Elution Buffer. The nanodrop readings of these were of 91.9 ul/ul with purity (260/280) of 1.96.<br />
</p><br />
<p class="body_text"><br />
<u>Preparative digest of amplified zeocin using EcoR1 and Pst1</u><br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Components</th><br />
<th>Volume (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>35</td><br />
</tr><br />
<tr><br />
<td>EcoR1</td><br />
<td>7</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>7</td><br />
</tr><br />
<tr><br />
<td>Buffer 2</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>4</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>37</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>100</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These reactions were Incubated for 2 hours at 37OC.<br />
After, the digest was purified with PCR purification kit. Nanodrop result was found to be 60.4 ng/ul (260/280=1.82, the purity).This was taken further for a ligation as per the following recipe:<br />
</p><br />
<p class="body_text"><br />
<u>Ligation 5 for zeocin and pSB1C3</u><br />
</p><br />
<p class="body_text"><br />
pSB1C3 concentration = 50 ng/ul<br />
</p><br />
<p class="body_text"><br />
Zeocin insert concentration = 25 ng/ul<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Component</th><br />
<th>Lig 1 (ul)</th><br />
<th>Lig 2 (ul)</th><br />
<th>Lig 3 (ul) control</th><br />
<th>Lig 4 (ul) control</th><br />
</tr><br />
<tr><br />
<td>pSB1C3</td><br />
<td>2</td><br />
<td>2</td><br />
<td>2</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Zeo</td><br />
<td>2</td><br />
<td>2.5</td><br />
<td>0</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>Quick T4 ligase</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>5</td><br />
<td>4.5</td><br />
<td>7</td><br />
<td>7</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>20</td><br />
<td>20</td><br />
<td>20</td><br />
<td>20</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These tubes were incubated at room temperature for 5 minutes then used 5 ul from each for transformation using W3110 cells. Plated 10 ul, 90 ul on 5xcmp selective plates and then incubated overnight at 37°C. Next day, there was no growth on neither plates<br />
</p><br />
<p class="body_text"><br />
Two gel were loaded using xba1 digests of recombinant zeo candidates<br />
</p><br />
<p class="body_text"><br />
Ten ul of sample 1(showed correct band pattern) was used from glycerol stock to make an inoculation in 4xcmp 10 ml LB broth.<br />
</p><br />
<br />
<p class="body_text"><br />
<u>Inoculation of MMP9 glycerol stocks</u><br />
</p><br />
<p class="body_text"><br />
These were made using 2 ml LB broth, 8 ul material from the glycerol stock and 4xcmp (8 ul cmp).<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Wednesday 18th September</b><br />
</p><br />
<p class="body_text"><br />
Carried out <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> miniprep</a><br />
of 16 MMP9 glycerol stocks candidates which were inoculated overnight the day before; another miniprep was set for 2 samples from zeocin ligation 1 (prepared the day before) which was inoculated overnight (10 ml LB and 40 ul cmp).<br />
Nanodrop results of the above<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Tube label</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>97.6</td><br />
<td>1.85</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>151.8</td><br />
<td>1.70</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>121.8</td><br />
<td>1.80</td><br />
</tr><br />
<tr><br />
<td>5</td><br />
<td>103.6/td><br />
<td>1.83</td><br />
</tr><br />
<tr><br />
<td>6</td><br />
<td>104.8</td><br />
<td>1.92/td><br />
</tr><br />
<tr><br />
<td>7</td><br />
<td>409.2</td><br />
<td>1.82</td><br />
</tr><br />
<tr><br />
<td>8</td><br />
<td>220.3</td><br />
<td>1.79</td><br />
</tr><br />
<tr><br />
<td>9</td><br />
<td>187.3</td><br />
<td>1.80</td><br />
</tr><br />
<tr><br />
<td>10</td><br />
<td>188.2</td><br />
<td>1.91</td><br />
</tr><br />
<tr><br />
<td>11</td><br />
<td>82.8/td><br />
<td>1.88</td><br />
</tr><br />
<tr><br />
<td>12</td><br />
<td>79.5</td><br />
<td>1.76</td><br />
</tr><br />
<tr><br />
<td>13</td><br />
<td>170.6/td><br />
<td>1.73</td><br />
</tr><br />
<tr><br />
<td>14</td><br />
<td>117.1</td><br />
<td>1.92</td><br />
</tr><br />
<tr><br />
<td>15</td><br />
<td>177.8</td><br />
<td>1.89</td><br />
</tr><br />
<tr><br />
<td>17</td><br />
<td>119.7</td><br />
<td>1.85</td><br />
</tr><br />
<tr><br />
<td>18</td><br />
<td>167.5</td><br />
<td>1.69</td><br />
</tr><br />
<tr><br />
<td>Zeo lig1A</td><br />
<td>419.6</td><br />
<td>1.94</td><br />
</tr><br />
<tr><br />
<td>Zeo lig1B</td><br />
<td>97.2</td><br />
<td>2.03</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Casey single digests recipes<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Component</th><br />
<th>Xba1 digest (ul)</th><br />
<th>EcoR1 digest (ul)</th><br />
<th>Pst1 digest (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>5</td><br />
<td>5</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Xba1</td><br />
<td>1</td><br />
<td>-</td><br />
<td>-</td><br />
</tr><br />
<tr><br />
<td>EcoR1</td><br />
<td>-</td><br />
<td>1</td><br />
<td>-</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>-</td><br />
<td>-</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 4/3</td><br />
<td>1 (buffer 5)</td><br />
<td>1 (EcoR1 buffer)</td><br />
<td>1 (buffer 3)</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
<td>0.5</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>2.5</td><br />
<td>2.5</td><br />
<td>2.5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
</total><br />
</p><br />
<br />
<p class="body_text"><br />
Gel loading order wells: 1x, 1E, 1P, 1u, 13x, 13E, 13P, 13u, 21x, 21E, 21P, 21u, 1.15x, 1.15E, 1.15P, 1.15u, 5.1x, 5.1E, 5.1P, 5.1u.<br />
</p><br />
<p class="body_text"><br />
Carried out gel extraction and purification of Weiling’s MMP9 amplification; total material loaded: 2x50 ul vials from past PCR and 6x50 ul PCR left at 4oC overnight on 17/09/13.<br />
After the purification procedure, the concentration of MMP9 was found to be 111 ng/ul and 260/280 indices = 2.13.<br />
</p><br />
<p class="body_text"><br />
<u>Prep digest of purified MMP9 with Dpn1, EcoR1 and Pst1</u><br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Components</th><br />
<th>Volumes (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>58</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>7</td><br />
</tr><br />
<tr><br />
<td>Ecor1</td><br />
<td>7</td><br />
</tr><br />
<tr><br />
<td>Dpn1</td><br />
<td>7</td><br />
</tr><br />
<tr><br />
<td>Buffer 2</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>100</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These were incubated for 2 hours at 37C. This was followed be a PCR purification. <br />
</p><br />
<p class="body_text"><br />
<u>Prep digest of pSB1C3 (the entire stock) with Dpn1, EcoR1, Pst1</u><br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Components</th><br />
<th>Volumes (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>70</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>Ecor1</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>Dpn1</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>Buffer 2</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>100</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
The digestion was incubated for 2 hours and then incubated for 20 min.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Thursday 19th September</b><br />
</p><br />
<p class="body_text"><br />
Focus on MMP-9<br />
- Minipreped samples from the 18 inoculations, only 16 of these had growth.<br />
- Took the 16 inoculations forward to minipreping.<br />
- Made glycerol stocks of the 16 inoculations.<br />
- Took nanodrop readings (range varied between 6 ng/ul -45 ng/ul).<br />
- Did an analytical digest with Xba1 using 5 samples of the highest concentrations (2, 4, 10, 14, 17).<br />
- Gel result didn’t show bands for 4, 2, 14, 17, only sample 10 showed visible bands for both cut and uncut.<br />
- Re-inoculated 16 samples from glycerol stocks for 16 hours to for minipreps the following day.<br />
- re-PCRed 6 tubes of MMP-9 with MMP9 4 bb RseFW primes (total of 8 tubes to gel extract and purify the following day).<br />
Nanodrop of PCR purified and E+P+D digest PSBIC3 (2013 High school iGEM )and MMP9 insert<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Tube</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>MMP9</td><br />
<td>184.4</td><br />
<td>1.87</td><br />
</tr><br />
<tr><br />
<td>pSB1C3</td><br />
<td>27.4</td><br />
<td>1.73</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Xba1 restriction sites in PSBIC3 – 1 restriction site<br />
</p><br />
<p class="body_text"><br />
the recombinant plasmid (with zeocin as an insert) is about 3-6-3.8 kb when cut with Xba1 or EcoR1.<br />
Instead the gel run for the xba1 digests have a strong 2 kb band in all the cuts. Possibly, some plasmids may have ligated to themselves.<br />
</p><br />
<p class="body_text"><br />
<u>Repeat digest of minipreps of AA1 ligations (transformation of only using EcoR1)</u><br />
</p><br />
<p class="body_text"><br />
Samples digested (9 in total): AA1 miniprep col 1à 8 & AA1 5xcmp miniprep<br />
10µl reaction volume<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Components</th><br />
<th>Volumes (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>EcoR1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>1</td><br />
</tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>2.5</td><br />
</tr><br />
<td>Total</td><br />
<td>10</td><br />
</tr><br />
</table><br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/0/04/Weilingyuan.png');height:315px;width:666px"></div><br />
<p class="body_text"><br />
Re-Inocubations from glycerol stocks from both batches of <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> transformation</a> with AA1 ligation pick using:<br />
- 2ml LB<br />
- 8µl amp<br />
- 10µl glycerol stock<br />
- Control pSecTag 2A in 8 ul of amp instead of cmp<br />
</p><br />
<p class="body_text"><br />
Glycerol stocks inoculated: 1.2, 1.6, 1.4, 1.18 (from second inoculation, in 4xcmp LB )<br />
All Batch I (from first inoculation, in 1xcmp for col.1-8 and 4xcmp for AA1 5xcmp) &Psectag 2A (Amp).<br />
</p><br />
<p class="body_text"><br />
Weiling: combined MMP-9 minipreps into one tube 11, 12, 13, 14, 17, 18, 1, 2, 4, 5, 6, 7, 10. This was labeled MMP9bbP00L, date, initials.<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Nanodrop of pooled</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>MMP9 sample</td><br />
<td>NO DATA</td><br />
<td>NO DATA</td><br />
</tr><br />
<tr><br />
<td>MMP9 bb P00L</td><br />
<td>169.8</td><br />
<td>1.91</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Saturday 21st September</b><br />
</p><br />
<p class="body_text"><br />
Minipred-ed 10 ml LB 4x CMP ZEC BB Sample 1 (2z.1) and 6 (7z.2) (from second attempt of zeocin and backbone ligation)<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Tube no.</th><br />
<th>ng/ul</th><br />
<th>Absorption</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>3.2</td><br />
<td>0.087</td><br />
<td>2.41</td><br />
</tr><br />
<tr><br />
<td>26</td><br />
<td>2.9</td><br />
<td>0.068</td><br />
<td>2.51</td><br />
</tr><br />
<tr><br />
<td>15</td><br />
<td>20.1</td><br />
<td>1.249</td><br />
<td>1.70</td><br />
</tr><br />
<tr><br />
<td>18</td><br />
<td>21.1</td><br />
<td>0.590</td><br />
<td>1.48</td><br />
</tr><br />
<tr><br />
<td>21</td><br />
<td>8.3</td><br />
<td>0.186</td><br />
<td>1.20</td><br />
</tr><br />
<tr><br />
<td>28</td><br />
<td>8.4</td><br />
<td>0.178</td><br />
<td>1.32</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Pooled all 6 samples + Yanika’s minipreped sample IA+IB<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th></th><br />
<th>ng/ul</th><br />
<th>Abs</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>Sample</td><br />
<td>12.3</td><br />
<td>0.337</td><br />
<td>1.82</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Inoculated 150µl of ZEC BB 1 into 4x cmp and 150 ml LB broth overnight.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Sunday 22nd September</b><br />
</p><br />
<p class="body_text"><br />
<a href="https://2013.igem.org/Team:UCL/Project/Protocols">Nanodrop</a> readings (after maxi-prep) of Zec sample 1, ng/ul = 40.7, 260/280 = 1.90<br />
</p><br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/Labbook/Week15Team:UCL/Labbook/Week152013-10-05T01:33:37Z<p>Naruto: </p>
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<p class="major_title">Lab Weeks</p><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="full_page"><br />
<p class="body_text"> <a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a> <br />
</p> <br />
</div><br />
<br />
<p class="minor_title">Week 15</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Bacterial Labs</b><br />
</p><br />
<p class="body_text"><br />
<b>Monday 9th September<b><br />
</p><br />
<p class="body_text"><br />
Contamination control was set using 15 ml Falcons with 3 ml LB broth and 3 ul cmp or 3 ul amp.<br />
</p><br />
<p class="body_text"><br />
Retransformation of zeocin pSB1C3 ligations from 4 September and 6:1 zeocin and cmv ligations from 31 of August.<br />
</p><br />
<p class="body_text"><br />
Tubes ligations used: 5z, 6z, 7z, 8z and 9, 10 controls from 4 of September and 2z from 31 of August. These were plated and left over-night.<br />
</p><br />
<p class="body_text"><br />
LIGATION 3:<br />
</p><br />
<p class="body_text"><br />
pSB1C3 digested and purified, concentration = 25 ng/ul <br />
<br />
</p><br />
<p class="body_text"><br />
zeo digested and purified conc = 77 ng/ul<br />
</p><br />
<p class="body_text"><br />
CMV digested and purified conc = 25 ng/ul<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Zeocin (77 ng/ul)</th><br />
<th>3 to 1 (mass ratio) [z1]</th><br />
<th>6 to 1 (mass ratio) [z2]</th><br />
</tr><br />
<tr><br />
<td>water (ul)</td><br />
<td>6</td><br />
<td>4</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>backbone (ul)</td><br />
<td>2</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>insert (ul)</td><br />
<td>2</td><br />
<td>4</td><br />
</tr><br />
<tr><br />
<td>ligase (ul)</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Cmv (25 ng/ul)</th><br />
<th>3 to 1 (mass ratio) [c3]</th><br />
<th>6 to 1 (mass ratio) [c4]</th><br />
</tr><br />
<tr><br />
<td>water (ul)</td><br />
<td>2</td><br />
<td>3</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>backbone (ul)</td><br />
<td>2</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>insert (ul)</td><br />
<td>6</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>ligase (ul)</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Controls</th><br />
<th>5 ctrl check no circular backbone</th><br />
<th>6 ctrl check digestion process</th><br />
<th>7 ctrl (uncut backbone)</th><br />
</tr><br />
<tr><br />
<td>water (ul)</td><br />
<td>7</td><br />
<td>18</td><br />
<td>18</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>backbone (ul)</td><br />
<td>3</td><br />
<td>3</td><br />
<td>3 of uncut backbone</td><br />
</tr><br />
<tr><br />
<td>insert (ul)</td><br />
<td>0</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>ligase (ul)</td><br />
<td>1</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These ligation reactions were stored at -20 degrees Celsius and used in the following day for <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> transformation</a>.<br />
</p><br />
<br />
<p class="body_text"><br />
Reinoculated K812014 from glycerol stocks in order to increase the stocks of backbone pSB1C3. 15 ml Falcon contained 3 ul cell culture, 3 ml LB broth and 3 ul chloramphenicol (cmp).<br />
</p><br />
<br />
<p class="body_text"><br />
These were incubated overnight and then <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> minipreped</a>.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Tuesday 10th September<b><br />
</p><br />
<br />
<p class="body_text"><br />
Colony counts of the re-transformation of ligations:<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Plate</th><br />
<th>Colony counts</th><br />
</tr><br />
<tr><br />
<td>2z from 31/08</td><br />
<td>100</td><br />
</tr><br />
<tr><br />
<td>5z from 4/09</td><br />
<td>150+</td><br />
</tr><br />
<tr><br />
<td>6z from 4/09</td><br />
<td>80+</td><br />
</tr><br />
<tr><br />
<td>7z from 4/09</td><br />
<td>100</td><br />
</tr><br />
<tr><br />
<td>8z from 4/09</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>9z - ctrl from 4/09</td><br />
<td>40</td><br />
</tr><br />
<tr><br />
<td>11z - ctrl from 4/09</td><br />
<td>0</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
MMP9 in cmv hygro plasmid was used to transform home-made <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> competent cells</a> in order to built a stock. <br />
</p><br />
<p class="body_text"><br />
Colony count - 90% - 100+ and 10% 80 colonies.<br />
</p><br />
<br />
<p class="body_text"><br />
Biobricks (K81 and J63) as well as potential recombinant plasmid transformations (transformations of 31 August) were inoculated from glycerol stocks and minipreped in order to achieve a strong stock of pSB1C3 backbone.<br />
</p><br />
<p class="body_text"><br />
The minipreps resulted showed an average concentration of about 30 ng/ul (highest concentrations were found for one of the miniprep of K812014 biobrick - 34.6 ng/ul and for a transformation of cell vial 28: 67.6 ng/ul.<br />
</p><br />
<p class="body_text"><br />
The following day, some of these, showing high concentrations, were E and P analytical digested in 30 ul reaction volumes.<br />
The nanodrop result of the MMP9 miniprep was 42 ng/ul with 260/280 = 2.05.<br />
</p><br />
<p class="body_text"><br />
Building up the competent cell stock using the home-made competent cells<br />
</p><br />
<p class="body_text"><br />
Using the cells of a remaining cell vial of homemade competent cells, 10 <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> LB agar plates</a> were spread<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Plate number</th><br />
<th>Drug</th><br />
<th>Volume competent cells (ul)</th><br />
<th>Colony count</th><br />
</tr><br />
<tr><br />
<td>I</td><br />
<td>1000 X CMP</td><br />
<td>20</td><br />
<td>60</td><br />
</tr><br />
<tr><br />
<td>II</td><br />
<td>500 X CMP</td><br />
<td>20</td><br />
<td>15</td><br />
</tr><br />
<tr><br />
<td>III</td><br />
<td>250 X CMP</td><br />
<td>20</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>IV</td><br />
<td>N/A</td><br />
<td>20</td><br />
<td>100+</td><br />
</tr><br />
<tr><br />
<td>V</td><br />
<td>1000 X CMP</td><br />
<td>20</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>VI</td><br />
<td>500 X CMP</td><br />
<td>20</td><br />
<td>3</td><br />
</tr><br />
<tr><br />
<td>VII</td><br />
<td>250 X CMP</td><br />
<td>20</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>VIII</td><br />
<td>N/A</td><br />
<td>20</td><br />
<td>100+</td><br />
</tr><br />
<tr><br />
<td>IX</td><br />
<td>1000 X CMP</td><br />
<td>100 from T vial</td><br />
<td>15</td><br />
</tr><br />
<tr><br />
<td>X</td><br />
<td>N/A</td><br />
<td>100 from T vial</td><br />
<td>100+</td><br />
</tr><br />
<tr><br />
<td>I</td><br />
<td>1000 X CMP</td><br />
<td>20</td><br />
<td>60</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Colonies from plate IX and X were picked and taken through the competent cell test using 250 x (4x) for drug plate.<br />
</p><br />
<p class="body_text"><br />
New inoculations (in 3 ml LB broth and 3 ul cmp) of the transformations from 4/09 and 9/09 (?) using 8 colonies from each of the following plates: 2 z, 5 z, 6 z, 7 z were prepared and left in the incu-shaker in the usual conditions over night.<br />
<br />
</p><br />
<p class="body_text"><br />
Transformation number 3:<br />
</p><br />
<p class="body_text"><br />
We transformed 7 homemade <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> competent cells</a> using 5 ul of each of the 7 ligation reactions set on the 9/09 (check date) as well as one more of these cell vials with pSecTag2A plasmid (concentration 30 ng/ul) as another control.<br />
</p><br />
<p class="body_text"><br />
The c3 ligation (1:3) was lost as one of the cell vials containing was taken by mistake by someone from the team.<br />
</p><br />
<p class="body_text"><br />
After the <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> transformation</a> procedures these cells were spread on agar plates containing 2x cmp and 2 x amp for the pSecTag2A biobrick.<br />
</p><br />
<p class="body_text"><br />
On the same day, 10 ul of z2, c4 and pSecTag2A (of potentially transformed cells) was inoculated with 2 ul cmp and 2 ml LB broth.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Wednesday 11th Sepetmber</b><br />
</p><br />
<br />
<p class="body_text"><br />
Re-inoculations of <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> glycerol stocks</a> of biobricks (those in pSB1C3 backbone - J63.. and K81 as well as that in pSecTag2A) and potential recombinant plasmids were made in an attempt to grow the stock of pSB1C3 backbone. <br />
There was no growth for 4 out of the 6 inoculations made for J63 biobrick and also no growth for transformed cells from original vial no. 28.<br />
</p><br />
<br />
<p class="body_text"><br />
Labeling of the 8 colony inoculations of each of the potential transformation with potentially recombinant pSB1C3 + zeocin (31 and 4/09 transformations):<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Falcon Tube no.</th><br />
<th>Content: Plate & Colony</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>2z.1</td><br />
<td>54.0</td><br />
<td>1.82</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>2z.2</td><br />
<td>28.3</td><br />
<td>2.21</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>2z.3</td><br />
<td>25.6</td><br />
<td>3.03</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>2z.4</td><br />
<td>20.3</td><br />
<td>3.16</td><br />
</tr><br />
<tr><br />
<td>5</td><br />
<td>2z.5</td><br />
<td>13.4</td><br />
<td>3.08</td><br />
</tr><br />
<tr><br />
<td>6</td><br />
<td>2z.6</td><br />
<td>15.7</td><br />
<td>2.61</td><br />
</tr><br />
<tr><br />
<td>7</td><br />
<td>2z.7</td><br />
<td>8.9</td><br />
<td>14.17</td><br />
</tr><br />
<tr><br />
<td>8</td><br />
<td>2z.8</td><br />
<td>14.8</td><br />
<td>4.08</td><br />
</tr><br />
<tr><br />
<td>9</td><br />
<td>5z.1</td><br />
<td>64.9</td><br />
<td>1.70</td><br />
</tr><br />
<tr><br />
<td>10</td><br />
<td>5z.2</td><br />
<td>36.5</td><br />
<td>1.93</td><br />
</tr><br />
<tr><br />
<td>11</td><br />
<td>5z.3</td><br />
<td>16.6</td><br />
<td>2.01</td><br />
</tr><br />
<tr><br />
<td>12</td><br />
<td>5z.4</td><br />
<td>32.1</td><br />
<td>2.22</td><br />
</tr><br />
<tr><br />
<td>13</td><br />
<td>5z.5</td><br />
<td>23.6</td><br />
<td>2.50</td><br />
</tr><br />
<tr><br />
<td>14</td><br />
<td>5z.6</td><br />
<td>53.3</td><br />
<td>1.87</td><br />
</tr><br />
<tr><br />
<td>15</td><br />
<td>5z.7</td><br />
<td>33.7</td><br />
<td>2.22</td><br />
</tr><br />
<tr><br />
<td>16</td><br />
<td>5z.8</td><br />
<td>50.1</td><br />
<td>2.06</td><br />
</tr><br />
<tr><br />
<td>17</td><br />
<td>6z.1</td><br />
<td>74.8</td><br />
<td>1.71</td><br />
</tr><br />
<tr><br />
<td>18</td><br />
<td>6z.2</td><br />
<td>32.6</td><br />
<td>1.82</td><br />
</tr><br />
<tr><br />
<td>19</td><br />
<td>6z.3</td><br />
<td>26.0</td><br />
<td>2.25</td><br />
</tr><br />
<tr><br />
<td>20</td><br />
<td>6z.4</td><br />
<td>42.5</td><br />
<td>2.15</td><br />
</tr><br />
<tr><br />
<td>21</td><br />
<td>6z.5</td><br />
<td>30.8</td><br />
<td>2.02</td><br />
</tr><br />
<tr><br />
<td>22</td><br />
<td>6z.6</td><br />
<td>27.3</td><br />
<td>2.23</td><br />
</tr><br />
<tr><br />
<td>23</td><br />
<td>6z.7</td><br />
<td>38.0</td><br />
<td>2.15</td><br />
</tr><br />
<tr><br />
<td>24</td><br />
<td>6z.8</td><br />
<td>43.5</td><br />
<td>2.10</td><br />
</tr><br />
<tr><br />
<td>25</td><br />
<td>7z.1</td><br />
<td>62.7</td><br />
<td>1.80</td><br />
</tr><br />
<tr><br />
<td>26</td><br />
<td>7z.2</td><br />
<td>31.7</td><br />
<td>1.89</td><br />
</tr><br />
<tr><br />
<td>27</td><br />
<td>7z.3</td><br />
<td>26.4</td><br />
<td>1.88</td><br />
</tr><br />
<tr><br />
<td>28</td><br />
<td>7z.4</td><br />
<td>21.4</td><br />
<td>2.12</td><br />
</tr><br />
<tr><br />
<td>29</td><br />
<td>7z.5</td><br />
<td>13.1</td><br />
<td>2.28</td><br />
</tr><br />
<tr><br />
<td>30</td><br />
<td>7z.6</td><br />
<td>27.2</td><br />
<td>2.22</td><br />
</tr><br />
<tr><br />
<td>31</td><br />
<td>7z.7</td><br />
<td>-</td><br />
<td>-</td><br />
</tr><br />
<tr><br />
<td>32</td><br />
<td>7z.8</td><br />
<td>33.0</td><br />
<td>2.28</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Colony counts of the transformed cells with the ligation prepared on the 9/09<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Plate</th><br />
<th>Colony Count</th><br />
</tr><br />
<tr><br />
<td>Z1</td><br />
<td>12</td><br />
</tr><br />
<tr><br />
<td>Z2</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>C4</td><br />
<td>1 (+small colonies)</td><br />
</tr><br />
<tr><br />
<td>5</td><br />
<td>50</td><br />
</tr><br />
<tr><br />
<td>6</td><br />
<td>50</td><br />
</tr><br />
<tr><br />
<td>7</td><br />
<td>17</td><br />
</tr><br />
<tr><br />
<td>pSecTag2A</td><br />
<td>10</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Inoculations for Z1 and C4 were prepared (3 colonies for Z1 plate were picked as well as the only colony on C4 plate; all inoculatons were made in 2 LB broth and 4 ul chloramphenicol and left for 16 hours in the incu-shaker at 200 rpm, 370 C).<br />
</p><br />
<p class="body_text"><br />
Prep digest of linearised pSB1C3 from the 2013 High school distribution kit with EcoR1 and Pst1 in a 40 ul volume reaction (using 25 ul DNA and 2 ul of each E and P). This was incubated for 2 hours and then freezed. Before it was used in another ligation, it was heat inactivated at 80 degrees Celsius for 20 min).<br />
</p><br />
<p class="body_text"><br />
Analytical digest of zeocin and K812014 biobrick with Stu1 restriction enzyme. Expected bands:<br />
- For zeocin 2 bands: one of 1037 bp and another of 783 bp<br />
- For K812014, 4 bands of 1589 bp, 877 bp, 311 bp and 175 bp<br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/4/44/Herpaderpa.png');height:527px;width:700px"></div><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Item</th><br />
<th>ul</th><br />
</tr><br />
<tr><br />
<td>DNA zeocin (55 ng/ul)/K812014 (34.6 ng/ul)</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Stu1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 4</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>3</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>10</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
The zeocin tube (PCR purified) of 77 ng/ul was diluted by adding 10 ul of RO water (new concentration around 55 ng/ul).<br />
These cuts were run on a gel, next to uncuts.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Thursday 12th September<b><br />
</p><br />
<p class="body_text"><br />
Prep digest of samples from <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> minipreped</a> inoculations 1, 14, 16, 17, 24.<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Contents</th><br />
<th>Volumes (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>14</td><br />
</tr><br />
<tr><br />
<td>Stu1</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>Buffer 2</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>30</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
After EcoR1 and Pst1 digestion and running a gel of the potential backbone sources for building up the pSB1C3 stocks we decided to avoid using biobrick K812014 due to the presence of 3 bands instead of 2. Instead we decided to use J632014 biobrick as a pSB1C3 source for now on because it shows the correct number of bands and lengths of the digested DNA material.<br />
</p><br />
<p class="body_text"><br />
Prep digest of pSB1C3 (E, P digested the day before) with Dpn1 – added 2 ul of Dpn1 to the volume of 40 ul of double digested pSB1C3 and incubated it for 1 hour at 37O C. This was done in order to prepare the backbone for ligation. Also, before ligation, the tube was heat inactivated at 80O C for 22 minutes.<br />
</p><br />
<br />
<p class="body_text"><br />
Ligation 4<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Ligations</th><br />
<th>AA1</th><br />
<th>BB1</th><br />
<th>CC1</th><br />
</tr><br />
<tr><br />
<td>EP cut, purified pAZEC fragment (53ng/ul)</td><br />
<td>2</td><br />
<td>0</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>*EPD cut pSB1C3</td><br />
<td>2</td><br />
<td>2</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>T4 DNA ligase buffer</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>**Vol. of dilute T4 ligase</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>RO Water</td><br />
<td>4</td><br />
<td>6</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>Cell Counts</td><br />
<td>0</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
* before EPD (E –EcoR1, P- Pst1, D-Dpn1) digestion it was in a concentration of 25 ng/ul;<br />
</p><br />
<p class="body_text"><br />
** Diluted by adding 4 ul of RO water to 4 ul of DNA T4 ligase from Alex.<br />
These were left on the bench for 30 minutes and then heat inactivated for 20 minutes at 80O C.<br />
</p><br />
<p class="body_text"><br />
<u>Transformation 4</u><br />
</p><br />
<p class="body_text"><br />
Two ul of each of these ligations were used to transform One Shot Top10 Competent Cells (2004) offered by Darren. The specific <a href="http://tools.lifetechnologies.com/content/sfs/manuals/oneshottop10_man.pdf" target="_blank"> transformation protocol</a> for this type of cells was followed apart from adjustments on step 8 where after incu-shaking at 225 rpm for one hour, the tubes were pelleted for 2 minutes and the supernatant was temporarily removed from each tube and each pellet was resuspened in 100 ul of the previously removed supernatant.<br />
3 LB agar plates with 5x chloramphenicol were prepared and the 3 <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> transformations</a> were spread on these.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Friday 13th September</b><br />
</p><br />
<p class="body_text"><br />
PCR of bb zeocin using <a href="https://www.neb.com/protocols/1/01/01/protocol-for-a-routine-taq-pcr-reaction" target="_blank"> Taq PCR kit New England Biolabs</a> in 50 ul reaction volume.<br />
</p><br />
<p class="body_text"><br />
Nanodrop readings of the miniprep of the inoculations of Z1 from the 11/09<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Sample</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>Z1.1</td><br />
<td>24.8</td><br />
<td>1.80</td><br />
</tr><br />
<tr><br />
<td>Z1.2</td><br />
<td>35.4</td><br />
<td>1.98</td><br />
</tr><br />
<tr><br />
<td>Z1.3</td><br />
<td>34.2</td><br />
<td>1.93</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Accomplished tasks asked by Darren:<br />
</p><br />
<p class="body_text"><br />
- Spread straight from the glycerol stock of J632014 on 4 plates (0 x cmp, 2 x cmp, 4 x cmp and 5 x cmp) in order to build up the stocks of pSB1C3 backbone source. There was no growth the following morning.<br />
</p><br />
<p class="body_text"><br />
- Spread the remainder of transformed cells (12/09): 50 % on 5 x cmp plate while the other half on 2 x cmp. These were pelleted for 4 minutes.<br />
</p><br />
<p class="body_text"><br />
- Plan MMP9 PCR with Taq DNA Polymerase: 2 reactions making use of the 2 types of primers as well as running 2 controls (with no template).<br />
</p><br />
<p class="body_text"><br />
- New transformation using One Shot Top10 Competent Cells. For each ligation type, spread the cells 50% on 1xcmp and the other half on 5xcmp.<br />
</p><br />
<p class="body_text"><br />
Candidate recombinant plasmid (zeo + pSB1C3) analytical single digest with Stu1 (expecting 4 kb band) and BSCr1 (expecting 2 bands: one of 3435 bp and the other of 508 bp) and double digest with EcoR1 and Pst1 (expecting 2 kb band). <br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/7/7b/Sep_13_UCLiGEM2013.png');height:554px;width:650px"></div><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Contents</th><br />
<th>E&P digest (ul)</th><br />
<th>BscR1 digest (ul)</th><br />
<th>Stu1 digest (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>5</td><br />
<td>5</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>EcoR1</td><br />
<td>1</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>1</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Stu1</td><br />
<td>0</td><br />
<td>0</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>BscR1</td><br />
<td>0</td><br />
<td>1</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>1</td><br />
<td>0</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 4</td><br />
<td>0</td><br />
<td>1</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
<td>0.5</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>1.5</td><br />
<td>2.5</td><br />
<td>2.5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Saturday 14th September</b><br />
</p><br />
<p class="body_text"><br />
Colony counts of the transformation prepared on 12th and 13th of September.<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Plate</th><br />
<th>AA1 5xcmp</th><br />
<th>AA1 2xcmp</th><br />
<th>AA1 1xcmp</th><br />
<th>BB1 5xcmp</th><br />
<th>BB1 2xcmp</th><br />
<th>BB1 1xcmp</th><br />
<th>CC1 5xcmp</th><br />
<th>CC1 2xcmp</th><br />
<th>CC1 1xcmp</th><br />
</tr><br />
<tr><br />
<td>12th Sep</td><br />
<td>1</td><br />
<td>0</td><br />
<td>-</td><br />
<td>3</td><br />
<td>28</td><br />
<td>-</td><br />
<td>0</td><br />
<td>0</td><br />
<td>-</td><br />
</tr><br />
<tr><br />
<td>13th Sep</td><br />
<td>0</td><br />
<td>-</td><br />
<td>30</td><br />
<td>0</td><br />
<td>-</td><br />
<td>3</td><br />
<td>0</td><br />
<td>-</td><br />
<td>0</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
8 inoculations in 2 ml LB and 1xcmp were made using picked colonies from plate AA1, 1xcmp. 1 inoculation was prepared in 4xcmp for the singular colony on AA1 5 x cmp which grew after more than 20 hours of incubation.<br />
</p><br />
<p class="body_text"><br />
Nanodrop result of these minipreps – data from the next day<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>AA1 1xcmp colony number</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>26.8</td><br />
<td>1.86</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>24.8</td><br />
<td>1.94</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>35.1</td><br />
<td>1.90</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>28.2</td><br />
<td>1.95</td><br />
</tr><br />
<tr><br />
<td>5</td><br />
<td>30.5</td><br />
<td>1.94</td><br />
</tr><br />
<tr><br />
<td>6</td><br />
<td>28.7</td><br />
<td>1.99</td><br />
</tr><br />
<tr><br />
<td>7</td><br />
<td>35.1</td><br />
<td>1.85</td><br />
</tr><br />
<tr><br />
<td>8</td><br />
<td>40.3</td><br />
<td>1.88</td><br />
</tr><br />
</table><br />
<p><br />
<br />
<p class="body_text"><br />
The inoculation in 4xcmp from the plate AA1 5xcmp showed a concentration of 40.1 ng/ul and 260/280 coefficient equal to 1.89.<br />
</p><br />
<p class="body_text"><br />
PCR of bb zeocin was set using Taq polymerase to make three 50 ul volume reactions and a control with no pSecTag2A template.<br />
</p><br />
<p class="body_text"><br />
A gel was run to check MMP9 PCR and zeocin PCR from 5/09 samples z1, z2, z5, z7 and control 9. The correct bands appeared apart from z2 and z7 which showed no DNA.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Sunday 15th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
Prepared 9 <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> glycerol stocks</a> from the 9 inoculations made the day before while the rest of each of these inoculations was <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> minipreped</a>.<br />
</p><br />
<p class="body_text"><br />
From the plates of the day before, other 20 colonies from AA1 1xcmp plate (falcons 1.1 – 1.20) and 3 colonies from AA1 5xcmp (falcons 5.1 to 5.3) were picked and inoculated in 4xcmp LB broth.<br />
</p><br />
<p class="body_text"><br />
Ligation and transformation of One Shot Top ten Competent cells with MMP9 and EPD triple digested pSB1C3 (form high school iGEM distribution kit – label hs):<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Volume (ul)</th><br />
<th>MMP9 4bb</th><br />
<th>MMP9 4bb</th><br />
<th>Control (no backbone)</th><br />
<th>Control (no insert)</th><br />
<th>MMP9 3</th><br />
</tr><br />
<tr><br />
<td>Water</td><br />
<td>2.5</td><br />
<td>2.5</td><br />
<td>5</td><br />
<td>7.5</td><br />
<td>2.5</td><br />
</tr><br />
<tr><br />
<td>pSB1C3</td><br />
<td>2.5 (hs)</td><br />
<td>2.5 (from K812014)</td><br />
<td>0</td><br />
<td>2.5 (hs)</td><br />
<td>2.5 (hs)</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>T4 ligase</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Insert - MMP9</td><br />
<td>5</td><br />
<td>5</td><br />
<td>5</td><br />
<td>0</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
<tr><br />
<td>Vial Labels</td><br />
<td>1</td><br />
<td>2</td><br />
<td>3</td><br />
<td>4</td><br />
<td>5</td><br />
</tr><br />
</table><br />
</p><br />
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<p class="body_text"><br />
<b>Mammalian Labs</b><br />
</p><br />
<p class="body_text"><br />
<b>Tuesday 10th September</b><br />
</p><br />
<p class="body_text"><br />
Threw away 12 over-confluent ( aprox. 150%) plates (P15).<br />
5 P16 plates with 90% confluency were passaged and then split 1:4. Made up 5 new p17 plates.<br />
</p><br />
<p class="body_text"><br />
Added FBS and PS intp DMEM media to make new solution.<br />
</p><br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/AchievementsTeam:UCL/Achievements2013-10-05T01:26:46Z<p>Naruto: </p>
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<p class="major_title">MEDAL CRITERIA</p><br />
<br />
<p class="minor_title">Bronze Medal</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have registered our <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">team</a> of eleven undergraduates. <br />
</p><br />
<p class="body_text"><br />
• We have completed the judging form.<br />
</p><br />
<p class="body_text"><br />
• We have created a beautiful team wiki in keeping with the iGEM guidelines.<br />
</p><br />
<p class="body_text"><br />
• We have a poster and presentation ready for the regional jamboree in Lyon.<br />
</p><br />
<p class="body_text"><br />
• We have developed two new <a href="https://2013.igem.org/Team:UCL/Project/Parts" target="_blank">BioBricks</a> and have submitted them to the iGEM Registry<br />
</div><br />
<br />
<p class="minor_title">Silver Medal</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have proved our zeocin resistance BioBrick <a href="https://2013.igem.org/Team:UCL/Project/Parts" target="_blank">worked as expected</a> by characterising it in HeLa cells. We did this by by creating a zeocin kill curve with and without our <a href="https://2013.igem.org/Team:UCL/Project/Marker" target="_blank">zeocin resistance</a> BioBrick. <br />
</p><br />
<p class="body_text"><br />
• Due to the gravity of Alzheimer's disease and the perceived sovereignty of the brain, we have taken the ethics of using synthetic biological treatments very seriously. We produced a <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">neuroethics</a> and a <a href="https://2013.igem.org/Team:UCL/Practice/Report" target="_blank">feasibility</a> report, consulted numerous experts and provided a concise but detailed <a href="https://2013.igem.org/Team:UCL/Background" target="_blank">background</a> to our project, which shows how our proposed <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">genetic circuit</a> is advised by multiple theories for the causation of Alzheimer's pathology.<br />
</p><br />
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<br />
<p class="minor_title"><br />
Gold Medal<br />
</p><br />
<div class="full_row"><br />
<div class="gap"><br />
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<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
• We collaborated with iGEM Westminster, by modelling how well their bed-bug killing device will operate in a bedroom as well advising them on how to run a speed debate using our format. One of our team members, Alex Bates, also attended as guest speaker at their speed debate event.<br />
</p><br />
<p class="body_text"><br />
Full details on collaboration with iGEM Westminster can be found below.<br />
</p><br />
<p class="body_text"><br />
• Our <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">Human Practices</a> deal with an entirely new area for iGEM and, indeed, almost a completely new avenue of research for synthetic biology as a field - the <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">fusion</a> of neuroscience and synthetic biology. We use a variety of strong methods for social and ethical analysis, and outreach. Please see the section below for a summary of what we achieved.<br />
</p><br />
<p class="body_text"><br />
• Outside of this theme, we also engaged in outreach by training and advising the UCL Academy iGEM high school team. This was the first time a British iGEM team has helped run an iGEM HS team.<br />
</p><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<p class="major_title">ABOVE AND BEYOND</p><br />
<br />
<p class="minor_title">Project</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• Developed and experimentally validated the first selectable marker (Zeocin) in a mammalian system (HeLa cells). <br />
</p><br />
</div><br />
<br />
<p class="minor_title">Human Practices</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have looked at an entirely new ethical area for iGEM that has also essentially not been covered in academia; the neuroethics of genetic engineering. We have dubbed this <a href="https://2013.igem.org/Team:UCL/Practice" target="_blank">'Neuro-genethics'</a>. <br />
</p><br />
<p class="body_text"><br />
• We have produced an extensive 20 page <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">report</a> that looks into neuro-genethics and what synthetic biology could achieve in neuroscience.<br />
</p><br />
<p class="body_text"><br />
• We have engaged the public on this topic by getting their opinions at the <a href="http://www.artscatalyst.org/" target="_blank">Arts Catalyst</a>, running a <a href="https://2013.igem.org/Team:UCL/Practice/Debate" target="_blank">speed debate</a> and a <a href="https://2013.igem.org/Team:UCL/Practice/TED" target="_blank">TED debate</a>, conducting an <a href="https://2013.igem.org/Team:UCL/Practice/Survey" target="_blank">online survey</a> and producing a <a href="https://2013.igem.org/Team:UCL/Practice/Documentary" target="_blank">documentary </a> on synthetic neurobiology.<br />
</p><br />
<p class="body_text"><br />
• We ran one of iGEM's first <a href="https://2013.igem.org/Team:UCL/Practice/Creative" target="_blank">creative writing competitions</a>, to gauge public opinion on brain modification and highligh5t the impact of fiction on society's views.<br />
</p><br />
<p class="body_text"><br />
• Petcha Kutcha style presentation to enguage prospective students of the UCL Engineering Department about iGEM.<br />
</p><br />
<p class="body_text"><br />
• We created an original memory bank, <a href="https://2013.igem.org/Team:UCL/Memories" target="_blank">Eternal Sunshine</a>, which highlights how precious memories are, indicating the desperate need to cure Alzheimer's disease. <br />
</p><br />
<p class="body_text"><br />
• We created a <a href="https://2013.igem.org/Team:UCL/Practice/Report" target="_blank">feasibility report</a> on implementing our treatment.<br />
</p><br />
<p class="body_text"><br />
• Attended YSB 1.0 along with other UK iGEM teams to discuss projects and potential collaborations. <br />
</p><br />
</div><br />
<br />
<p class="minor_title">Modelling</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We are the first iGEM team to use a <a href="https://2013.igem.org/Team:UCL/Modeling" target="_blank">protein network analysis approach</a>. Network based bioinformatics can feedback into synthetic biology by informing the choice of parts in therapeutic genetic circuits.<br />
</p><br />
</div><br />
<br />
<p class="minor_title">Wiki</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have developed an, original attractive wiki using art work by our artists in residence, <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Fong Yi Khoo</a> and <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Oran Maguire</a>.<br />
</p><br />
<p class="body_text"><br />
• We have included an extensive neuroscience background section, which explains and compares multiple theories for the causation of Alzheimer's disease, so that readers can fully understand the pros and cons of our genetic circuit. <br />
</p><br />
<p class="body_text"><br />
• We have included a full complement of citations that link to PubMed pages so that it is easy to see from where our ideas and explanations have been drawn, and which papers have inspired us.<br />
</p><br />
</div><br />
<br />
<p class="minor_title">Collaboration with Westminster iGEM</p><br />
<div class="full_row"><br />
<div class="gap"><br />
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<p class="body_text"><br />
<b>Model (ROBIN WRITES HERE)</b><br />
</p><br />
<p class="body_text"><br />
Movement of bed-bugs towards blood meal<br />
</div><br />
<br />
<p class="minor_title">Mentoring iGEM HS</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Two members of the UCL iGEM team volunteered as advisers to the UCL Academy iGEM team – <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Ruxi</a> and <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Khaicheng</a>, under the guidance of Aurelija Grigonyte, a member of the UCL iGEM 2012 team. During the high school team’s brainstorming process, we provided them with guidance and resources for their research. We also supervised their lab work in the UCL Biochemical Engineering department.<br />
</p><br />
<p class="body_text"><br />
UCL is the first university in the UK to be the sole sponsor of an academy – a non-selective mixed state school in our home borough of Camden. UCL Academy represents a unique opportunity to blur the boundaries between secondary and higher education.<br />
</p><br />
<p class="body_text"><br />
The academy is one of the first UK high schools to participate in iGEM this year, and is the only UK team so far to have attended the <a href="https://2013hs.igem.org/Main_Page" target="_blank">High School iGEM Jamboree</a> at MIT, Boston. The team aimed to revolutionise the recycling industry by proposing a home system that converts cellulose into glucose, allowing the up-cycling of paper into a commercial product of bioplastic - polyhydroxybutyrate (PHB) <br />
</p><br />
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</p><br />
<p class="body_text"><br />
For information about their iGEM project, check out their wiki <a href="https://2013hs.igem.org/Team:UCL_Academy" target="_blank">here</a>. <br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/Labbook/Week14Team:UCL/Labbook/Week142013-10-05T01:24:11Z<p>Naruto: </p>
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<p class="major_title">Lab Weeks</p><br />
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<p class="body_text"> <a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a> <br />
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<br />
<p class="minor_title">Week 14</p><br />
<div class="full_row"><br />
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<b>Bacterial Lab</b><br />
</p><br />
<p class="body_text"><br />
<b>Monday 2nd September</b><br />
</p><br />
<br />
<p class="body_text"><br />
We re-autoclaved water due to our suspicion it was nuclease contaminated.<br />
New inoculations (in 3 ml LB) of the transformations were prepared: 5 using cmv ligation and 5 using the zeocin ligation. These were left over night into the incushaker.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Tuesday 3rd September</b><br />
</p><br />
<br />
<p class="body_text"><br />
The falcons were removed from the incu-shaker. <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> Glycerol stocks</a> were prepared using 0.5 ml while the rest was kept for <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> miniprepping</a>.<br />
</p><br />
<br />
<p class="body_text"><br />
Preparative digest of zeocin and K812014 plasmid<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Components (ul)</th><br />
<th>Prep digest of Zeocin</th><br />
<th>Prep digest of K812014</th><br />
</tr><br />
<tr><br />
<td>Zeo/K812014</td><br />
<td>80+</td><br />
<td>20+</td><br />
</tr><br />
<tr><br />
<td>EcoRI</td><br />
<td>10</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>PstI</td><br />
<td>10</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>15</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>3</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>22</td><br />
<td>14</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>150</td><br />
<td>50</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These were incubated for 1 hour at 37ºC and for 20 minute heat inactivated.<br />
</p><br />
<p class="body_text"><br />
Nanodrop of the minipreps from the new inoculations.<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>pDNA new</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>51/1</td><br />
<td>9.5</td><br />
<td>2.2</td><br />
</tr><br />
<tr><br />
<td>51/2</td><br />
<td>9.9</td><br />
<td>1.91</td><br />
</tr><br />
<tr><br />
<td>51/3</td><br />
<td>8.9</td><br />
<td>1.93</td><br />
</tr><br />
<tr><br />
<td>51/4</td><br />
<td>8.6</td><br />
<td>2.31</td><br />
</tr><br />
<tr><br />
<td>51/5</td><br />
<td>13.2</td><br />
<td>2.49</td><br />
</tr><br />
<tr><br />
<td>45/1</td><br />
<td>8.2</td><br />
<td>1.89</td><br />
</tr><br />
<tr><br />
<td>45/2</td><br />
<td>8.7</td><br />
<td>2.65</td><br />
</tr><br />
<tr><br />
<td>45/3</td><br />
<td>10.7</td><br />
<td>2.13</td><br />
</tr><br />
<tr><br />
<td>45/4</td><br />
<td>9.1</td><br />
<td>2.00</td><br />
</tr><br />
<tr><br />
<td>45/5</td><br />
<td>9.7</td><br />
<td>2.34</td><br />
</tr><br />
<tr><br />
<td>28 glycerol stock</td><br />
<td>40.4</td><br />
<td>2.23</td><br />
</tr><br />
<tr><br />
<td>45 glycerol stock</td><br />
<td>36.7</td><br />
<td>2.24</td><br />
</tr><br />
<tr><br />
<td>51 glycerol stock</td><br />
<td>45.4</td><br />
<td>2.18</td><br />
</tr><br />
<tr><br />
<td>52 glycerol stock</td><br />
<td>28.4</td><br />
<td>1.98</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These samples of minipreps and from glycerol stocks were run on a gel in order to check if the circularised potentially recombinant plasmid is present. Again, there were no bands for any of these samples, hence we tend to believe the ligations were not successful OR/AND the transformation was faulty.<br />
</p><br />
<p class="body_text"><br />
Gel extraction <br />
- of digested E and P - K812014 biobrick plasmid<br />
After gel extraction purification of the corresponding 2 kb band, the concentration showed up to around 6.6 ng/ul and 260/280 = 1.73. Given the very small concentration, a second attempt of obtaining pure pSB1C3 was performed, not successful though, only 4.9 ng/ul concentration.<br />
</p><br />
<p class="body_text"><br />
- of PCR purified zeocin, 2 kb band. This was purified and the nanodrop showed a concentation of 77.6 ng/ul and 260/280 = 1.82.<br />
</p><br />
<p class="body_text"><br />
Nanodrop of E, P digested cmv (from 30 of August) gave a concentration of 25.1 ng/ul, 260/280 = 1.86.<br />
</p><br />
<p class="body_text"><br />
New inoculations of K812014 biobrick were prepared and left for incubation over night in the incu-shaker (37°C, at 200 rpm).<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Wednesday 4th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
Miniprep of the K812014 biobrick inoculations showed the following concentrations:<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Tube number</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>151</td><br />
<td>1.93</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>80.8</td><br />
<td>1.93</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>111.5</td><br />
<td>1.82</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>219.1</td><br />
<td>1.94</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Samples 1 and 2 went through a preparative digest<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>item</th><br />
<th>ul</th><br />
</tr><br />
<tr><br />
<td>pDNA</td><br />
<td>50</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>EcoR1</td><br />
<td>7</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>7</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>Water</td><br />
<td>24</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>100</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Ligation 2<br />
</p><br />
<br />
<table><br />
<tr><br />
<th>Contents</th><br />
<th>ng/ul</th><br />
</tr><br />
<tr><br />
<td>pSB1C3 digested & pruified</td><br />
<td>25</td><br />
</tr><br />
<td>seo digested & purified</td><br />
<td>77</td><br />
</tr><br />
<tr><br />
<td>cmv digested & purified</td><br />
<td>25</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>CMV (25 ng/ul)</th><br />
<th>1 c (3:1 molar ratio)</th><br />
<th>2 c (2:1 molar ratio)</th><br />
<th>3 c (1:1 molar ratio)</th><br />
<th>4 c (3:1 molar ratio)</th><br />
</tr><br />
<tr><br />
<td>Water (ul)</td><br />
<td>1.8</td><br />
<td>3.2</td><br />
<td>8</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>Backbone (ul)</td><br />
<td>4</td><br />
<td>4</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>*Insert (ul)</td><br />
<td>4.2</td><br />
<td>2.8</td><br />
<td>1</td><br />
<td>3</td><br />
</tr><br />
<tr><br />
<td>Ligase (ul)</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
<tr><br />
<td>Colony count</td><br />
<td>50</td><br />
<td>100+</td><br />
<td>100+</td><br />
<td>150+</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Zeocin (77 ng/ul)</th><br />
<th>5 z (3:1 molar ratio)</th><br />
<th>6 z (2:1 molar ratio)</th><br />
<th>7 z (1:1 molar ratio)</th><br />
<th>8 z (3:1 molar ratio)</th><br />
</tr><br />
<tr><br />
<td>Water (ul)</td><br />
<td>2.1</td><br />
<td>3.3</td><br />
<td>8</td><br />
<td>8</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>Backbone (ul)</td><br />
<td>4</td><br />
<td>4</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>*Insert (ul)</td><br />
<td>3.9 from *</td><br />
<td>2.6 from *</td><br />
<td>1 from *</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Ligase (ul)</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
<tr><br />
<td>Colony count</td><br />
<td>30</td><br />
<td>0</td><br />
<td>1</td><br />
<td>0+</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
* we prepared a tube of concentration 25 ng/ul<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Controls</th><br />
<th>9</th><br />
<th>10</th><br />
<th>11</th><br />
</tr><br />
<tr><br />
<td>Water (ul)</td><br />
<td>6</td><br />
<td>7</td><br />
<td>7</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>Backbone (ul)</td><br />
<td>4</td><br />
<td>4</td><br />
<td>4 of uncut</td><br />
</tr><br />
<tr><br />
<td>*Insert (ul)</td><br />
<td>0</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Ligase (ul)</td><br />
<td>1</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
<tr><br />
<td>Comments</td><br />
<td>Check no circular backbone</td><br />
<td>Check digestion</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>Colony count</td><br />
<td>0</td><br />
<td>0</td><br />
<td>0+</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
5 ul of each ligation was used to transform our home made competent cells. After the transformation procedure, these cell vials were entirely spread on 2 x cmp plates.<br />
</p><br />
<br />
<p class="body_text"><br />
The 2 ordered oligonucleotides (1gm Oxn LF and 1gm ox LF) were annealed according to the open wet lab protocol. The thermocycler was busy at the time we booked therefore we thermocylced in the evening left in the machine at 4 degrees Celsius over night to collect the following morning.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Thursday 5th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
The pSB1C3 obtained from the postgrads was digested.<br />
</p><br />
<p class="body_text"><br />
1 colony per each plate of transformation was picked and inoculated in 2 ml LB with 4 ul cmp. These were incu-shaked over night (37 degrees Celsius and 200 rpm).<br />
</p><br />
<p class="body_text"><br />
PCR of zeocin and cmv was performed: 8 same reactions for zeocin, 3 same reactions for cmv and 2 controls (no pSecTag2A template). The zeocin and cmv reaction tubes contained a volume of 1.5 ul template. Primers used for zeocin: zec bb F,R and for cmv: bFW and bbRE.<br />
</p><br />
<p class="body_text"><br />
Ran out of any Phusion DNA polymerase.<br />
</p><br />
<p class="body_text"><br />
OxLF and OxLF Annealing oligos<br />
The annealed oligos were retrieved from the thermocycler in the morning. The entire tube contents (30 ul) underwent PCR purification - producing aprox. 30 ul pure DNA.<br />
5 ul from this volume was run on a 3% agarose gel with both a 50 bp and 1 kb DNA ladder (120 V for 1 hour). The gel showed the annealed oligos at the right length.<br />
The left 24 ul of the pure DNA tube were prep digested while 1 ul was used for nanodroping ( 318.6 ng/ul, 260/280 = 1.84).<br />
</p><br />
<p class="body_text"><br />
Prep digest of purified DNA oligos.<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Component</th><br />
<th>Volume added (ul)</th><br />
</tr><br />
<tr><br />
<td>Sample</td><br />
<td>24</td><br />
</tr><br />
<tr><br />
<td>EcoRI</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>PstI</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>4</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>50</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These digests were run on a gel and the correct bands were extracted. <br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/f/f1/Sep_5_oligos.png');height:651px;width:650px"></div><br />
<br />
<p class="body_text"><br />
<b>Friday 6th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
Did glycerol stocks and minipreps of the new transformations.<br />
</p><br />
<br />
<p class="body_text"><br />
Nanodrop results of the miniprep:<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th></th><br />
<th>1 c</th><br />
<th>2 c</th><br />
<th>3 c</th><br />
<th>4 c</th><br />
<th>5 z</th><br />
<th>7 z</th><br />
</tr><br />
<tr><br />
<td>ng/ul</td><br />
<td>7</td><br />
<td>12.2</td><br />
<td>17.3</td><br />
<td>14.1</td><br />
<td>18.6</td><br />
<td>13.8</td><br />
</tr><br />
<tr><br />
<td>260/280</td><br />
<td>1.78</td><br />
<td>1.69</td><br />
<td>1.87</td><br />
<td>1.98</td><br />
<td>1.88</td><br />
<td>1.68</td></tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Analytical digest of the above minipreps:<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Component</th><br />
<th>Volume added (ul)</th><br />
</tr><br />
<tr><br />
<td>pDNA</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>EcoRI</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>PstI</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>1.5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>10</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These digestions were run next to uncuts but again, no bands on the gel.<br />
</p><br />
<p class="body_text"><br />
Gel extraction and purification of: <br />
EcoR1 and Pst1 (E and P) digested K812014 (2 kb band corresponding to pSB1C3) was purified and the nanodrop results were: 51.1 ng/ul and 260/280 = 1.88 <br />
OxLF and OxLF annealed. Nanodrop result: 44.6 ng/ul and 0.08 for 260/280.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Saturday 7th September<b><br />
</p><br />
<br />
<p class="body_text"><br />
Picked a total of 15 colonies from plates of potentially ligated cmv and zeocin in pSB1C3 - from both transformations (31st of August and 4th of September).<br />
Incubated overnight for 16 hours in 2 ml LB broth and 4 ul chloramphenicol (cmp).<br />
Made new glycerols stocks using 0.5 ml of the inoculated volume.<br />
Minipreped the 15 inoculations but the nanodrop of these showed concentrations of below 20 ng/ul.<br />
</p><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Mammalian Lab</b><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Monday 2nd September<b><br />
</p><br />
<p class="body_text"><br />
All 6 dishes have 100% confluency. Split 3 dishes 3:1 obtaining a total of 9 dishes. Discarded 3 dishes.<br />
</p><br />
<p class="body_text"><br />
<b>Thursday 5th September<b><br />
</p><br />
<p class="body_text"><br />
All 9 dishes are over-confluent, some with floating dumps. One dish flamentous contamination.<br />
Discarded 5 dishes, including contaminated dish.<br />
Split 4 dishes in 1:4. 16 dishes in total (P15).<br />
</p><br />
<p class="body_text"><br />
<b>Friday 6th September</b><br />
</p><br />
<p class="body_text"><br />
The incubator was decontaminated. Confluency was between 30-90%. <br />
Split 4 dishes in 4:1, 5 dishes in total (P16).<br />
Discarded 1 dish (not enough tryptophan). Other 11 are kept incubated.<br />
</p><br />
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<div class="gap"><br />
</div><br />
<br />
<div class="full_page"><br />
<p class="body_text"> <a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a> <br />
</p> <br />
</div><br />
<br />
<p class="minor_title">Week 13</p><br />
<br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Bacterial Lab</b><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Tuesday 27th August</b> - <br />
</p><br />
<p class="body_text"><br />
Purify zeocin CMV from zeo FB primers<br />
<br />
</p><br />
<p class="body_text"><br />
Zeocin and CMV purification<br />
<br />
</p><br />
<p class="body_text"><br />
Tube:<br />
</p><br />
<p class="body_text"><br />
1 - CMV 1ul template bb F, R<br />
</p><br />
<p class="body_text"><br />
2 - CMV 2ul template bb F, R<br />
</p><br />
<p class="body_text"><br />
3 - Zeocin 2ul template zecF, R<br />
</p><br />
<p class="body_text"><br />
4 - Zeocin 1ul template zecF, R<br />
</p><br />
<p class="body_text"><br />
5 - Zeocin 2ul template zecbbF, R<br />
</p><br />
<p class="body_text"><br />
6 - Zeocin 1ul template zecbbF, R<br />
<br />
</p><br />
<p class="body_text"><br />
<b>Wednesday 28th August</b><br />
</p><br />
<p class="body_text"><br />
3x <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> glycerol stocks</a> of K812014 were prepared from inoculum. 1.5mL of culture was used for <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> miniprep</a>.<br />
</p><br />
<br />
<p class="body_text"><br />
Nanodrop of Zec and CMV was recorded:<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th></th><br />
<th>260/280</th><br />
<th>ng/ul</th><br />
</tr><br />
<tr><br />
<td>ZEC</td><br />
<td>1.68</td><br />
<td>21.1</td><br />
</tr><br />
<tr><br />
<td>CMV</td><br />
<td>1.96</td><br />
<td>82.1</td><br />
</tr><br />
</table><br />
<br />
</p><br />
<p class="body_text"><br />
A gel was run on 100ul of prep digest K812014 + 20ul dye. 2000bp was scooped in <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> gel extraction</a>. <br />
<br />
</p><br />
<p class="body_text"><br />
<a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Chloramphenicol</a> was tested at x2 and x4 concentration with 50ul cells spread on each plate, results from the next day still showed significant colony growth (100+).<br />
<br />
</p><br />
<p class="body_text"><br />
<b>Thursday 29th August</b><br />
</p><br />
<p class="body_text"><br />
Nanodrop of pSB1C3 purified from gel extract, 260/280: -25/21, ng/ul: 13.5<br />
</p><br />
<br />
<p class="body_text"><br />
5ul was run on a gel, however extract of pSB1C3 was unsuccessful as no bands were visible.<br />
</p><br />
<br />
<p class="body_text"><br />
PCR:<br />
<br />
</p><br />
<p class="body_text"><br />
3 <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> PCR</a> reactions of zeocin using zec BB F, R primers were performed. First reaction: 1 ul pSecTag2A template, second 2 ul pSecTag2A and third reaction - negative control - no template.<br />
</p><br />
<p class="body_text"><br />
3 PCR reactions for cmv promoter were prepared using the same variation of template volume as above. These were tested the next day and were successful. Gel photo (30/08).<br />
<br />
</p><br />
<p class="body_text"><br />
Prep digest+gel+extract+purify+nanodrop+5ul gel of zeocin and CMV were carried out<br />
<br />
</p><br />
<p class="body_text"><br />
Prep digest:<br />
<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Sample (ul)</th><br />
<th>Zeo</th><br />
<th>CMV</th><br />
<th>Cyc100</th><br />
<th>Cyc100</th><br />
<th>Cyc70</th><br />
<th>Cyc70</th><br />
<th>Cyc28</th><br />
<th>Cyc28</th><br />
</tr><br />
<tr><br />
<td>V. Sample</td><br />
<td>48</td><br />
<td>45</td><br />
<td>17.5</td><br />
<td>17.5</td><br />
<td>23</td><br />
<td>23</td><br />
<td>23.5</td><br />
<td>23.5</td><br />
</tr><br />
<tr><br />
<tr><br />
<td>B3</td><br />
<td>10</td><br />
<td>10</td><br />
<td>5</td><br />
<td>5</td><br />
<td>5</td><br />
<td>5</td><br />
<td>5</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>E</td><br />
<td>7</td><br />
<td>7</td><br />
<td>5</td><br />
<td></td><br />
<td>5</td><br />
<td></td><br />
<td>5</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>P</td><br />
<td>7</td><br />
<td>7</td><br />
<td>5</td><br />
<td>5</td><br />
<td>5</td><br />
<td>5</td><br />
<td>5</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>X</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td>5</td><br />
<td></td><br />
<td>5</td><br />
<td></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>2</td><br />
<td>2</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<th>H2O</th><br />
<td>26</td><br />
<td>29</td><br />
<td>16.5</td><br />
<td>16.5</td><br />
<td>11</td><br />
<td>11</td><br />
<td>10.5</td><br />
<td>10.5</td><br />
</tr><br />
<tr><br />
<th>Total</th><br />
<td>100</td><br />
<td>100</td><br />
<td>50</td><br />
<td>50</td><br />
<td>50</td><br />
<td>50</td><br />
<td>50</td><br />
<td>50</td><br />
</tr><br />
</table><br />
<br />
</p><br />
<p class="body_text"><br />
<a href="https://2013.igem.org/Team:UCL/Project/Protocols"> Glycerol stocks</a> (x15) of <a href="http://parts.igem.org/Part:BBa_J63009" target="_blank"> J63009</a>, <a href="http://parts.igem.org/Part:BBa_K105027" target="_blank"> K105027</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K105028" target="_blank"> K105028</a> and <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K105030" target="_blank"> K105030</a> were made.<br />
<br />
</p><br />
<p class="body_text"><br />
Additionally, nanodrops were recorded:<br />
<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Sample</th><br />
<th>260/280</th><br />
<th>Absorbance</th><br />
<th>ng/ul</th><br />
</tr><br />
<tr><br />
<td>J63009</td><br />
<td>2.10</td><br />
<td>0.774</td><br />
<td>88.7</td><br />
</tr><br />
<tr><br />
<td>K105030</td><br />
<td>209.00</td><br />
<td>0.653</td><br />
<td>60</td><br />
</tr><br />
<tr><br />
<td>K105028</td><br />
<td>2.14</td><br />
<td>0.408</td><br />
<td>50</td><br />
</tr><br />
<tr><br />
<td>K105029</td><br />
<td>2.18</td><br />
<td>0.585</td><br />
<td>77.0</td><br />
</tr><br />
<tr><br />
<td>K218014</td><br />
<td>2.12</td><br />
<td>0.946</td><br />
<td>104.9</td><br />
</tr><br />
</table><br />
<br />
</p><br />
<p class="body_text"><br />
A prep digest was conducted on <a href="http://parts.igem.org/Part:BBa_K812014" target="_blank"> K812014</a> to extract <a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a>.<br />
<br />
</p><br />
<p class="body_text"><br />
<a href="https://2013.igem.org/Team:UCL/Project/Protocols"> Chloramphenicol</a> was tested again but this time with x8 cmp (80ul cmp to 10mL LB agar). Overgrad, undergrad and Yanika chloramphenicol stocks were used and 50ul <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> competent cells</a> were spread. Incubated at 37°C overnight. <br />
</p><br />
<br />
<p class="body_text"><br />
Zec and CMV nanodrops were recorded:<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Sample</th><br />
<th>260/280</th><br />
<th>Absorbance</th><br />
<th>ng/ul</th><br />
</tr><br />
<tr><br />
<td>ZEC</td><br />
<td>1.87</td><br />
<td>9.538</td><br />
<td>2.5</td><br />
</tr><br />
<tr><br />
<td>CMV</td><br />
<td>2.06</td><br />
<td>11.048</td><br />
<td>25.4</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Friday 30th August</b><br />
</p><br />
<br />
<p class="body_text"><br />
<a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Chloramphenicol</a> test - plate colony growth observation and count: all 3 plates prepared using the chloramphenicol from postgrads, us and Yanika had 30+ very small colonies.<br />
</p><br />
<br />
<p class="body_text"><br />
After overnight incubation, from each of the the 10 ml inoculations of K812014 (I), pSecTag2A and second (different) sample of K812014 (II), 2.5 ml were <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> minipreped</a> while the rest of 7.5 ml from each was used to make <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> glycerol stocks</a>.<br />
</p><br />
<br />
<p class="body_text"><br />
Nanodrop of these minipreps:<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Sample</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
<th>260/230</th><br />
<th>Absorbance</th><br />
</tr><br />
<tr><br />
<td>K812014 (I)</td><br />
<td>117.8</td><br />
<td>2.10</td><br />
<td>2.49</td><br />
<td>0.944</td><br />
</tr><br />
<tr><br />
<td>pSecTag2A</td><br />
<td>35.9</td><br />
<td>1.81</td><br />
<td>1.44</td><br />
<td>0.496</td><br />
</tr><br />
<tr><br />
<td>K812014 (II)</td><br />
<td>10.7</td><br />
<td>1.71</td><br />
<td>2.55</td><br />
<td>0.084</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Prep digest of K812014 (I) - miniprep sample prepared by Andy. Contents of reaction:<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Sample K812014 (I)</th><br />
<th>30 ul</th><br />
</tr><br />
<tr><br />
<td>EcoRI (E)</td><br />
<td>5 ul</td><br />
</tr><br />
<tr><br />
<td>PstI (P)</td><br />
<td>5 ul</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>1 ul</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>5 ul</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>4 ul</td><br />
</tr><br />
<tr><br />
<td>Total Volume of Reaction</td><br />
<td>50 ul</td><br />
</tr><br />
</table><br />
<br />
</p><br />
<p class="body_text"><br />
A gel was run with the E, P cut K812014 and the corresponding band of liniarised pSB1C3 (backbone of K812014) was extracted for purification.<br />
<br />
</p><br />
<p class="body_text"><br />
Nanodrop of the purified pSB1C3 DNA showed a concentration of 8.4 ng/ul (260/290 = 2.91; 260/230 = 0.02, Absorbance = 7.379) while the nanodrop of the pSB1C3 DNA offered by the postgrads showed 13.01 ng/ul (260/280=1.34).<br />
<br />
</p><br />
<p class="body_text"><br />
PCR:<br />
<p class="body_text"><br />
8 new reactions were prepared for zeocin - same primers bb F,R as used previously. <br />
<p class="body_text"><br />
A checking (analytical) gel was performed revealing no bands for reaction tubes 6 and 7 (each of these with 1 ul template)- PCR unsuccessful.<br />
</p><br />
<p class="body_text"><br />
These amplified zeocin together with the 2 reaction tubes of amplified zeocin f aa cmv from a day before and a were loaded onto the gel and the bands corresponding to 2 kb were extracted for purification.<br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/3/34/Aug_30_C3_%2B_zeo.png');height:400px;width:650px"></div><br />
<br />
</p><br />
<p class="body_text"><br />
<b>Saturday 31st August</b> -<br />
<p class="body_text"><br />
Gel extraction purification of zeocin and cmv. We are expecting that through this procedure the Phusion polymerase, DMSO, buffer, template DNA will be washed away, thus achieving purification of zeocin and cmv DNA (which are to be digested hence, prepared for ligation in the pSB1C3 backbone).<br />
<br />
</p><br />
<p class="body_text"><br />
PCR:<br />
<p class="body_text"><br />
Zeocin was amplified again, in 9 tubes (same reaction) -> a total volume of 50 ul x 9 = 450 ul.<br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/f/f3/Aug_31_UCLiGEM2013.png');height:508px;width:650px"></div><br />
<p class="body_text"><br />
Prep digest of zeocin and cmv straight after PCR.<br />
<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Components</th><br />
<th>Prep digest Cmv</th><br />
<th>Prep digest Zeocin</th><br />
</tr><br />
<tr><br />
<td>DNA sample</td><br />
<td>60 ul</td><br />
<td>60 ul</td><br />
</tr><br />
<tr><br />
<td>EcoRI</td><br />
<td>7 ul</td><br />
<td>7 ul</td><br />
</tr><br />
<tr><br />
<td>PstI</td><br />
<td>7 ul</td><br />
<td>7 ul</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>10 ul</td><br />
<td>10 ul</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>2 ul</td><br />
<td>2 ul</td><br />
</tr><br />
<tr><br />
<td>Water</td><br />
<td>14 ul</td><br />
<td>14 ul</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>100 ul</td><br />
<td>100 ul</td><br />
</tr><br />
</table><br />
<br />
</p><br />
<p class="body_text"><br />
These digests were used for LIGATION 1<br />
</p><br />
<p class="body_text"><br />
LIGATION 1: using<br />
<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>pSB1C3 digested and purified</th><br />
<th>10 ng/ul</th><br />
</tr><br />
<tr><br />
<th>Zeocin digested and purified</th><br />
<th>55 ng/ul</th><br />
</tr><br />
<tr><br />
<th>Cmv digested and purified</th><br />
<th>20 ng/ul</th><br />
</tr><br />
</table><br />
<br />
</p><br />
<p class="body_text"><br />
Ligation of zeocin to pSB1C3:<br />
<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Zeocin (55 ng/ul)</th><br />
<th>3 to 1</th><br />
<th>6 to 1</th><br />
</tr><br />
<tr><br />
<td>Water (ul)</td><br />
<td>2.1</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>Backbone (ul)</td><br />
<td>5</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Insert (ul)</td><br />
<td>2.7</td><br />
<td>5.5</td><br />
</tr><br />
<tr><br />
<td>Ligase (ul)</td><br />
<td>1 ul</td><br />
<td>1 ul</td><br />
</tr><br />
<tr><br />
<td>Total (ul)</td><br />
<td>21</td><br />
<td>21.5</td><br />
</tr><br />
</table><br />
<br />
</p><br />
<p class="body_text"><br />
Ligation of cmv to pSB1C3:<br />
<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>(20 ng/ul)</th><br />
<th>3 to 1</th><br />
<th>6 to 1</th><br />
</tr><br />
<tr><br />
<td>Water (ul)</td><br />
<td>2.4</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>Backbone (ul)</td><br />
<td>5</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Insert (ul)</td><br />
<td>2.6</td><br />
<td>5.3</td><br />
</tr><br />
<tr><br />
<td>Ligase (ul)</td><br />
<td>1 ul</td><br />
<td>1 ul</td><br />
</tr><br />
<tr><br />
<td>Total (ul)</td><br />
<td>21</td><br />
<td>21.3</td><br />
</tr><br />
</table><br />
<br />
</p><br />
<p class="body_text"><br />
Controls used:<br />
<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Controls</th><br />
<th>1 check no circular backbone</th><br />
<th>2 check no digestion process</th><br />
<th>3 control (uncut backbone)</th><br />
</tr><br />
<tr><br />
<td>Water (ul)</td><br />
<td>5</td><br />
<td>6</td><br />
<td>9</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>Backbone (ul)</td><br />
<td>5</td><br />
<td>5</td><br />
<td>2 of uncut backbone</td><br />
</tr><br />
<tr><br />
<td>Insert (ul)</td><br />
<td>0</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Ligase (ul)</td><br />
<td>1</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Total (ul)</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
</table><br />
<br />
</p><br />
<p class="body_text"><br />
5 ul of each of each ligation reaction were used to transform our home-made competent cells (4 vials - all ligations apart from the 3 controls) as well as Yanika’s top 10 cells (7 vials - all ligations).<br />
<br />
</p><br />
<p class="body_text"><br />
T10 - Top 10 cells<br />
</p><br />
<p class="body_text"><br />
HM - home made competent cells<br />
<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th></th><br />
<th>T10</th><br />
<th>T10</th><br />
<th>T10</th><br />
<th>T10</th><br />
<th>T10</th><br />
<th>T10</th><br />
<th>T10</th><br />
<th>HM</th><br />
<th>HM</th><br />
<th>HM</th><br />
<th>HM</th><br />
</tr><br />
<tr><br />
<th>Cell vial no.</th><br />
<th>1</th><br />
<th>2</th><br />
<th>3</th><br />
<th>4</th><br />
<th>5</th><br />
<th>6</th><br />
<th>7</th><br />
<th>28</th><br />
<th>45</th><br />
<th>51</th><br />
<th>52</th><br />
</tr><br />
<tr><br />
<th>Vol. ligation plated (ul)</th><br />
<th>90 & 10</th><br />
<th>90 & 10</th><br />
<th>100</th><br />
<th>100</th><br />
<th>100</th><br />
<th>90 & 10</th><br />
<th>90 & 10</th><br />
<th>100</th><br />
<th>90 & 10</th><br />
<th>100</th><br />
<th>100</th><br />
</tr><br />
<tr><br />
<th>Ligation containing</th><br />
<th>zeo 3:1</th><br />
<th>zeo 6:1</th><br />
<th>control 1</th><br />
<th>control 2</th><br />
<th>control 3</th><br />
<th>zeo 3:1</th><br />
<th>zeo 6:1</th><br />
<th>zeo 3:1</th><br />
<th>zeo 6:1</th><br />
<th>zeo 3:1</th><br />
<th>zeo 6:1</th><br />
</tr><br />
<tr><br />
<th>Cell counts</th><br />
<th>0</th><br />
<th>0</th><br />
<th>0</th><br />
<th>0</th><br />
<th>0</th><br />
<th>0</th><br />
<th>0</th><br />
<th>15</th><br />
<th>0 & 10*</th><br />
<th>15</th><br />
<th>15</th><br />
</tr><br />
</table><br />
<br />
</p><br />
<p class="body_text"><br />
* 0 colonies in plate with 10 ul inoculum spread and 10 colonies for the 90 ul inoculum spread.<br />
<br />
</p><br />
<p class="body_text"><br />
After the transformation protocol we spread the transformed cells on 4x cmp (?) agar plates using both 90 ul and 10 ul cell inoculum in the case of cell vials 1,2,6,7 (Yanika’s) and 45 (ours).<br />
<br />
</p><br />
<p class="body_text"><br />
We picked 5 colonies from each plated cell vials 28, 45, 51 and 52 (2 ml and 2 ul cmp) and incubated for 10 hours. These inoculations were then used to make glycerol stocks and minipreps.<br />
<br />
</p><br />
<p class="body_text"><br />
Gel extraction purification<br />
<br />
</p><br />
<p class="body_text"><br />
The 9 tubes of amplified zeocin performed were loaded on a gel and the 2 kb bands were subsequently extracted (6.2 g of gel). This zeocin DNA was purified using gel extraction purification kit and 3 tubes of each 60 ul purified zeocin resulted (labelling zeo g.e.p.).<br />
<br />
</p><br />
<p class="body_text"><br />
Miniprep of K812014 cell-containing-biobrick: in order to make stocks of pSB1C3, the backbone into which this biobrick is inserted.<br />
<br />
</p><br />
<p class="body_text"><br />
Nanodrop of purified zeocin (from above):<br />
<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Zeo g.e.p. tube</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>36.1</td><br />
<td>1.95</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>38.9</td><br />
<td>1.90</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>42.9</td><br />
<td>1.90</td><br />
</tr><br />
</table><br />
<br />
</p><br />
<br />
<p class="body_text"><br />
Nanodrop of miniprep of K812014: 197 ng/ul (260/280 = 1.94)<br />
</p><br />
</div><br />
<br />
<p class="major_title">September</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Sunday 1st September</b><br />
</p><br />
<br />
<p class="body_text"><br />
The 20 inoculations were retrieved from the incu-shaker. All falcons showed growth apart from one inoculation from cell vial 28 (ligation 3:1 zeo).<br />
</p><br />
<br />
<p class="body_text"><br />
1.5 ml of each tube was used for <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> miniprep</a> while the other 0.5 ml was used to make <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> glycerol stocks</a>.<br />
<br />
</p><br />
<p class="body_text"><br />
Prep digest of K812014 (miniprep prepared on the 31st August)<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Component</th><br />
<th>Volume added (ul)</th><br />
</tr><br />
<tr><br />
<td>K812014 DNA</td><br />
<td>25</td><br />
</tr><br />
<tr><br />
<td>EcoRI</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>PstI</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>9</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>50</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Nanodrop result of the miniprep of transformations:<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Cell vial/colony no.</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>28/1</td><br />
<td>84.2</td><br />
<td>1.65</td><br />
</tr><br />
<tr><br />
<td>28/2</td><br />
<td>58.5</td><br />
<td>1.77</td><br />
</tr><br />
<tr><br />
<td>28/3</td><br />
<td>49.6</td><br />
<td>1.83</td><br />
</tr><br />
<tr><br />
<td>28/4</td><br />
<td>14.6</td><br />
<td>1.97</td><br />
</tr><br />
<tr><br />
<td>45/1</td><br />
<td>130.9</td><br />
<td>1.72</td><br />
</tr><br />
<tr><br />
<td>45/2</td><br />
<td>18.7</td><br />
<td>2.34</td><br />
</tr><br />
<tr><br />
<td>45/3</td><br />
<td>49.6</td><br />
<td>1.73</td><br />
</tr><br />
<tr><br />
<td>45/4</td><br />
<td>111.3</td><br />
<td>1.64</td><br />
</tr><br />
<tr><br />
<td>45/5</td><br />
<td>23.9</td><br />
<td>1.96</td><br />
</tr><br />
<tr><br />
<td>51/1</td><br />
<td>69.5</td><br />
<td>1.70</td><br />
</tr><br />
<tr><br />
<td>51/2</td><br />
<td>60.9</td><br />
<td>1.78</td><br />
</tr><br />
<tr><br />
<td>51/3</td><br />
<td>41.2</td><br />
<td>1.85</td><br />
</tr><br />
<tr><br />
<td>51/4</td><br />
<td>50.9</td><br />
<td>1.79</td><br />
</tr><br />
<tr><br />
<td>51/5</td><br />
<td>23.9</td><br />
<td>1.96</td><br />
</tr><br />
<tr><br />
<td>52/1</td><br />
<td>40.4</td><br />
<td>1.83</td><br />
</tr><br />
<tr><br />
<td>52/2</td><br />
<td>40.2</td><br />
<td>1.87</td><br />
</tr><br />
<tr><br />
<td>52/3</td><br />
<td>78.1</td><br />
<td>1.94</td><br />
</tr><br />
<tr><br />
<td>52/4</td><br />
<td>127.2</td><br />
<td>1.62</td><br />
</tr><br />
<tr><br />
<td>52/5</td><br />
<td>16.5</td><br />
<td>2.11</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Analytical digest of the above minipreps - incubated at 37 degrees C for 1 hour<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Volume (ul)</th><br />
<th>Single Digest (PstI)</th><br />
<th>Double Digest (EcoRI + PstI)</th><br />
</tr><br />
<tr><br />
<td>Miniprep</td><br />
<td>5</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>EcoRI</td><br />
<td>0</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>PstI</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>2.5</td><br />
<td>1.5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These digests were loaded into a gel in the following order: single digest, double digest, uncut.<br />
The result of this gel was negative, there were no bands for the loaded DNAs (the 1 kb DNA ladder was on the gel), the procedures of achieving recombinant zeocin and cmv plasmid were unsuccessful.<br />
</p><br />
<br />
<p class="body_text"><br />
We suspect that the water was contaminated with nuclease. <br />
</p><br />
<br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Mammalian Lab</b><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Saturday 31st August</b> - HeLa cells confluency: 100% and 40%. Split 100% confluency dish in 1:4, 40% confluency dish in 1:2<br />
</p><br />
<br />
</div><br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/AchievementsTeam:UCL/Achievements2013-10-05T01:09:52Z<p>Naruto: </p>
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<br />
<p class="major_title">MEDAL CRITERIA</p><br />
<br />
<p class="minor_title">Bronze Medal</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have registered our <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">team</a> of eleven undergraduates. <br />
</p><br />
<p class="body_text"><br />
• We have completed the judging form.<br />
</p><br />
<p class="body_text"><br />
• We have created a beautiful team wiki in keeping with the iGEM guidelines.<br />
</p><br />
<p class="body_text"><br />
• We have a poster and presentation ready for the regional jamboree in Lyon.<br />
</p><br />
<p class="body_text"><br />
• We have developed two new <a href="https://2013.igem.org/Team:UCL/Project/Parts" target="_blank">BioBricks</a> and have submitted them to the iGEM Registry<br />
</div><br />
<br />
<p class="minor_title">Silver Medal</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have proved our zeocin resistance BioBrick <a href="https://2013.igem.org/Team:UCL/Project/Parts" target="_blank">worked as expected</a> by characterising it in HeLa cells. We did this by by creating a zeocin kill curve with and without our <a href="https://2013.igem.org/Team:UCL/Project/Marker" target="_blank">zeocin resistance</a> BioBrick. <br />
</p><br />
<p class="body_text"><br />
• Due to the gravity of Alzheimer's disease and the perceived sovereignty of the brain, we have taken the ethics of using synthetic biological treatments very seriously. We produced a <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">neuroethics</a> and a <a href="https://2013.igem.org/Team:UCL/Practice/Report" target="_blank">feasibility</a> report, consulted numerous experts and provided a concise but detailed <a href="https://2013.igem.org/Team:UCL/Background" target="_blank">background</a> to our project, which shows how our proposed <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">genetic circuit</a> is advised by multiple theories for the causation of Alzheimer's pathology.<br />
</p><br />
</div><br />
<br />
<p class="minor_title"><br />
Gold Medal<br />
</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
• We collaborated with iGEM Westminster, by modelling how well their bed-bug killing device will operate in a bedroom as well advising them on how to run a speed debate using our format. One of our team members, Alex Bates, also attended as guest speaker at their speed debate event.<br />
</p><br />
<p class="body_text"><br />
Full details on collaboration with iGEM Westminster can be found below.<br />
</p><br />
<p class="body_text"><br />
• Our <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">Human Practices</a> deal with an entirely new area for iGEM and, indeed, almost a completely new avenue of research for synthetic biology as a field - the <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">fusion</a> of neuroscience and synthetic biology. We use a variety of strong methods for social and ethical analysis, and outreach. Please see the section below for a summary of what we achieved.<br />
</p><br />
<p class="body_text"><br />
• Outside of this theme, we also engaged in outreach by training and advising the UCL Academy iGEM high school team. This was the first time a British iGEM team has helped run an iGEM HS team.<br />
</p><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<p class="major_title">ABOVE AND BEYOND</p><br />
<br />
<p class="minor_title">Project</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text">project stuff<br />
</p><br />
</div><br />
<br />
<p class="minor_title">Human Practices</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have looked at an entirely new ethical area for iGEM that has also essentially not been covered in academia; the neuroethics of genetic engineering. We have dubbed this <a href="https://2013.igem.org/Team:UCL/Practice" target="_blank">'Neuro-genethics'</a>. <br />
</p><br />
<p class="body_text"><br />
• We have produced an extensive 20 page <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">report</a> that looks into neuro-genethics and what synthetic biology could achieve in neuroscience.<br />
</p><br />
<p class="body_text"><br />
• We have engaged the public on this topic by getting their opinions at the <a href="http://www.artscatalyst.org/" target="_blank">Arts Catalyst</a>, running a <a href="https://2013.igem.org/Team:UCL/Practice/Debate" target="_blank">speed debate</a> and a <a href="https://2013.igem.org/Team:UCL/Practice/TED" target="_blank">TED debate</a>, conducting an <a href="https://2013.igem.org/Team:UCL/Practice/Survey" target="_blank">online survey</a> and producing a <a href="https://2013.igem.org/Team:UCL/Practice/Documentary" target="_blank">documentary </a> on synthetic neurobiology.<br />
</p><br />
<p class="body_text"><br />
• We ran one of iGEM's first <a href="https://2013.igem.org/Team:UCL/Practice/Creative" target="_blank">creative writing competitions</a>, to gauge public opinion on brain modification and highligh5t the impact of fiction on society's views.<br />
</p><br />
<p class="body_text"><br />
• Petcha Kutcha style presentation to enguage prospective students of the UCL Engineering Department about iGEM.<br />
</p><br />
<p class="body_text"><br />
• We created an original memory bank, <a href="https://2013.igem.org/Team:UCL/Memories" target="_blank">Eternal Sunshine</a>, which highlights how precious memories are, indicating the desperate need to cure Alzheimer's disease. <br />
</p><br />
<p class="body_text"><br />
• We created a <a href="https://2013.igem.org/Team:UCL/Practice/Report" target="_blank">feasibility report</a> on implementing our treatment.<br />
</p><br />
<p class="body_text"><br />
• Attended YSB 1.0 along with other UK iGEM teams to discuss projects and potential collaborations. <br />
</p><br />
</div><br />
<br />
<p class="minor_title">Modelling</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We are the first iGEM team to use a <a href="https://2013.igem.org/Team:UCL/Modeling" target="_blank">protein network analysis approach</a>. Network based bioinformatics can feedback into synthetic biology by informing the choice of parts in therapeutic genetic circuits.<br />
</p><br />
</div><br />
<br />
<p class="minor_title">Wiki</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have developed an, original attractive wiki using art work by our artists in residence, <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Fong Yi Khoo</a> and <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Oran Maguire</a>.<br />
</p><br />
<p class="body_text"><br />
• We have included an extensive neuroscience background section, which explains and compares multiple theories for the causation of Alzheimer's disease, so that readers can fully understand the pros and cons of our genetic circuit. <br />
</p><br />
<p class="body_text"><br />
• We have included a full complement of citations that link to PubMed pages so that it is easy to see from where our ideas and explanations have been drawn, and which papers have inspired us.<br />
</p><br />
</div><br />
<br />
<p class="minor_title">Collaboration with Westminster iGEM</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Model (ROBIN WRITES HERE)</b><br />
</p><br />
<p class="body_text"><br />
Movement of bed-bugs towards blood meal<br />
</div><br />
<br />
<p class="minor_title">Mentoring iGEM HS</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Two members of the UCL iGEM team volunteered as advisers to the UCL Academy iGEM team – <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Ruxi</a> and <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Khaicheng</a>, under the guidance of Aurelija Grigonyte, a member of the UCL iGEM 2012 team. During the high school team’s brainstorming process, we provided them with guidance and resources for their research. We also supervised their lab work in the UCL Biochemical Engineering department.<br />
</p><br />
<p class="body_text"><br />
UCL is the first university in the UK to be the sole sponsor of an academy – a non-selective mixed state school in our home borough of Camden. UCL Academy represents a unique opportunity to blur the boundaries between secondary and higher education.<br />
</p><br />
<p class="body_text"><br />
The academy is one of the first UK high schools to participate in iGEM this year, and is the only UK team so far to have attended the <a href="https://2013hs.igem.org/Main_Page" target="_blank">High School iGEM Jamboree</a> at MIT, Boston. The team aimed to revolutionise the recycling industry by proposing a home system that converts cellulose into glucose, allowing the up-cycling of paper into a commercial product of bioplastic - polyhydroxybutyrate (PHB) <br />
</p><br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/b0/IMG_4769-1024x68211.jpg');height:280px;width:650px;"><br />
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</p><br />
<p class="body_text"><br />
For information about their iGEM project, check out their wiki <a href="https://2013hs.igem.org/Team:UCL_Academy" target="_blank">here</a>. <br />
</p><br />
</div><br />
<!-- END CONTENT ------------------------------------------------------------------------------------------------------><br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/AchievementsTeam:UCL/Achievements2013-10-05T01:03:11Z<p>Naruto: </p>
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<br />
<p class="major_title">MEDAL CRITERIA</p><br />
<br />
<p class="minor_title">Bronze Medal</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have registered our <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">team</a> of eleven undergraduates. <br />
</p><br />
<p class="body_text"><br />
• We have completed the judging form.<br />
</p><br />
<p class="body_text"><br />
• We have created a beautiful team wiki in keeping with the iGEM guidelines.<br />
</p><br />
<p class="body_text"><br />
• We have a poster and presentation ready for the regional jamboree in Lyon.<br />
</p><br />
<p class="body_text"><br />
• We have developed two new <a href="https://2013.igem.org/Team:UCL/Project/Parts" target="_blank">BioBricks</a> and have submitted them to the iGEM Registry<br />
</div><br />
<br />
<p class="minor_title">Silver Medal</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have proved our zeocin resistance BioBrick <a href="https://2013.igem.org/Team:UCL/Project/Parts" target="_blank">worked</a> by characterising it in HeLa cells. We did this by by creating a zeocin kill curve with and without our <a href="https://2013.igem.org/Team:UCL/Project/Marker" target="_blank">zeocin resistance</a> BioBrick. <br />
</p><br />
<p class="body_text"><br />
• Due to the gravity of Alzheimer's disease and the perceived sovereignty of the brain, we have taken the ethics of using synthetic biological treatments very seriously. We produced a <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">neuroethics</a> and a <a href="https://2013.igem.org/Team:UCL/Practice/Report" target="_blank">feasibility</a> report, consulted numerous experts and provided a concise but detailed <a href="https://2013.igem.org/Team:UCL/Background" target="_blank">background</a> to our project, which shows how our proposed <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">genetic circuit</a> is advised by multiple theories for the causation of Alzheimer's pathology.<br />
</p><br />
</div><br />
<br />
<p class="minor_title"><br />
Gold Medal<br />
</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
• We collaborated with iGEM Westminster, by modelling how well their bed-bug killing device will operate in a bedroom as well advising them on how to run a speed debate using our format. One of our team members, Alex Bates, also attended as guest speaker at their speed debate event.<br />
</p><br />
<p class="body_text"><br />
Full details on collaboration with iGEM Westminster can be found below.<br />
</p><br />
<p class="body_text"><br />
• Our <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">Human Practices</a> deal with an entirely new area for iGEM and, indeed, almost a completely new avenue of research for synthetic biology as a field - the <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">fusion</a> of neuroscience and synthetic biology. We use a variety of strong methods for social and ethical analysis, and outreach. Please see the section below for a summary of what we achieved.<br />
</p><br />
<p class="body_text"><br />
• Outside of this theme, we also engaged in outreach by training and advising the UCL Academy iGEM high school team. This was the first time a British iGEM team has helped run an iGEM HS team.<br />
</p><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<p class="major_title">ABOVE AND BEYOND</p><br />
<br />
<p class="minor_title">Project</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text">project stuff<br />
</p><br />
</div><br />
<br />
<p class="minor_title">Human Practices</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have looked at an entirely new ethical area for iGEM that has also essentially not been covered in academia; the neuroethics of genetic engineering. We have dubbed this <a href="https://2013.igem.org/Team:UCL/Practice" target="_blank">'Neuro-genethics'</a>. <br />
</p><br />
<p class="body_text"><br />
• We have produced an extensive 20 page <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">report</a> that looks into neuro-genethics and what synthetic biology could achieve in neuroscience.<br />
</p><br />
<p class="body_text"><br />
• We have engaged the public on this topic by getting their opinions at the <a href="http://www.artscatalyst.org/" target="_blank">Arts Catalyst</a>, running a <a href="https://2013.igem.org/Team:UCL/Practice/Debate" target="_blank">speed debate</a> and a <a href="https://2013.igem.org/Team:UCL/Practice/TED" target="_blank">TED debate</a>, conducting an <a href="https://2013.igem.org/Team:UCL/Practice/Survey" target="_blank">online survey</a> and producing a <a href="https://2013.igem.org/Team:UCL/Practice/Documentary" target="_blank">documentary </a> on synthetic neurobiology.<br />
</p><br />
<p class="body_text"><br />
• We ran one of iGEM's first <a href="https://2013.igem.org/Team:UCL/Practice/Creative" target="_blank">creative writing competitions</a>, to gauge public opinion on brain modification and highligh5t the impact of fiction on society's views.<br />
</p><br />
<p class="body_text"><br />
• Petcha Kutcha style presentation to enguage prospective students of the UCL Engineering Department about iGEM.<br />
</p><br />
<p class="body_text"><br />
• We created an original memory bank, <a href="https://2013.igem.org/Team:UCL/Memories" target="_blank">Eternal Sunshine</a>, which highlights how precious memories are, indicating the desperate need to cure Alzheimer's disease. <br />
</p><br />
<p class="body_text"><br />
• We created a <a href="https://2013.igem.org/Team:UCL/Practice/Report" target="_blank">feasibility report</a> on implementing our treatment.<br />
</p><br />
<p class="body_text"><br />
• Attended YSB 1.0 along with other UK iGEM teams to discuss projects and potential collaborations. <br />
</p><br />
</div><br />
<br />
<p class="minor_title">Modelling</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We are the first iGEM team to use a <a href="https://2013.igem.org/Team:UCL/Modeling" target="_blank">protein network analysis approach</a>. Network based bioinformatics can feedback into synthetic biology by informing the choice of parts in therapeutic genetic circuits.<br />
</p><br />
</div><br />
<br />
<p class="minor_title">Wiki</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have developed an, original attractive wiki using art work by our artists in residence, <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Fong Yi Khoo</a> and <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Oran Maguire</a>.<br />
</p><br />
<p class="body_text"><br />
• We have included an extensive neuroscience background section, which explains and compares multiple theories for the causation of Alzheimer's disease, so that readers can fully understand the pros and cons of our genetic circuit. <br />
</p><br />
<p class="body_text"><br />
• We have included a full complement of citations that link to PubMed pages so that it is easy to see from where our ideas and explanations have been drawn, and which papers have inspired us.<br />
</p><br />
</div><br />
<br />
<p class="minor_title">Collaboration with Westminster iGEM</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Model (ROBIN WRITES HERE)</b><br />
</p><br />
<p class="body_text"><br />
Movement of bed-bugs towards blood meal<br />
</div><br />
<br />
<p class="minor_title">Mentoring iGEM HS</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Two members of the UCL iGEM team volunteered as advisers to the UCL Academy iGEM team – <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Ruxi</a> and <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Khaicheng</a>, under the guidance of Aurelija Grigonyte, a member of the UCL iGEM 2012 team. During the high school team’s brainstorming process, we provided them with guidance and resources for their research. We also supervised their lab work in the UCL Biochemical Engineering department.<br />
</p><br />
<p class="body_text"><br />
UCL is the first university in the UK to be the sole sponsor of an academy – a non-selective mixed state school in our home borough of Camden. UCL Academy represents a unique opportunity to blur the boundaries between secondary and higher education.<br />
</p><br />
<p class="body_text"><br />
The academy is one of the first UK high schools to participate in iGEM this year, and is the only UK team so far to have attended the <a href="https://2013hs.igem.org/Main_Page" target="_blank">High School iGEM Jamboree</a> at MIT, Boston. The team aimed to revolutionise the recycling industry by proposing a home system that converts cellulose into glucose, allowing the up-cycling of paper into a commercial product of bioplastic - polyhydroxybutyrate (PHB) <br />
</p><br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/b0/IMG_4769-1024x68211.jpg');height:280px;width:650px;"><br />
</div><br />
</p><br />
<p class="body_text"><br />
For information about their iGEM project, check out their wiki <a href="https://2013hs.igem.org/Team:UCL_Academy" target="_blank">here</a>. <br />
</p><br />
</div><br />
<!-- END CONTENT ------------------------------------------------------------------------------------------------------><br />
</div><br />
<br />
</div><br />
<br />
<script type="text/javascript" src="https://2013.igem.org/Team:UCL/static/footer.js?action=raw&ctype=text/javascript"> <br />
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</body><br />
</html></div>Narutohttp://2013.igem.org/Team:UCL/AchievementsTeam:UCL/Achievements2013-10-05T01:01:13Z<p>Naruto: </p>
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<div id="container"><br />
<!-- START CONTENT ----------------------------------------------------------------------------------------------------><br />
<div class="gap"><br />
</div><br />
<br />
<p class="major_title">MEDAL CRITERIA</p><br />
<br />
<p class="minor_title">Bronze Medal</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have registered our <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">team</a> of eleven undergraduates. <br />
</p><br />
<p class="body_text"><br />
• We have completed the judging form.<br />
</p><br />
<p class="body_text"><br />
• We have created a beautiful team wiki in keeping with the iGEM guidelines.<br />
</p><br />
<p class="body_text"><br />
• We have a poster and presentation ready for the regional jamboree in Lyon.<br />
</p><br />
<p class="body_text"><br />
• We have developed two new <a href="https://2013.igem.org/Team:UCL/Project/Parts" target="_blank">BioBricks</a> and have submitted them to the iGEM Registry<br />
</div><br />
<br />
<p class="minor_title">Silver Medal</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have proved our zeocin resistance BioBrick <a href="https://2013.igem.org/Team:UCL/Project/Parts" target="_blank">worked</a> by characterising it in HeLa cells. We did this by by creating a zeocin kill curve with and without our <a href="https://2013.igem.org/Team:UCL/Project/Marker" target="_blank">zeocin resistance</a> BioBrick. <br />
</p><br />
<p class="body_text"><br />
• Due to the gravity of Alzheimer's disease and the perceived sovereignty of the brain, we have taken the ethics of using synthetic biological treatments very seriously. We produced a <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">neuroethics</a> and a <a href="https://2013.igem.org/Team:UCL/Practice/Report" target="_blank">feasibility</a> report, consulted numerous experts and provided a concise but detailed <a href="https://2013.igem.org/Team:UCL/Background" target="_blank">background</a> to our project, which shows how our proposed <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">genetic circuit</a> is advised by multiple theories for the causation of Alzheimer's pathology.<br />
</p><br />
</div><br />
<br />
<p class="minor_title"><br />
Gold Medal<br />
</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
• We collaborated with iGEM Westminster, by modelling how well their bed-bug killing device will operate in a bedroom as well advising them on how to run a speed debate using our format. One of our team members, Alex Bates, also attended as guest speaker at their speed debate event.<br />
</p><br />
<p class="body_text"><br />
Full details on collaboration with iGEM Westminster can be found below.<br />
</p><br />
<p class="body_text"><br />
• Our <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">Human Practices</a> deal with an entirely new area for iGEM and, indeed, almost a completely new avenue of research for synthetic biology as a field - the <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">fusion</a> of neuroscience and synthetic biology. We use a variety of strong methods for social and ethical analysis, and outreach. Please see the section below for a summary of what we achieved.<br />
</p><br />
<p class="body_text"><br />
• Outside of this theme, we also engaged in outreach by training and advising the UCL Academy iGEM high school team. This was the first time a British iGEM team has helped run an iGEM HS team.<br />
</p><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<p class="major_title">ABOVE AND BEYOND</p><br />
<br />
<p class="minor_title">Project</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text">project stuff<br />
</p><br />
</div><br />
<br />
<p class="minor_title">Human Practices</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have looked at an entirely new ethical area for iGEM that has also essentially not been covered in academia; the neuroethics of genetic engineering. We have dubbed this <a href="https://2013.igem.org/Team:UCL/Practice" target="_blank">'Neuro-genethics'</a>. <br />
</p><br />
<p class="body_text"><br />
• We have produced an extensive 20 page <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">report</a> that looks into neuro-genethics and what synthetic biology could achieve in neuroscience.<br />
</p><br />
<p class="body_text"><br />
• We have engaged the public on this topic by getting their opinions at the <a href="http://www.artscatalyst.org/" target="_blank">Arts Catalyst</a>, running a <a href="https://2013.igem.org/Team:UCL/Practice/Debate" target="_blank">speed debate</a> and a <a href="https://2013.igem.org/Team:UCL/Practice/TED" target="_blank">TED debate</a>, conducting an <a href="https://2013.igem.org/Team:UCL/Practice/Survey" target="_blank">online survey</a> and producing a <a href="https://2013.igem.org/Team:UCL/Practice/Documentary" target="_blank">documentary </a> on synthetic neurobiology.<br />
</p><br />
<p class="body_text"><br />
• We ran one of iGEM's first <a href="https://2013.igem.org/Team:UCL/Practice/Creative" target="_blank">creative writing competitions</a>, to gauge public opinion on brain modification and highligh5t the impact of fiction on society's views.<br />
</p><br />
<p class="body_text"><br />
• Petcha Kutcha style presentation to enguage prospective students of the UCL Engineering Department about iGEM.<br />
</p><br />
<p class="body_text"><br />
• We created an original memory bank, <a href="https://2013.igem.org/Team:UCL/Memories" target="_blank">Eternal Sunshine</a>, which highlights how precious memories are, indicating the desperate need to cure Alzheimer's disease. <br />
</p><br />
<p class="body_text"><br />
• We created a <a href="https://2013.igem.org/Team:UCL/Practice/Report" target="_blank">feasibility report</a> on implementing our treatment.<br />
</p><br />
<br />
</div><br />
<br />
<p class="minor_title">Modelling</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We are the first iGEM team to use a <a href="https://2013.igem.org/Team:UCL/Modeling" target="_blank">protein network analysis approach</a>. Network based bioinformatics can feedback into synthetic biology by informing the choice of parts in therapeutic genetic circuits.<br />
</p><br />
</div><br />
<br />
<p class="minor_title">Wiki</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
• We have developed an, original attractive wiki using art work by our artists in residence, <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Fong Yi Khoo</a> and <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Oran Maguire</a>.<br />
</p><br />
<p class="body_text"><br />
• We have included an extensive neuroscience background section, which explains and compares multiple theories for the causation of Alzheimer's disease, so that readers can fully understand the pros and cons of our genetic circuit. <br />
</p><br />
<p class="body_text"><br />
• We have included a full complement of citations that link to PubMed pages so that it is easy to see from where our ideas and explanations have been drawn, and which papers have inspired us.<br />
</p><br />
</div><br />
<br />
<p class="minor_title">Collaboration with Westminster iGEM</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Model (ROBIN WRITES HERE)</b><br />
</p><br />
<p class="body_text"><br />
Movement of bed-bugs towards blood meal<br />
</div><br />
<br />
<p class="minor_title">Mentoring iGEM HS</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Two members of the UCL iGEM team volunteered as advisers to the UCL Academy iGEM team – <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Ruxi</a> and <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Khaicheng</a>, under the guidance of Aurelija Grigonyte, a member of the UCL iGEM 2012 team. During the high school team’s brainstorming process, we provided them with guidance and resources for their research. We also supervised their lab work in the UCL Biochemical Engineering department.<br />
</p><br />
<p class="body_text"><br />
UCL is the first university in the UK to be the sole sponsor of an academy – a non-selective mixed state school in our home borough of Camden. UCL Academy represents a unique opportunity to blur the boundaries between secondary and higher education.<br />
</p><br />
<p class="body_text"><br />
The academy is one of the first UK high schools to participate in iGEM this year, and is the only UK team so far to have attended the <a href="https://2013hs.igem.org/Main_Page" target="_blank">High School iGEM Jamboree</a> at MIT, Boston. The team aimed to revolutionise the recycling industry by proposing a home system that converts cellulose into glucose, allowing the up-cycling of paper into a commercial product of bioplastic - polyhydroxybutyrate (PHB) <br />
</p><br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/b0/IMG_4769-1024x68211.jpg');height:280px;width:650px;"><br />
</div><br />
</p><br />
<p class="body_text"><br />
For information about their iGEM project, check out their wiki <a href="https://2013hs.igem.org/Team:UCL_Academy" target="_blank">here</a>. <br />
</p><br />
</div><br />
<!-- END CONTENT ------------------------------------------------------------------------------------------------------><br />
</div><br />
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</div><br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/AchievementsTeam:UCL/Achievements2013-10-05T01:00:52Z<p>Naruto: </p>
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<p class="major_title">MEDAL CRITERIA</p><br />
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<p class="minor_title">Bronze Medal</p><br />
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• We have registered our <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">team</a> of eleven undergraduates. <br />
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• We have completed the judging form.<br />
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• We have created a beautiful team wiki in keeping with the iGEM guidelines.<br />
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• We have a poster and presentation ready for the regional jamboree in Lyon.<br />
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• We have developed two new <a href="https://2013.igem.org/Team:UCL/Project/Parts" target="_blank">BioBricks</a> and have submitted them to the iGEM Registry<br />
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<p class="minor_title">Silver Medal</p><br />
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• We have proved our zeocin resistance BioBrick <a href="https://2013.igem.org/Team:UCL/Project/Parts" target="_blank">worked</a> by characterising it in HeLa cells. We did this by by creating a zeocin kill curve with and without our <a href="https://2013.igem.org/Team:UCL/Project/Marker" target="_blank">zeocin resistance</a> BioBrick. <br />
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• Due to the gravity of Alzheimer's disease and the perceived sovereignty of the brain, we have taken the ethics of using synthetic biological treatments very seriously. We produced a <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">neuroethics</a> and a <a href="https://2013.igem.org/Team:UCL/Practice/Report" target="_blank">feasibility</a> report, consulted numerous experts and provided a concise but detailed <a href="https://2013.igem.org/Team:UCL/Background" target="_blank">background</a> to our project, which shows how our proposed <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">genetic circuit</a> is advised by multiple theories for the causation of Alzheimer's pathology.<br />
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Gold Medal<br />
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• We collaborated with iGEM Westminster, by modelling how well their bed-bug killing device will operate in a bedroom as well advising them on how to run a speed debate using our format. One of our team members, Alex Bates, also attended as guest speaker at their speed debate event.<br />
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Full details on collaboration with iGEM Westminster can be found below.<br />
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• Our <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">Human Practices</a> deal with an entirely new area for iGEM and, indeed, almost a completely new avenue of research for synthetic biology as a field - the <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">fusion</a> of neuroscience and synthetic biology. We use a variety of strong methods for social and ethical analysis, and outreach. Please see the section below for a summary of what we achieved.<br />
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• Outside of this theme, we also engaged in outreach by training and advising the UCL Academy iGEM high school team. This was the first time a British iGEM team has helped run an iGEM HS team.<br />
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<p class="major_title">ABOVE AND BEYOND</p><br />
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<p class="minor_title">Project</p><br />
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<p class="body_text">project stuff<br />
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<p class="minor_title">Human Practices</p><br />
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• We have looked at an entirely new ethical area for iGEM that has also essentially not been covered in academia; the neuroethics of genetic engineering. We have dubbed this <a href="https://2013.igem.org/Team:UCL/Practice" target="_blank">'Neuro-genethics'</a>. <br />
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• We have produced an extensive 20 page <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">report</a> that looks into neuro-genethics and what synthetic biology could achieve in neuroscience.<br />
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• We have engaged the public on this topic by getting their opinions at the <a href="http://www.artscatalyst.org/" target="_blank">Arts Catalyst</a>, running a <a href="https://2013.igem.org/Team:UCL/Practice/Debate" target="_blank">speed debate</a> and a <a href="https://2013.igem.org/Team:UCL/Practice/TED" target="_blank">TED debate</a>, conducting an <a href="https://2013.igem.org/Team:UCL/Practice/Survey" target="_blank">online survey</a> and producing a <a href="https://2013.igem.org/Team:UCL/Practice/Documentary" target="_blank">documentary </a> on synthetic neurobiology.<br />
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• We ran one of iGEM's first <a href="https://2013.igem.org/Team:UCL/Practice/Creative" target="_blank">creative writing competitions</a>, to gauge public opinion on brain modification and highligh5t the impact of fiction on society's views.<br />
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•Petcha Kutcha style presentation to enguage prospective students of the UCL Engineering Department about iGEM.<br />
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• We created an original memory bank, <a href="https://2013.igem.org/Team:UCL/Memories" target="_blank">Eternal Sunshine</a>, which highlights how precious memories are, indicating the desperate need to cure Alzheimer's disease. <br />
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• We created a <a href="https://2013.igem.org/Team:UCL/Practice/Report" target="_blank">feasibility report</a> on implementing our treatment.<br />
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<p class="minor_title">Modelling</p><br />
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• We are the first iGEM team to use a <a href="https://2013.igem.org/Team:UCL/Modeling" target="_blank">protein network analysis approach</a>. Network based bioinformatics can feedback into synthetic biology by informing the choice of parts in therapeutic genetic circuits.<br />
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<p class="minor_title">Wiki</p><br />
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• We have developed an, original attractive wiki using art work by our artists in residence, <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Fong Yi Khoo</a> and <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Oran Maguire</a>.<br />
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• We have included an extensive neuroscience background section, which explains and compares multiple theories for the causation of Alzheimer's disease, so that readers can fully understand the pros and cons of our genetic circuit. <br />
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• We have included a full complement of citations that link to PubMed pages so that it is easy to see from where our ideas and explanations have been drawn, and which papers have inspired us.<br />
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<p class="minor_title">Collaboration with Westminster iGEM</p><br />
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<b>Model (ROBIN WRITES HERE)</b><br />
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Movement of bed-bugs towards blood meal<br />
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<p class="minor_title">Mentoring iGEM HS</p><br />
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Two members of the UCL iGEM team volunteered as advisers to the UCL Academy iGEM team – <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Ruxi</a> and <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Khaicheng</a>, under the guidance of Aurelija Grigonyte, a member of the UCL iGEM 2012 team. During the high school team’s brainstorming process, we provided them with guidance and resources for their research. We also supervised their lab work in the UCL Biochemical Engineering department.<br />
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UCL is the first university in the UK to be the sole sponsor of an academy – a non-selective mixed state school in our home borough of Camden. UCL Academy represents a unique opportunity to blur the boundaries between secondary and higher education.<br />
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The academy is one of the first UK high schools to participate in iGEM this year, and is the only UK team so far to have attended the <a href="https://2013hs.igem.org/Main_Page" target="_blank">High School iGEM Jamboree</a> at MIT, Boston. The team aimed to revolutionise the recycling industry by proposing a home system that converts cellulose into glucose, allowing the up-cycling of paper into a commercial product of bioplastic - polyhydroxybutyrate (PHB) <br />
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For information about their iGEM project, check out their wiki <a href="https://2013hs.igem.org/Team:UCL_Academy" target="_blank">here</a>. <br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/Project/SafetyTeam:UCL/Project/Safety2013-10-05T00:58:01Z<p>Naruto: </p>
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<p class="major_title">KEEPING SAFE</p><br />
<p class="minor_title">It's Just Good Lab Practice</p><br />
<p class="body_text"><br />
The health and safety considerations of the project are vital for the well-being of the project members and the laboratory environment. Some of the key considerations to be made involve handling bio-hazardous materials, process chemicals and mechanical operations; requiring planning and awareness in order prevent hazards from occurring, as well as appropriate plans of action in order to deal with any situation that may occur.<br />
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The Escherichia coli strain used is not considered pathogenic, and thus not of considerable risk to the environment or team members. Despite this, there must still be a level of Good Laboratory Practice (GLP) to reduce risk associated with performing experiments on a daily basis. As a minimum requirement, all members will wear some form of protective clothing, which generally consists of a lab coat and goggles, as well as single use gloves.<br />
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For disposal of spent materials, any item should be isolated and sealed in a container which prevents physical contact with any of the facility. The material should then be removed from the room through the waste corridor and disposed of accordingly (chemical or steam treatment for example).<br />
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Continual monitoring and safety reviews of the facility is required to maintain GLP and the condition of the plant. By undertaking continual assessments of laboratory safety, the risk of a hazard occurring in the plant can be reduced. Training of team members in safe operation in general safety protocols is essential to reduce risk; so safety training and education in good laboratory practise was undertaken by all team members before experiments could begin.<br />
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The main principle behind performing experiments in a safe environment is to minimise risk. Risk is considered as the amalgamation of the potential damage that a hazard could cause, combined with the likelihood of said hazard occurring. The damage a hazard could cause is normally set by the equipment present in the laboratory, whilst laboratory team members can often decrease risk by decreasing the likelihood of a hazard occurring. Below the three main categories (Microbiological, Chemical and Mechanical) are defined with tables showing which hazard fall into said category, and how the risk is minimised in each case.<br />
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<p class="minor_title">Microbiological Hazards</p><br />
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The key focus of microbiological hazard control concerns the Escherichia coli expression system being used. The majority of Escherichia coli strains are categorised into Bio-safety levels 1 or 2, with non-pathogenic strains placed into level 1 under World Health Organisation classifications. Therefore the Escherichia coli strain used in the facility is considered to be Bio-safety level one, which often requires less stringent safety standards. The genetically engineered strains also may require more safety regulation requirements than wild type micro-organisms; however advances in synthetic biology are allowing more control over the expression system in terms of pathogenicity and also the ability to express suicide genes, which can prevent any live Escherichia coli from surviving outside of the facility environment. Regardless, the facility must ensure and validate that all of the Escherichia coli is killed and effectively disposed of. Pressure systems and directed ventilation are in place to prevent escape of any strains, although this may lead to possible contaminants entering the halls, which dictates that the live Escherichia coli must be sealed in containers or vessels at all times where possible in order to prevent infection. The use of mammalian systems in the project also present a degree of hazard, although the associated risk is lower when compared to using bacteria.<br />
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<table><br />
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<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
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<tr><br />
<td>Escape of Escherichia coli from lab</td><br />
<td>The strain(s) exit the laboratory environment, thus allowing the possibility of contamination of exterior objects and/or persons.</td><br />
<td>Pressure of lab is lower than of corridor, thus causing airflow into the lab as opposed of out, which minimises the chance of airborne or aerosol escape of E. coli. Laboratory coats are worn over clothes which do not leave the lab, thus reducing possibility of a team member inadvertently acting as a carrier out of the lab.</td><br />
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<td>Contamination of E coli/ HeLa/ Microglia</td><br />
<td>Contamination of the desired strain leads to competition between several strains of bacteria, resulting in inaccurate stocks and therefore unusable data. Note that bacterial and mammalian experiments are undertaken in separate parts of the facility.</td><br />
<td>Exposure of strain to open air is minimised, which is standard practise for any container that may be involved with the desired E. coli strain. Single use gloves are used to minimise possible contamination when experiments are performed. Ethanol is applied on work surfaces before and after experimental procedures in order to minimise contamination on work surfaces etc.</td><br />
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<tr><br />
<td>Contamination of team members</td><br />
<td>Illness could ensue from working in the laboratory environment.</td><br />
<td>Team members are not advised to work when feeling unwell. Gloves, lab coat and goggles are also worn to minimise contact with organisms. Personal hygiene standards are advised to be upheld in particular for laboratory workers.</td><br />
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<tr><br />
<td>HeLa Cells</td><br />
<td>Level 2 biosafety class cells</td><br />
<td>Cell experiments always performed in a sterile environment and a fume hood, with a supervisor providing assistance to the experiment when necessary</td><br />
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<td>Microglia cells</td><br />
<td>Level 2 biosafety class cells</td><br />
<td>Dr. Darren Nesbeth will perform the neccesary assays in place of iGEm members due to the safety regulations</td><br />
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<p class="minor_title">Chemical Hazards</p><br />
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When performing experiments, it may be necessary to use dangerous chemicals during certain processes. Whilst using hazardous chemicals is avoided where necessary, in some cases it is required, so in each case where this occurs the procedure must be executed in a fashion which is as safe as reasonably possible. Therefore standard protocols such as wearing protective clothing may be added to by performing experiments in fume hoods or other such devices to minimise any chance of contact via evaporation etc.<br />
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<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
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<td>Ethidium Bromide (EtBr)</td><br />
<td>Mutagenic.<br />
Minor toxicity issue</td><br />
<td>All operations using EtBr are carried out in a fume cupboard - separate equipment (pipettes etc.) are used only in the fume cupboard specifically for EtBr.<br />
Gloves and long sleeved protective gowns MUST be worn, disposal of items in contact with EtBr are disposed of separately to other wastes.<br />
Wash hands after gloves are removed.</td><br />
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<td>High concentration Ethanol (EtOH) and other alcohols</td><br />
<td>Flammable.</td><br />
<td>Securely stored (glass container) in a separate cupboard in volumes less than 2 Litres.</td><br />
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<td>Dimethyl sulfoxide & Zeocin</td><br />
<td>Irritant to operator if contact with skin is made</td><br />
<td>Chemical handled with extreme caution, with particular attention to no skin exposed when performing an experiment, particularly goggles must be worn</td><br />
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<p class="minor_title">Mechanical Hazards</p><br />
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For this project, mechanical hazards pose the lowest probability of occurring out of the three and thus may be considered as the lowest risk group. However, there are hazards present which must be minimised. The predominant issue here is the use of the desktop centrifuge system, which poses considerable danger if a blowout occurs.<br />
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<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
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<td>Centrifuge</td><br />
<td>Centrifuge mechanical failure (eg. of rotor) can cause severe damage to the machine, and possibly personnel in the nearby vicinity.</td><br />
<td><br />
Regular inspections and maintenance of the machine is required (performed by external engineers). <br />
Machine not to be used if in non optimal condition.<br />
Centrifuge must be balanced to minimise chance of rotor failure.<br />
Centrifuge must be properly closed before use (required for the machine to work.)<br />
Attention must be paid to ensure that nothing is spilled in the centrifuge bowl, or a build up of dust or aerosol should also be avoided.</td><br />
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<p class="minor_title">Concluding Comments</p><br />
<p class="body_text"><br />
The safety of the team members, produced material and environment are key aspects to the smooth running of the project, and it must be ensured that adequate measures are in place to provide protection and ensuring that guidelines can be followed proceeding mandatory safety training. Current Good Laboratory Practice must also be adhered to at all times. By following guidelines and acting in an appropriate manner whilst in the laboratory, the general risk in terms of micro-biological, chemical and mechanical aspects will be reduced to a level which is as low as reasonably possible for all project members involved.<br />
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Due to the relatively small scale of the experiments performed, the quantities used of material is not large, but still must be handled and respected properly at all times. Proper containment and labelling of materials in sealed containers is necessary, especially when of particular hazard such as live material or Ethidium Bromide. Flammability concerns are also present with alcohol and other flammable materials stored in the lab, although the volumes (generally less than 2L) are not considerably large enough, although should still be used and handled properly when used, with fire extinguishers available and also emergency exits present if required. Protective clothing also should be worn where appropriate to minimise the possibility of skin contact, with wash and first aid station available in key areas where allowable due to the area classification.<br />
</p><br />
<p class="body_text"><br />
The mechanical safety predominantly concerns the proper maintenance of the centrifuge system, which can cause a considerable risk if a failure occurs. Regular inspections should be made, with the centrifuge often restricted access when it was considered unfit for use. This room is also placed away from mammalian and bacterial processing centres. Prevention methods should also be in place such as emergency shut-off procedures.<br />
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By following all guidelines set out in safety training and following supervisor instructions, the project may be performed in a safe and effective manner. Continual training and inspection of the facility will aid in ensuring safety levels are maintained, with review protocols in place to investigate any faults. Team members have been trained in general safety, with supervisors having first aid experience and training. These considerations combined will ensure the safety risk to the personnel; product and environment is as low as reasonably possible. For further reading, the Institute of Chemical Engineering (IChemE) has relevant information, as well as other safety guidance documents relating to the relevant country where the university is located.<br />
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</html></div>Narutohttp://2013.igem.org/Team:UCL/Project/SafetyTeam:UCL/Project/Safety2013-10-05T00:57:11Z<p>Naruto: </p>
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<br />
<p class="major_title">KEEPING SAFE</p><br />
<p class="minor_title">It's Just Good Lab Practice</p><br />
<p class="body_text"><br />
The health and safety considerations of the project are vital for the well-being of the project members and the laboratory environment. Some of the key considerations to be made involve handling bio-hazardous materials, process chemicals and mechanical operations; requiring planning and awareness in order prevent hazards from occurring, as well as appropriate plans of action in order to deal with any situation that may occur.<br />
</p><br />
<p class="body_text"><br />
The Escherichia coli strain used is not considered pathogenic, and thus not of considerable risk to the environment or team members. Despite this, there must still be a level of Good Laboratory Practice (GLP) to reduce risk associated with performing experiments on a daily basis. As a minimum requirement, all members will wear some form of protective clothing, which generally consists of a lab coat and goggles, as well as single use gloves.<br />
</p><br />
<p class="body_text"><br />
For disposal of spent materials, any item should be isolated and sealed in a container which prevents physical contact with any of the facility. The material should then be removed from the room through the waste corridor and disposed of accordingly (chemical or steam treatment for example).<br />
</p><br />
<p class="body_text"><br />
Continual monitoring and safety reviews of the facility is required to maintain GLP and the condition of the plant. By undertaking continual assessments of laboratory safety, the risk of a hazard occurring in the plant can be reduced. Training of team members in safe operation in general safety protocols is essential to reduce risk; so safety training and education in good laboratory practise was undertaken by all team members before experiments could begin.<br />
</p><br />
<p class="body_text"><br />
The main principle behind performing experiments in a safe environment is to minimise risk. Risk is considered as the amalgamation of the potential damage that a hazard could cause, combined with the likelihood of said hazard occurring. The damage a hazard could cause is normally set by the equipment present in the laboratory, whilst laboratory team members can often decrease risk by decreasing the likelihood of a hazard occurring. Below the three main categories (Microbiological, Chemical and Mechanical) are defined with tables showing which hazard fall into said category, and how the risk is minimised in each case.<br />
</p><br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">Microbiological Hazards</p><br />
<p class="body_text"><br />
The key focus of microbiological hazard control concerns the Escherichia coli expression system being used. The majority of Escherichia coli strains are categorised into Bio-safety levels 1 or 2, with non-pathogenic strains placed into level 1 under World Health Organisation classifications. Therefore the Escherichia coli strain used in the facility is considered to be Bio-safety level one, which often requires less stringent safety standards. The genetically engineered strains also may require more safety regulation requirements than wild type micro-organisms; however advances in synthetic biology are allowing more control over the expression system in terms of pathogenicity and also the ability to express suicide genes, which can prevent any live Escherichia coli from surviving outside of the facility environment. Regardless, the facility must ensure and validate that all of the Escherichia coli is killed and effectively disposed of. Pressure systems and directed ventilation are in place to prevent escape of any strains, although this may lead to possible contaminants entering the halls, which dictates that the live Escherichia coli must be sealed in containers or vessels at all times where possible in order to prevent infection. The use of mammalian systems in the project also present a degree of hazard, although the associated risk is lower when compared to using bacteria.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Escape of Escherichia coli from lab</td><br />
<td>The strain(s) exit the laboratory environment, thus allowing the possibility of contamination of exterior objects and/or persons.</td><br />
<td>Pressure of lab is lower than of corridor, thus causing airflow into the lab as opposed of out, which minimises the chance of airborne or aerosol escape of E. coli. Laboratory coats are worn over clothes which do not leave the lab, thus reducing possibility of a team member inadvertently acting as a carrier out of the lab.</td><br />
</tr><br />
<tr><br />
<td>Contamination of E coli/ HeLa/ Microglia</td><br />
<td>Contamination of the desired strain leads to competition between several strains of bacteria, resulting in inaccurate stocks and therefore unusable data. Note that bacterial and mammalian experiments are undertaken in separate parts of the facility.</td><br />
<td>Exposure of strain to open air is minimised, which is standard practise for any container that may be involved with the desired E. coli strain. Single use gloves are used to minimise possible contamination when experiments are performed. Ethanol is applied on work surfaces before and after experimental procedures in order to minimise contamination on work surfaces etc.</td><br />
</tr><br />
<tr><br />
<td>Contamination of team members</td><br />
<td>Illness could ensue from working in the laboratory environment.</td><br />
<td>Team members are not advised to work when feeling unwell. Gloves, lab coat and goggles are also worn to minimise contact with organisms. Personal hygiene standards are advised to be upheld in particular for laboratory workers.</td><br />
</tr><br />
<tr><br />
<td>HeLa Cells</td><br />
<td>Level 2 biosafety class cells</td><br />
<td>Cell experiments always performed in a sterile environment and a fume hood, with a supervisor providing assistance to the experiment when necessary</td><br />
</tr><br />
<tr><br />
<td>Microglia cells</td><br />
<td>Level 2 biosafety class cells</td><br />
<td>Dr. Darren Nesbeth will perform the neccesary assays in place of iGEm members due to the safety regulations</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Chemical Hazards</p><br />
<p class="body_text"><br />
When performing experiments, it may be necessary to use dangerous chemicals during certain processes. Whilst using hazardous chemicals is avoided where necessary, in some cases it is required, so in each case where this occurs the procedure must be executed in a fashion which is as safe as reasonably possible. Therefore standard protocols such as wearing protective clothing may be added to by performing experiments in fume hoods or other such devices to minimise any chance of contact via evaporation etc.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Ethidium Bromide (EtBr)</td><br />
<td>Mutagenic.<br />
Minor toxicity issue</td><br />
<td>All operations using EtBr are carried out in a fume cupboard - separate equipment (pipettes etc.) are used only in the fume cupboard specifically for EtBr.<br />
Gloves and long sleeved protective gowns MUST be worn, disposal of items in contact with EtBr are disposed of separately to other wastes.<br />
Wash hands after gloves are removed.</td><br />
</tr><br />
<tr><br />
<td>High concentration Ethanol (EtOH) and other alcohols</td><br />
<td>Flammable.</td><br />
<td>Securely stored (glass container) in a separate cupboard in volumes less than 2 Litres.</td><br />
</tr><br />
<tr><br />
<td>Dimethyl sulfoxide</td><br />
<td>Irritant to operator if contact with skin is made</td><br />
<td>Chemical handled with extreme caution, with particular attention to no skin exposed when performing an experiment, particularly goggles must be worn</td><br />
</tr><br />
<tr><br />
<td>Zeocin</td><br />
<td>Irritant to operator if contact with skin is made</td><br />
<td>Chemical handled with extreme caution, with particular attention to no skin exposed when performing an experiment, particularly goggles must be worn</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Mechanical Hazards</p><br />
<p class="body_text"><br />
For this project, mechanical hazards pose the lowest probability of occurring out of the three and thus may be considered as the lowest risk group. However, there are hazards present which must be minimised. The predominant issue here is the use of the desktop centrifuge system, which poses considerable danger if a blowout occurs.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Centrifuge</td><br />
<td>Centrifuge mechanical failure (eg. of rotor) can cause severe damage to the machine, and possibly personnel in the nearby vicinity.</td><br />
<td><br />
Regular inspections and maintenance of the machine is required (performed by external engineers). <br />
Machine not to be used if in non optimal condition.<br />
Centrifuge must be balanced to minimise chance of rotor failure.<br />
Centrifuge must be properly closed before use (required for the machine to work.)<br />
Attention must be paid to ensure that nothing is spilled in the centrifuge bowl, or a build up of dust or aerosol should also be avoided.</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Concluding Comments</p><br />
<p class="body_text"><br />
The safety of the team members, produced material and environment are key aspects to the smooth running of the project, and it must be ensured that adequate measures are in place to provide protection and ensuring that guidelines can be followed proceeding mandatory safety training. Current Good Laboratory Practice must also be adhered to at all times. By following guidelines and acting in an appropriate manner whilst in the laboratory, the general risk in terms of micro-biological, chemical and mechanical aspects will be reduced to a level which is as low as reasonably possible for all project members involved.<br />
</p><br />
<p class="body_text"><br />
Due to the relatively small scale of the experiments performed, the quantities used of material is not large, but still must be handled and respected properly at all times. Proper containment and labelling of materials in sealed containers is necessary, especially when of particular hazard such as live material or Ethidium Bromide. Flammability concerns are also present with alcohol and other flammable materials stored in the lab, although the volumes (generally less than 2L) are not considerably large enough, although should still be used and handled properly when used, with fire extinguishers available and also emergency exits present if required. Protective clothing also should be worn where appropriate to minimise the possibility of skin contact, with wash and first aid station available in key areas where allowable due to the area classification.<br />
</p><br />
<p class="body_text"><br />
The mechanical safety predominantly concerns the proper maintenance of the centrifuge system, which can cause a considerable risk if a failure occurs. Regular inspections should be made, with the centrifuge often restricted access when it was considered unfit for use. This room is also placed away from mammalian and bacterial processing centres. Prevention methods should also be in place such as emergency shut-off procedures.<br />
</p><br />
<p class="body_text"><br />
By following all guidelines set out in safety training and following supervisor instructions, the project may be performed in a safe and effective manner. Continual training and inspection of the facility will aid in ensuring safety levels are maintained, with review protocols in place to investigate any faults. Team members have been trained in general safety, with supervisors having first aid experience and training. These considerations combined will ensure the safety risk to the personnel; product and environment is as low as reasonably possible. For further reading, the Institute of Chemical Engineering (IChemE) has relevant information, as well as other safety guidance documents relating to the relevant country where the university is located.<br />
</p><br />
<div class="gap"></div><br />
<br />
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<br />
<p class="major_title">KEEPING SAFE</p><br />
<p class="minor_title">It's Just Good Lab Practice</p><br />
<p class="body_text"><br />
The health and safety considerations of the project are vital for the well-being of the project members and the laboratory environment. Some of the key considerations to be made involve handling bio-hazardous materials, process chemicals and mechanical operations; requiring planning and awareness in order prevent hazards from occurring, as well as appropriate plans of action in order to deal with any situation that may occur.<br />
</p><br />
<p class="body_text"><br />
The Escherichia coli strain used is not considered pathogenic, and thus not of considerable risk to the environment or team members. Despite this, there must still be a level of Good Laboratory Practice (GLP) to reduce risk associated with performing experiments on a daily basis. As a minimum requirement, all members will wear some form of protective clothing, which generally consists of a lab coat and goggles, as well as single use gloves.<br />
</p><br />
<p class="body_text"><br />
For disposal of spent materials, any item should be isolated and sealed in a container which prevents physical contact with any of the facility. The material should then be removed from the room through the waste corridor and disposed of accordingly (chemical or steam treatment for example).<br />
</p><br />
<p class="body_text"><br />
Continual monitoring and safety reviews of the facility is required to maintain GLP and the condition of the plant. By undertaking continual assessments of laboratory safety, the risk of a hazard occurring in the plant can be reduced. Training of team members in safe operation in general safety protocols is essential to reduce risk; so safety training and education in good laboratory practise was undertaken by all team members before experiments could begin.<br />
</p><br />
<p class="body_text"><br />
The main principle behind performing experiments in a safe environment is to minimise risk. Risk is considered as the amalgamation of the potential damage that a hazard could cause, combined with the likelihood of said hazard occurring. The damage a hazard could cause is normally set by the equipment present in the laboratory, whilst laboratory team members can often decrease risk by decreasing the likelihood of a hazard occurring. Below the three main categories (Microbiological, Chemical and Mechanical) are defined with tables showing which hazard fall into said category, and how the risk is minimised in each case.<br />
</p><br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">Microbiological Hazards</p><br />
<p class="body_text"><br />
The key focus of microbiological hazard control concerns the Escherichia coli expression system being used. The majority of Escherichia coli strains are categorised into Bio-safety levels 1 or 2, with non-pathogenic strains placed into level 1 under World Health Organisation classifications. Therefore the Escherichia coli strain used in the facility is considered to be Bio-safety level one, which often requires less stringent safety standards. The genetically engineered strains also may require more safety regulation requirements than wild type micro-organisms; however advances in synthetic biology are allowing more control over the expression system in terms of pathogenicity and also the ability to express suicide genes, which can prevent any live Escherichia coli from surviving outside of the facility environment. Regardless, the facility must ensure and validate that all of the Escherichia coli is killed and effectively disposed of. Pressure systems and directed ventilation are in place to prevent escape of any strains, although this may lead to possible contaminants entering the halls, which dictates that the live Escherichia coli must be sealed in containers or vessels at all times where possible in order to prevent infection. The use of mammalian systems in the project also present a degree of hazard, although the associated risk is lower when compared to using bacteria.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Escape of Escherichia coli from lab</td><br />
<td>The strain(s) exit the laboratory environment, thus allowing the possibility of contamination of exterior objects and/or persons.</td><br />
<td>Pressure of lab is lower than of corridor, thus causing airflow into the lab as opposed of out, which minimises the chance of airborne or aerosol escape of E. coli. Laboratory coats are worn over clothes which do not leave the lab, thus reducing possibility of a team member inadvertently acting as a carrier out of the lab.</td><br />
</tr><br />
<tr><br />
<td>Contamination of E coli/ HeLa/ Microglia</td><br />
<td>Contamination of the desired strain leads to competition between several strains of bacteria, resulting in inaccurate stocks and therefore unusable data. Note that bacterial and mammalian experiments are undertaken in separate parts of the facility.</td><br />
<td>Exposure of strain to open air is minimised, which is standard practise for any container that may be involved with the desired E. coli strain. Single use gloves are used to minimise possible contamination when experiments are performed. Ethanol is applied on work surfaces before and after experimental procedures in order to minimise contamination on work surfaces etc.</td><br />
</tr><br />
<tr><br />
<td>Contamination of team members</td><br />
<td>Illness could ensue from working in the laboratory environment.</td><br />
<td>Team members are not advised to work when feeling unwell. Gloves, lab coat and goggles are also worn to minimise contact with organisms. Personal hygiene standards are advised to be upheld in particular for laboratory workers.</td><br />
</tr><br />
<tr><br />
<td>HeLa Cells</td><br />
<td>Level 2 biosafety class cells</td><br />
<td>Cell experiments always performed in a sterile environment and a fume hood, with a supervisor providing assistance to the experiment when necessary</td><br />
</tr><br />
<tr><br />
<td>Microglia cells</td><br />
<td>Level 2 biosafety class cells</td><br />
<td>Dr. Darren Nesbeth will perform the neccesary assays in place of iGEm members due to the safety regulations</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Chemical Hazards</p><br />
<p class="body_text"><br />
When performing experiments, it may be necessary to use dangerous chemicals during certain processes. Whilst using hazardous chemicals is avoided where necessary, in some cases it is required, so in each case where this occurs the procedure must be executed in a fashion which is as safe as reasonably possible. Therefore standard protocols such as wearing protective clothing may be added to by performing experiments in fume hoods or other such devices to minimise any chance of contact via evaporation etc.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Ethidium Bromide (EtBr)</td><br />
<td>Mutagenic.<br />
Minor toxicity issue</td><br />
<td>All operations using EtBr are carried out in a fume cupboard - separate equipment (pipettes etc.) are used only in the fume cupboard specifically for EtBr.<br />
Gloves and long sleeved protective gowns MUST be worn, disposal of items in contact with EtBr are disposed of separately to other wastes.<br />
Wash hands after gloves are removed.</td><br />
</tr><br />
<tr><br />
<td>High concentration Ethanol (EtOH) and other alcohols</td><br />
<td>Flammable.</td><br />
<td>Securely stored (glass container) in a separate cupboard in volumes less than 2 Litres.</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Mechanical Hazards</p><br />
<p class="body_text"><br />
For this project, mechanical hazards pose the lowest probability of occurring out of the three and thus may be considered as the lowest risk group. However, there are hazards present which must be minimised. The predominant issue here is the use of the desktop centrifuge system, which poses considerable danger if a blowout occurs.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Centrifuge</td><br />
<td>Centrifuge mechanical failure (eg. of rotor) can cause severe damage to the machine, and possibly personnel in the nearby vicinity.</td><br />
<td><br />
Regular inspections and maintenance of the machine is required (performed by external engineers). <br />
Machine not to be used if in non optimal condition.<br />
Centrifuge must be balanced to minimise chance of rotor failure.<br />
Centrifuge must be properly closed before use (required for the machine to work.)<br />
Attention must be paid to ensure that nothing is spilled in the centrifuge bowl, or a build up of dust or aerosol should also be avoided.</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Concluding Comments</p><br />
<p class="body_text"><br />
The safety of the team members, produced material and environment are key aspects to the smooth running of the project, and it must be ensured that adequate measures are in place to provide protection and ensuring that guidelines can be followed proceeding mandatory safety training. Current Good Laboratory Practice must also be adhered to at all times. By following guidelines and acting in an appropriate manner whilst in the laboratory, the general risk in terms of micro-biological, chemical and mechanical aspects will be reduced to a level which is as low as reasonably possible for all project members involved.<br />
</p><br />
<p class="body_text"><br />
Due to the relatively small scale of the experiments performed, the quantities used of material is not large, but still must be handled and respected properly at all times. Proper containment and labelling of materials in sealed containers is necessary, especially when of particular hazard such as live material or Ethidium Bromide. Flammability concerns are also present with alcohol and other flammable materials stored in the lab, although the volumes (generally less than 2L) are not considerably large enough, although should still be used and handled properly when used, with fire extinguishers available and also emergency exits present if required. Protective clothing also should be worn where appropriate to minimise the possibility of skin contact, with wash and first aid station available in key areas where allowable due to the area classification.<br />
</p><br />
<p class="body_text"><br />
The mechanical safety predominantly concerns the proper maintenance of the centrifuge system, which can cause a considerable risk if a failure occurs. Regular inspections should be made, with the centrifuge often restricted access when it was considered unfit for use. This room is also placed away from mammalian and bacterial processing centres. Prevention methods should also be in place such as emergency shut-off procedures.<br />
</p><br />
<p class="body_text"><br />
By following all guidelines set out in safety training and following supervisor instructions, the project may be performed in a safe and effective manner. Continual training and inspection of the facility will aid in ensuring safety levels are maintained, with review protocols in place to investigate any faults. Team members have been trained in general safety, with supervisors having first aid experience and training. These considerations combined will ensure the safety risk to the personnel; product and environment is as low as reasonably possible. For further reading, the Institute of Chemical Engineering (IChemE) has relevant information, as well as other safety guidance documents relating to the relevant country where the university is located.<br />
</p><br />
<div class="gap"></div><br />
<br />
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<br />
<p class="major_title">KEEPING SAFE</p><br />
<p class="minor_title">It's Just Good Lab Practice</p><br />
<p class="body_text"><br />
The health and safety considerations of the project are vital for the well-being of the project members and the laboratory environment. Some of the key considerations to be made involve handling bio-hazardous materials, process chemicals and mechanical operations; requiring planning and awareness in order prevent hazards from occurring, as well as appropriate plans of action in order to deal with any situation that may occur.<br />
</p><br />
<p class="body_text"><br />
The Escherichia coli strain used is not considered pathogenic, and thus not of considerable risk to the environment or team members. Despite this, there must still be a level of Good Laboratory Practice (GLP) to reduce risk associated with performing experiments on a daily basis. As a minimum requirement, all members will wear some form of protective clothing, which generally consists of a lab coat and goggles, as well as single use gloves.<br />
</p><br />
<p class="body_text"><br />
For disposal of spent materials, any item should be isolated and sealed in a container which prevents physical contact with any of the facility. The material should then be removed from the room through the waste corridor and disposed of accordingly (chemical or steam treatment for example).<br />
</p><br />
<p class="body_text"><br />
Continual monitoring and safety reviews of the facility is required to maintain GLP and the condition of the plant. By undertaking continual assessments of laboratory safety, the risk of a hazard occurring in the plant can be reduced. Training of team members in safe operation in general safety protocols is essential to reduce risk; so safety training and education in good laboratory practise was undertaken by all team members before experiments could begin.<br />
</p><br />
<p class="body_text"><br />
The main principle behind performing experiments in a safe environment is to minimise risk. Risk is considered as the amalgamation of the potential damage that a hazard could cause, combined with the likelihood of said hazard occurring. The damage a hazard could cause is normally set by the equipment present in the laboratory, whilst laboratory team members can often decrease risk by decreasing the likelihood of a hazard occurring. Below the three main categories (Microbiological, Chemical and Mechanical) are defined with tables showing which hazard fall into said category, and how the risk is minimised in each case.<br />
</p><br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">Microbiological Hazards</p><br />
<p class="body_text"><br />
The key focus of microbiological hazard control concerns the Escherichia coli expression system being used. The majority of Escherichia coli strains are categorised into Bio-safety levels 1 or 2, with non-pathogenic strains placed into level 1 under World Health Organisation classifications. Therefore the Escherichia coli strain used in the facility is considered to be Bio-safety level one, which often requires less stringent safety standards. The genetically engineered strains also may require more safety regulation requirements than wild type micro-organisms; however advances in synthetic biology are allowing more control over the expression system in terms of pathogenicity and also the ability to express suicide genes, which can prevent any live Escherichia coli from surviving outside of the facility environment. Regardless, the facility must ensure and validate that all of the Escherichia coli is killed and effectively disposed of. Pressure systems and directed ventilation are in place to prevent escape of any strains, although this may lead to possible contaminants entering the halls, which dictates that the live Escherichia coli must be sealed in containers or vessels at all times where possible in order to prevent infection. The use of mammalian systems in the project also present a degree of hazard, although the associated risk is lower when compared to using bacteria.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Escape of Escherichia coli from lab</td><br />
<td>The strain(s) exit the laboratory environment, thus allowing the possibility of contamination of exterior objects and/or persons.</td><br />
<td>Pressure of lab is lower than of corridor, thus causing airflow into the lab as opposed of out, which minimises the chance of airborne or aerosol escape of E. coli. Laboratory coats are worn over clothes which do not leave the lab, thus reducing possibility of a team member inadvertently acting as a carrier out of the lab.</td><br />
</tr><br />
<tr><br />
<td>Contamination of E coli/ HeLa/ Microglia</td><br />
<td>Contamination of the desired strain leads to competition between several strains of bacteria, resulting in inaccurate stocks and therefore unusable data. Note that bacterial and mammalian experiments are undertaken in separate parts of the facility.</td><br />
<td>Exposure of strain to open air is minimised, which is standard practise for any container that may be involved with the desired E. coli strain. Single use gloves are used to minimise possible contamination when experiments are performed. Ethanol is applied on work surfaces before and after experimental procedures in order to minimise contamination on work surfaces etc.</td><br />
</tr><br />
<tr><br />
<td>Contamination of team members</td><br />
<td>Illness could ensue from working in the laboratory environment.</td><br />
<td>Team members are not advised to work when feeling unwell. Gloves, lab coat and goggles are also worn to minimise contact with organisms. Personal hygiene standards are advised to be upheld in particular for laboratory workers.</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Chemical Hazards</p><br />
<p class="body_text"><br />
When performing experiments, it may be necessary to use dangerous chemicals during certain processes. Whilst using hazardous chemicals is avoided where necessary, in some cases it is required, so in each case where this occurs the procedure must be executed in a fashion which is as safe as reasonably possible. Therefore standard protocols such as wearing protective clothing may be added to by performing experiments in fume hoods or other such devices to minimise any chance of contact via evaporation etc.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Ethidium Bromide (EtBr)</td><br />
<td>Mutagenic.<br />
Minor toxicity issue</td><br />
<td>All operations using EtBr are carried out in a fume cupboard - separate equipment (pipettes etc.) are used only in the fume cupboard specifically for EtBr.<br />
Gloves and long sleeved protective gowns MUST be worn, disposal of items in contact with EtBr are disposed of separately to other wastes.<br />
Wash hands after gloves are removed.</td><br />
</tr><br />
<tr><br />
<td>High concentration Ethanol (EtOH) and other alcohols</td><br />
<td>Flammable.</td><br />
<td>Securely stored (glass container) in a separate cupboard in volumes less than 2 Litres.</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Mechanical Hazards</p><br />
<p class="body_text"><br />
For this project, mechanical hazards pose the lowest probability of occurring out of the three and thus may be considered as the lowest risk group. However, there are hazards present which must be minimised. The predominant issue here is the use of the desktop centrifuge system, which poses considerable danger if a blowout occurs.<br />
</p><br />
<br />
<table><br />
<tr><br />
<th><b>Hazard Type</b></th><br />
<th>Hazard Explanation</th><br />
<th>How Hazard Is Addressed</th><br />
</tr><br />
<tr><br />
<td>Centrifuge</td><br />
<td>Centrifuge mechanical failure (eg. of rotor) can cause severe damage to the machine, and possibly personnel in the nearby vicinity.</td><br />
<td><br />
Regular inspections and maintenance of the machine is required (performed by external engineers). <br />
Machine not to be used if in non optimal condition.<br />
Centrifuge must be balanced to minimise chance of rotor failure.<br />
Centrifuge must be properly closed before use (required for the machine to work.)<br />
Attention must be paid to ensure that nothing is spilled in the centrifuge bowl, or a build up of dust or aerosol should also be avoided.</td><br />
</tr><br />
</table><br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Concluding Comments</p><br />
<p class="body_text"><br />
The safety of the team members, produced material and environment are key aspects to the smooth running of the project, and it must be ensured that adequate measures are in place to provide protection and ensuring that guidelines can be followed proceeding mandatory safety training. Current Good Laboratory Practice must also be adhered to at all times. By following guidelines and acting in an appropriate manner whilst in the laboratory, the general risk in terms of micro-biological, chemical and mechanical aspects will be reduced to a level which is as low as reasonably possible for all project members involved.<br />
</p><br />
<p class="body_text"><br />
Due to the relatively small scale of the experiments performed, the quantities used of material is not large, but still must be handled and respected properly at all times. Proper containment and labelling of materials in sealed containers is necessary, especially when of particular hazard such as live material or Ethidium Bromide. Flammability concerns are also present with alcohol and other flammable materials stored in the lab, although the volumes (generally less than 2L) are not considerably large enough, although should still be used and handled properly when used, with fire extinguishers available and also emergency exits present if required. Protective clothing also should be worn where appropriate to minimise the possibility of skin contact, with wash and first aid station available in key areas where allowable due to the area classification.<br />
</p><br />
<p class="body_text"><br />
The mechanical safety predominantly concerns the proper maintenance of the centrifuge system, which can cause a considerable risk if a failure occurs. Regular inspections should be made, with the centrifuge often restricted access when it was considered unfit for use. This room is also placed away from mammalian and bacterial processing centres. Prevention methods should also be in place such as emergency shut-off procedures.<br />
</p><br />
<p class="body_text"><br />
By following all guidelines set out in safety training and following supervisor instructions, the project may be performed in a safe and effective manner. Continual training and inspection of the facility will aid in ensuring safety levels are maintained, with review protocols in place to investigate any faults. Team members have been trained in general safety, with supervisors having first aid experience and training. These considerations combined will ensure the safety risk to the personnel; product and environment is as low as reasonably possible. For further reading, the Institute of Chemical Engineering (IChemE) has relevant information, as well as other safety guidance documents relating to the relevant country where the university is located.<br />
</p><br />
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</p> <br />
</div><br />
<br />
<p class="major_title">September</p><br />
<div class="full_row"> <br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">1st September</p><br />
<p class="body_text"><br />
The Bacteria Team is living some intense moments! The first transformation of the zeocin ligation took place yesterday and we're all very optimistic! We're about to know the results of this zeocin cloning on the 2nd, the latest the 3rd.<br />
<br />
<p class="minor_title">2nd September</p><br />
<p class="body_text"><br />
We finally received the oligonucleotides needed for the linker region! We can now start the cloning plan for this biobrick.<br />
<br />
<p class="minor_title">3rd September</p><br />
<p class="body_text"><br />
We started to think which type of poster would be the best idea for the Jamboree presentation.<br />
</p><br />
We met Darren at 4 pm to discuss about the cloning strategy for MMP9.<br />
<br />
<p class="minor_title">4th September</p><br />
<p class="body_text"><br />
We used SurveryMonkey in order to make a decision on who should present at the Jamboree. <br />
</p><br />
We reached a consensus for Alex, Tom and Casey to carry out this precious job for the team.<br />
</p><br />
<br />
<p class="minor_title">5th September</p><br />
<p class="body_text"><br />
We decided that the best option as the background colour for the T-shirts would be white.<br />
</p><br />
<br />
<p class="minor_title">6th September</p><br />
<p class="body_text"><br />
HQ replied about zeocin resistance biobrick. It will count as a new part. They also confirmed our attendance to the Regional Jamboree. Lyon, here we come!<br />
</p><br />
Alex produced a first draft of the poster while the other gave him feedback and FYi offered to take care of the actual design.<br />
</p><br />
<br />
<p class="minor_title">9th September</p><br />
<p class="body_text"><br />
Today Darren visited us at the lab and brought us MMP9 which was used to transform our competent cells. <br />
</p><br />
A new ligation for zeocin was prepared and competent cells were transformed with it.<br />
</p><br />
<br />
<p class="minor_title">10th September</p><br />
<p class="body_text"><br />
All the photos of the team members and supervisors were mounted on wiki.<br />
</p><br />
We had another discussion with Darren who advised us to test again the chloramphenicol and also to prepare more competent cells. He also reminded us to always use pSecTag2A as a positive control when minipreping.<br />
</p><br />
<br />
<p class="minor_title">11th September</p><br />
<br />
<p class="minor_title">12th September</p><br />
<p class="body_text"><br />
We agreed on the final details for the T-shirts.<br />
</p><br />
Robin released the updated modelling. Yey!<br />
</p><br />
Darren gave us some OneShot Top 10 competent cells from 2004 in order to continue with the transformations.<br />
</p><br />
<br />
<p class="minor_title">13th September</p><br />
<br />
<p class="minor_title">14th September</p><br />
<p class="body_text"><br />
We decided not to use K812014 biobrick anymore because of the inconsistent digestion. We're always obtaining 3 bands instead of 2 when digesting with EcoR1 and Pst1.<br />
<br />
<br />
<p class="minor_title">15th September</p><br />
<p class="body_text"><br />
After many minipreps of the stock of 4 transformations and subsequent digestions of these DNAs, we finally identified the ligated zeocin into pSB1C3 (origin, second ligation and transformation set).<br />
</p><br />
<br />
<p class="minor_title">16th September</p><br />
<p class="body_text"><br />
Weiling set ligations of MMP9 in pSB1C3 after pcr-ing it and digesting it with EcoR1, Pst1 and Dpn1.<br />
</p><br />
<br />
<p class="minor_title">17th September</p><br />
<br />
<p class="minor_title">18th September</p><br />
<p class="body_text"><br />
Work is being done on the presentation preparation. A first draft of the powerpoint was produced and people invited to give feedback on it.<br />
</p><br />
<br />
<p class="minor_title">19th September</p><br />
<p class="body_text"><br />
Bacteria Lab worked on maxipreping the recombinant zeocin plamid as well as on the MMP9 recombinant plasmid.<br />
</p><br />
<br />
<p class="minor_title">20th September</p><br />
<p class="body_text">><br />
Today is the deadline for sending our biobrick. Casey prepared for shipping and sent the zeocin biobrick.<br />
</p><br />
<br />
<p class="minor_title">21st September</p><br />
<p class="minor_title">22nd September</p><br />
<p class="minor_title">23rd September</p><br />
<br />
<p class="minor_title">24thSeptember</p><br />
<p class="body_text"><br />
Transformation of HeLa cells with the recombinant zeocin plasmid was performed today under the assistance of Alex Kinna. Thanks Alex!<br />
<br />
<p class="minor_title">25th September</p><br />
<p class="body_text"><br />
The company to print our T-shirts was chosen. We're going with Image Scotland.<br />
</p><br />
<br />
<p class="minor_title">26th September</p><br />
<p class="body_text"><br />
Two representatives of Source Biosciences gave us a visit in the tissue culture lab at 2pm. They discussed transfection methods with the us and advertised their reagents.<br />
</p><br />
<br />
<p class="minor_title">27th September</p><br />
<p class="body_text"><br />
Darren confirmed with us the funding for the trip to come! Friday, the 11th of October, in the afternoon, we're flying to Lyon!<br />
</p><br />
<br />
<p class="minor_title">28th September</p><br />
<p class="minor_title">29th September</p><br />
<p class="minor_title">30th September</p><br />
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
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<br />
<p class="major_title">August</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">1st August</p><br />
<p class="body_text"><br />
Bacterial lab had good results today in the preparation of a new stock of competent cells.<br />
In the evening we celebrated the success of the speed debate.<br />
</p><br />
<p class="minor_title">2nd August</p><br />
<p class="body_text"><br />
Stjohn designed the linkers for the Mammalian Oxidative Stress Inducible Promoter.<br />
The team met to discuss fundraising ideas somehow making use of [kickstarter.com]. A starting idea: brain-with-plaques-for-sale.<br />
</p><br />
<p class="body_text"><br />
We came up with the idea of a Memory Lane, where people could upload a photo of one of their memories and write a small description about it.<br />
</p><br />
<p class="body_text"><br />
Alex suggested a collaboration with Westminster iGEM team regarding the speed debate idea.<br />
</p><br />
<br />
<p class="minor_title">5th August</p><br />
<p class="body_text"><br />
Snapshots of the team members were taken!<br />
</p><br />
<p class="body_text"><br />
The team worked on the abstract which must be uploaded shortly on wiki as the deadline is on the 9th.<br />
</p><br />
<p class="body_text"><br />
Alex contacted the Imperial iGEM team regarding an eventual collaboration. <br />
</p><br />
<br />
<p class="minor_title">6th August</p><br />
<p class="body_text"><br />
Rob invited the team at 12 noon in the Anatomy Hub to discuss about the wiki design in order to make sure that all the ideas about this matter are taken into account.<br />
</p><br />
<br />
<p class="minor_title">7th August</p><br />
<p class="body_text"><br />
Barbeque evening, venue Wilkins Roof Garden!<br />
</p><br />
<p class="body_text"><br />
Prof. Eli Keshavarz-Moore was our guest and at 3 pm we also had the chance to present our project. (venue: Malet Place Engineering LT 1.03)<br />
</p><br />
<br />
<p class="minor_title">8th August</p><br />
<p class="body_text"><br />
The team discussed about the work on zeocin,pA-f1-Zec biobrick, which will indeed be an improvement of BBa_J176124 because:<br />
</p><br />
<p class="body_text"><br />
i) it gives most of the functionality of BBa_J176124 but is compatible with standard assembly<br />
</p><br />
<p class="body_text"><br />
ii) it allows people to simply insert a PROMOTER-ORF fragment upstream of a pA to give an expression cassette for the ORF of interest, and a ZEC to select stable transfectants. <br />
</p><br />
<br />
<p class="minor_title">9th August</p><br />
<p class="body_text"><br />
Project description is up on Wiki!<br />
</p><br />
<p class="body_text"><br />
Darren gave us a visit at the lab to check if everything is O.K. with our work and enthusiasm.<br />
The requested batch of biobricks arrived as glycerol stocks.<br />
</p><br />
<p class="body_text"><br />
The team discussed about Kickstarter crowdfunding and planned to launch the Memory Lane/Map thing WITHOUT getting people to pay. We will get people to upload their best memories in different forms and potentially do some beautiful art with it like the Memory Palace FYi suggested. <br />
</p><br />
<br />
<p class="minor_title">12th August</p><br />
<p class="body_text"><br />
We had a strategy chat at the lab with Darren. <br />
</p><br />
FYi drawn the wiki background for the diary section. She also made the illustrations for the T-shirts.<br />
</p><br />
The team also debated on the wiki design and a consensus was reached regarding the site map, default banner, logo.<br />
</p><br />
In 'Memory Lane', we are going to ask people to 'leave one strong memory' on one page whether in text or pictures. These will be done anonymously but they will leave their emails with us so they will be notified when the 'compilation' is up. <br />
</p><br />
The website came to life today!<br />
</p><br />
<br />
<br />
<p class="minor_title">13th August</p><br />
<p class="body_text"><br />
Alex and Oran came up with the idea of a Creative writing competition. <br />
</p><br />
FYi, Robin, Alex and Stjohn and Oran focused on wiki building for the weeks to come while the rest of the team worked in the Bacterial Labs.<br />
</p><br />
<br />
<br />
<p class="minor_title">14th August</p><br />
<p class="body_text"><br />
The advertisement for the competition was written and the competition was launched. More details about the outcome can be found on the ‘Competition’ subsection.<br />
</p><br />
Met the Westminister team to discuss about the potential modelling collaboration. It was a nice gathering.<br />
</p><br />
<br />
<p class="minor_title">15th August</p><br />
<p class="body_text"><br />
Continued intensively planning and brainstorming for the design of our wiki, especially on the front page design. <br />
</p><br />
<br />
<p class="minor_title">16th August</p><br />
<br />
<br />
<br />
<p class="minor_title">17th August</p><br />
<br />
<p class="minor_title">18th August</p><br />
<br />
<p class="minor_title">19th August</p><br />
<p class="body_text"><br />
Alex advertised the writing competition on prizemagic.co.uk.<br />
</p><br />
Stjohn released a new set of rules for managing wiki content in order to make work easier before the wiki freeze.<br />
</p><br />
<br />
<p class="minor_title">20th August</p><br />
<p class="body_text"><br />
The actual work on the main poster on the frontal page started. FYi produced the first sketch and the team gave feedback.<br />
</p><br />
The members’ Profiles are ready to be uploaded on wiki!<br />
</p><br />
<br />
<p class="minor_title">21th August</p><br />
<p class="body_text"><br />
The lab was closed in the morning, however in the afternoon the Bacteria Team prepared selective plates and selective media in order to culture the last arrived biobricks from the HQ. Darren assisted us.<br />
</p><br />
The linkers designed by Stjohn: IGM Ox L1, L2, L3, L4 as well primers for cmv promoter were ordered.<br />
</p><br />
<br />
<p class="minor_title">22th August</p><br />
<p class="body_text"><br />
The first Creative Competition Entry! Yey! Thank you!<br />
</p><br />
The atmosphere in the Bacterial Lab became slightly more cheerful. The amplification of zeocin from the 2 types of ordered primers was successful as well as the digestion of K812014 and pSB1C3 and pSB1A3. We decided to use the zec bb F,R primers for the further amplification of zeocin. <br />
</p><br />
The Zeo kill curve was derived, a concentration of 150 ug/ml was used.<br />
</p><br />
<br />
<p class="minor_title">23th August</p><br />
<p class="body_text"><br />
The main poster for the front page was finalised. Well done FYi!<br />
</p><br />
New submissions for the Creative writing! <br />
Lonza confirmed a sponsorship of £1, 207. Happy Happy Joy Joy! Well done Weiling!<br />
</p><br />
<br />
<br />
<p class="minor_title">26th August</p><br />
<p class="body_text"><br />
The lab was closed today hence we all focused on the wiki content.<br />
</p><br />
The front page poster background - wasteland was completed.<br />
</p><br />
<br />
<br />
<p class="minor_title">27th August</p><br />
<br />
<p class="minor_title">28th August</p><br />
<p class="body_text"><br />
The Biosafety forms were filled in as necessary. These must be signed by Darren before the 30th.<br />
</p><br />
We met Darren at 4 pm in the lab to discuss about the biobrick processing.<br />
</p><br />
<br />
<p class="minor_title">29th August</p><br />
<p class="body_text"><br />
We considered the strategy to deal with the linker region. First step is to achieve the annealing of the oligonucleotides making up this linker. We're still waiting for these sequences.<br />
</p><br />
Agreed on the final design of the T-shirts. We're aiming to order them as soon as possible.<br />
</p><br />
<br />
<p class="minor_title">30th August</p><br />
<p class="body_text"><br />
We uploaded the first samples of memories on the Memory Lane page.<br />
</p><br />
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
</p> <br />
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<br />
<p class="major_title">July</p><br />
<div class="full_row"><br />
<div class="gap"><br />
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<p class="minor_title">1st July</p><br />
<p class="body_text"><br />
Tom – Extracted information from private wiki and shutdown performed by Philipp Boeing. Prepared for narration of stop-motion. Also discussed project proposals with StJohn and Ruxi.<br />
</p><br />
<p class="body_text"><br />
Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
StJohn & Ruxi – Formed project proposals for the laboratory experiments.<br />
</p><br />
<p class="minor_title">2nd July</p><br />
<p class="body_text"><br />
The team had a meeting with Philipp Boeing, primarily about Human Practice and which direction should be taken in terms of gaining awareness and also funding for the project. Ruxi and StJohn then continued working on experimental protocol preparation while the rest of the team visited the Science Museum to look at their Alzheimer's exhibit for inspiration on both project development and artistic direction that our human practices should take.<br />
</p><br />
<p class="minor_title">3rd July</p><br />
<p class="body_text"><br />
The Majority of the group continued to work on the proposals as some of the components were found to be difficult to obtain or not feasible. Tom began the YSB poster design, Robin continued on the modelling proposal.<br />
</p><br />
<br />
<p class="minor_title">4th July</p><br />
<p class="body_text"><br />
The entire group attended safety training demonstrated by Brian O’Sullivan. A meeting was also held with experts in the field concerning microglia, Jenny Reagen amongst others.<br />
</p><br />
<p class="body_text"><br />
Tom continued on poster design with Catrin looking at previous posters for inspiration. Andy met with Bethan Wolfenden to talk about debating, the rest of the group. <br />
</p><br />
<br />
<p class="minor_title">5th July</p><br />
</p><br />
<p class="body_text"><br />
Tom & Catrin – Worked on the poster and finished it, as well as the presentation<br />
</p><br />
<p class="body_text"><br />
Alex, Andy and Weiling – Focussed on human practises, pafrticularly essay writing and documentary planning.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi and StJohn – Continued work on proposals and sent completed documents to Darren.<br />
</p><br />
<p class="minor_title">8th July</p><br />
<p class="body_text"><br />
Meeting with Darren leads to more work on proposals, particularly procurement and logistics of items required for laboratory work. The group also spent a lot of time discussing titles for the project, with ‘Plaque Buster’ and ‘Memory Guardian’ being the more popular names in an alternate voting system.<br />
</p><br />
<br />
<p class="minor_title">9th July</p><br />
<br />
<p class="body_text"><br />
Following the meeting with Darren yesterday, the group met and rectified the experiments system to make it clearer and more achievable to obtain bronze, silver and gold medals, reducing the number of new parts required from 12 to 3 essential ones, for example.<br />
</p><br />
<br />
<p class="minor_title">10th July</p><br />
<p class="body_text"><br />
The group sent the new proposal to Dr. Darren Nesbeth, and are to wait for a response before continuing with specific inventory/experiment write ups. Instead, the group allocated roles for this should the proposal be accepted, and then went to the gallery of surgery to investigate cranial injections, and the implications and feasibility of this form of surgery.<br />
</p><br />
<p class="minor_title">11th July</p><br />
<p class="body_text"><br />
The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.<br />
</p><br />
<p class="minor_title">12th July</p><br />
<p class="body_text"><br />
The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.<br />
</p><br />
<p class="minor_title">13th July</p><br />
<p class="body_text"><br />
YSB Day 2: Collaboration continued between teams for feedback and suggestion purposes. Tom and Alex initiated the creation of a national SynbioSoc so it easier for iGEM teams to communicate ideas and generally collaborate for both this year and the future. Tom also announced the iGEM football tournament, which was met with enthusiasm by other teams.<br />
</p><br />
<p class="minor_title">15th July</p><br />
<p class="body_text"><br />
First day of lab, under instruction by Dr. Darren Nesbeth and Yanika Borg, the team were shown various items in the labs and how to use them, with emphasis on good laboratory practice at all times. The team also met up with Oran and FongYi to discuss how the artistic side of the project will be undertaken. Oran and FongYi joined the team.<br />
</p><br />
<p class="minor_title">16th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team created ‘minimal agar’ plates to grow W3110 E. coli cells on. The cells were left to incubate overnight for a 16 hour period.<br />
</p><br />
<p class="body_text"><br />
KC, Alex & StJohn – Worked on primer design for the PCR reactions planned. Difficulties with finding flanking DNA sequences were encountered.<br />
</p><br />
<p class="minor_title">17th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team looked at the cell cultures in the morning and discovered that the cells had not grown, so came back in the afternoon and noticed growth on 2 of the 5 plates. Further incubation of 17 hours was agreed upon.<br />
</p><br />
<p class="body_text"><br />
KC & Alex – Started mammalian cell lab induction.<br />
</p><br />
<p class="body_text"><br />
The team then met with artists to further develop the branding of the whole project.<br />
</p><br />
<p class="minor_title">18th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling – Lab experiment with Yanika Borg – Selection of colonies then resuspension into growth media, followed by incubation until 10:00 tomorrow.<br />
</p><br />
<p class="body_text"><br />
StJohn & KC – Primer design for the PCR protocols<br />
</p><br />
<p class="body_text"><br />
Alex – Continued work on ethics and feasibility report & bioinformatics<br />
</p><br />
<p class="minor_title">19th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling – Continued Lab experiments with Yanika Borg – Re-suspension & centrifugation of colonies.<br />
</p><br />
<p class="body_text"><br />
StJohn & KC – Primer design for the PCR protocols<br />
</p><br />
<p class="body_text"><br />
Alex – Continued work on ethics and feasibility report & bioinformatics<br />
</p><br />
<br />
<p class="minor_title">22nd July</p><br />
<p class="body_text"><br />
Meeting with Darren reveals that primer design needs to be reconfigured, and that the strategy for Gold is currently not acceptable, so this will be worked on. We won the inter-UCL award for best wiki of July. StJohn worked on primers and KC worked on protocols.<br />
</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Emily performed bacterial labs, using transformation skills.<br />
</p><br />
<p class="minor_title">23rd July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Emily performed bacterial labs once more, repeating yesterday’s experiments due to a failed transformation.<br />
</p><br />
<p class="body_text"><br />
StJohn did more rectification work on primer design. KC searched for any possible molecules which could be used as an alternative molecules that naturally exist in the brain as replacements for auxin detection system.<br />
</p><br />
<p class="body_text"><br />
Weiling & Alex went to KCL (Institute of Psychiatry) to interview professor John Powell, an expert in the field of Alzheimer’s diseases, and other brain related diseases.<br />
</p><br />
<br />
<p class="minor_title">24th July</p><br />
<p class="body_text"><br />
Until the 26th of July the bacterial lab work did not get any further. Several transformations were performed but neither was successful. After these trials, the decision of making new competent cells was taken.<br />
</p><br />
<p class="body_text"><br />
The entire team was sent the information regarding mammalian lab aseptic techniques.<br />
StJohn analised an <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277080/" target="_blank"> article</a> on Microglia function in Alzheimer’s disease.<br />
</p><br />
<p class="body_text"><br />
Alex gathered more <a href="http://www.scielo.br/pdf/bjmbr/v38n7/v38n7a03.pdf" target="_blank"> information</a> regarding main transcription factors/promotors we could use for detecting the oxidative stress caused near plaques.<br />
</p><br />
<p class="body_text"><br />
The team decided to meet over for a barbeque on the 7th of August.<br />
</p><br />
<br />
<p class="minor_title">25th July</p><br />
<p class="body_text"><br />
Oran came to the lab and was introduced to the lab routine and to the activities on going.<br />
The team met again in the Student Anatomy hub to continue research on useful articles.<br />
</p><br />
<br />
<p class="minor_title">26th July</p><br />
<p class="body_text"><br />
A summary of the week lab work:<br />
</p><br />
<p class="body_text"><br />
- We have made stocks of all constituents needed to grow cells (E. coli W3110) and have a stock in the -80C cold storage.<br />
</p><br />
<p class="body_text"><br />
- We attempted transformation (p1313) on three separate occasions but it failed each time (although controls worked as expected).<br />
</p><br />
<p class="body_text"><br />
- We used Yanika's personal cell stock of W3110 and performed the transformation successfully.<br />
</p><br />
<p class="body_text"><br />
- Therefore today we remade the constituents needed at the start, we will perform plate streaking etc. after the weekend, and hopefully have more success with transformation as well.<br />
</p><br />
<p class="body_text"><br />
The following biobricks were ordered BBa_1712004, BBa_K812014, BBa_J63008. They’re supposed to arrive through UPS service by the 31st of July.<br />
</p><br />
<br />
<p class="minor_title">29th July</p><br />
<p class="body_text"><br />
An important day for our team! The project name “Spotless mind” was chosen!<br />
</p><br />
<p class="body_text"><br />
The MathWorks license for the 2013 iGEM student competition has been created.<br />
</p><br />
<p class="body_text"><br />
The Biobricks from the iGEM HQ arrived today, which includes a mammalian plasmid backbone and 2 auxin signalling parts.<br />
</p><br />
<br />
<p class="minor_title">30th July</p><br />
<p class="body_text"><br />
The entire team is involved in organising the speed debate taking place tomorrow, 31st.<br />
FYi and Oran produced a nice <a href="https://scontent-b.xx.fbcdn.net/hphotos-prn1/q71/s720x720/1098138_10151827937531617_373872629_n.jpg" target="_blank"> debate poster</a> and a new logo!<br />
</p><br />
<br />
<p class="minor_title">31st July</p><br />
<p class="body_text"><br />
We organised a neuroethics themed Speed Debate at Print Room Cafe, UCL. We started preparation such as buying refreshments, setting up the venue, printing survey sheets and poster at 4pm. At 7pm, guests started to arrive. Over 90 participants attended the speed debate. Dr. Howard Boland, Alex Bates, Philipp Boeing and Shirley Nurock from the Alzheimer's Society spoke at the speed debate.<br />
</p><br />
<p class="body_text"><br />
The event was a success, many guests stayed to discuss further and alot of interests were received regarding the progress of our project. We cleaned the venue and wrapped up at 10.30pm<br />
</p><br />
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
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<br />
<p class="major_title">June</p><br />
<div class="full_row"><br />
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<p class="minor_title">5th June</p><br />
<p class="body_text"><br />
Group discussion concerning the project idea to be carried forward - favouring the 'Anti cancer project'. Roles were then assigned to team members present for intial research roles for the week:<br />
</p><br />
<p class="body_text"><br />
Cancer research roles:<br />
</p><br />
<p class="body_text"><br />
1. Ruxi Comisel - Proteins upregulated in cancer of the intestines. Specifically in the outer epithelial cell (enterocytes) – in microvilli. Also, what actually is... gut cancer? A general overview would be useful…<br />
</p><br />
<p class="body_text"><br />
2. Khaicheng Kiew - Our chassis (bearing in mind that we will also build it in E. coli as a backup). We need to think what would make a good chassis in our case (ie. naturally found in the gut in an obvious one), and how well does the chassis fit.<br />
</p><br />
<p class="body_text"><br />
3. Alex Bates - What will the killing mechanism be? A broad overview of cancer treatments is required, specifically detailing how a bacterium can administer the treatment.<br />
</p><br />
<p class="body_text"><br />
Considerations:<br />
</p><br />
<p class="body_text"><br />
a. The bacteria may secrete a toxin etc – how will we ensure that it doesn’t simply diffuse through the gut? <br />
b. If it is a toxin, what sort of biosynthetic pathway is required?<br />
c. Does the bacteria trigger apoptosis in the cancer cells (ie. an intracellular killing mechanism)? How can this be done from an extracellular bacterium? Perhaps beta-arrestin?<br />
d. Are there any treatments which we can take advantage of specifically because we are using bacteria? <br />
e. For example, a protein which creates holes in the cancer cells? Does using a bacterium open up the possibility of using a different cure that currently isn’t in use because we cannot target it to cancer cells – could the use of bacteria allow this?<br />
</p><br />
<p class="body_text"><br />
4. Weiling Yuan - Targeting – do we use antibodies? What previous projects have used bacteria expressing antibodies? Are there any other ways of doing this? Perhaps the latching and initiation mechanisms can be incorporated into one protein?<br />
</p><br />
<p class="body_text"><br />
5. StJohn Townsend - Initiation – mechanoreceptor activated upon latching? What other ways are there of doing this?<br />
</p><br />
<p class="body_text"><br />
6. Tom Johnson - Past iGEM projects which we could incorporate into our own: Cancer projects, Gut projects, Protein engineering, Antibodies expressed in bacteria etc.<br />
</p><br />
<br />
<p class="minor_title">7th June</p><br />
<p class="body_text"><br />
The team discusses findings from the initial research - further agreement that the 'Anti Cancer' project seemed to be the best idea, preparation of 'project sheets' to be sent to Dr. Darren Nesbeth for review and subsequent meetings.<br />
</p><br />
<p class="minor_title">11th June</p><br />
<p class="body_text"><br />
looked a bit at the possible chassis species: salmonella, clostridium, helicobacter, E. coli. according to the tissue type/cancer type we shall decide which works with which. We start with E. coli in the lab.<br />
</p><br />
<p class="body_text"><br />
We considered a pro-drug approach - bacterially directed enzyme pro-drug therapy which suggests that we may establish a transformed bacterial population with an enzyme capable to activate an ingested prodrug. This pro-drug would be connected to an antibody (possibly part of the tail) and would also have linking consensus sequence targeted by the enzyme produced locally by our bacteria.<br />
</p><br />
<p class="body_text"><br />
From this above point Alex distinguished 2 scenarios built on the circuit sketch that he and Laia posted a while ago. These would be:<br />
</p><br />
<p class="body_text"><br />
1) Kill unit produces tailed protein pro-drug (possibly tailed perforin) and signaling molecule, A. When A reaches a threshold amount, perforin and a protease to remove the confounding tail is produced, bacteria lyses and activated pro-drug acts on surrounding cells.<br />
</p><br />
<p class="body_text"><br />
2) No protease is produced, because the tail can be cleaved off by matrix metalloproteases.<br />
</p><br />
<p class="body_text"><br />
Goals for the end of this week: <br />
</p><br />
<p class="body_text"><br />
- Alex, Andy and Weiling continue investigating possible candidates to fill in the parts for the scenarios<br />
</p><br />
<p class="body_text"><br />
-Tom, KC and Ruxi make sure we have everything set up to start the work in the lab: protocol, parts etc.<br />
</p><br />
<p class="minor_title">12th June</p><br />
<p class="body_text"><br />
Ruxi and Tom went through a general cloning protocol but then realised that the best way to prepare for the lab is to get familiarised with the iGEM distribution kits. We discovered that we are given almost everything we need in order to get it right.<br />
</p><br />
<p class="body_text"><br />
Alex filled in the form with our proposal requested by Darren - we have the sequences and details of potential new biobricks. <br />
</p><br />
<p class="body_text"><br />
We formulated a new proposal regarding the Alzheimer’s disease amyloid plaque degradation.<br />
</p><br />
<p class="body_text"><br />
Andy searched potential cancer killer molecules:<br />
</p><br />
<p class="body_text"><br />
- CD95 - Fas agonist (http://www.nature.com/cdd/journal/v14/n4/full/4402051a.html)<br />
- Tumor Necrosis Factor, Histamine - induces inflammation<br />
- HAMLET (human a-lactalbumin) - induces apoptosis <br />
- endostatin, thrombospondin - reduce cancer growth<br />
</p><br />
<p class="body_text"><br />
Weiling looked at potential promotors: <br />
</p><br />
<p class="body_text"><br />
- RacA (based on increased DNA damaged due to radiation) to start the killing cascade and CD95 as a potential killer molecule<br />
- Lux pR promotor<br />
- Lld promoter<br />
- Vgb promotor <br />
- HIP-1<br />
</p><br />
<p class="body_text"><br />
(about gastric Oxygen levels: http://www.biomedcentral.com/1471-2180/11/96) <br />
</p><br />
<p class="body_text"><br />
For promoter 1 (switches on the pro-drug and signaling molecule transcription), a very <br />
good candidate is HIP 1 promoter - hypoxia-inducible promoter which drives reporter gene expression under both acute and chronic hypoxia. It was developed in attenuated Salmonella species. Take a look here: http://www.landesbioscience.com/journals/cbt/article/2951/mengesha5-9.pdf<br />
</p><br />
<p class="body_text"><br />
We need to register this part!<br />
</p><br />
<br />
<p class="minor_title">13th June</p><br />
<p class="body_text"><br />
Alex sent the 3 main project proposals to Dr. Darren Nesbeth for review.<br />
</p><br />
<p class="body_text"><br />
Tom and Andy edited the wiki page adding various sections and elaborating on previously created pages.<br />
</p><br />
<p class="body_text"><br />
Weiling researched on killing mechanisms being able to target hypoxic regions of solid tumors and promoters in hypoxia environments.<br />
</p><br />
<p class="body_text"><br />
Catrin - General project research<br />
</p><br />
<p class="body_text"><br />
Ruxi - Further researched the potential promoters esp HIP 1 and the Fas regulated programmed apoptosis.<br />
</p><br />
<p class="body_text"><br />
We attended a Synthetic Biology talk by Neil Dixon, University of Manchester (Tom and Andy).<br />
</p><br />
<p class="body_text"><br />
Had a general meeting for discussion of what has been accomplished so far, and the subsequent actions, which are to be undertaken by team members. Further documents were also submitted to Dr. Darren Nesbeth concerning 'team roles'. The team then began to do individual research or other activity:<br />
</p><br />
<p class="body_text"><br />
Tom and Robin - Edited the iGEM wiki, added team information and removed the unnecessary tutorial information, replacing it with more useful information and streamlining the whole interface.<br />
</p><br />
<p class="body_text"><br />
Weiling and Alex - Further development of circuit ideas, taking inspiration from previous iGEM ideas as well as further research into the CD95L molecule.<br />
</p><br />
<p class="body_text"><br />
Ruxi and Catrin - Research into latching molecules for a bacteria to tumour interface to increase target specificity. Idea encounted from Hong Kong 2012 where Colon Cancer was targeted.<br />
</p><br />
<br />
<p class="minor_title">14th June</p><br />
<p class="body_text"><br />
Tom - Website design for: Main Page, UCL information, Team based pages and Notebook pages<br />
</p><br />
<p class="body_text"><br />
Robin - Coding in HTML for website<br />
</p><br />
<p class="body_text"><br />
Ruxi, Catrin, Weiling - Further investigation of Hong Kong 2010 to see what parts may be improved or of use to the project, these were: a blue light activated promoter, how can the quorum sensing and CagA be exploited, a negative regulatory system for drug secretion.<br />
</p><br />
<p class="body_text"><br />
Alex - searched for potential bacterial receptor to be modified in order to be a good target for something else in the environment/cancer cell surface.<br />
</p><br />
<p class="minor_title">17th June</p><br />
<p class="body_text"><br />
The group had a meeting to discuss what had been achieved so far and what needed to be done today. <br />
</p><br />
<p class="body_text"><br />
Tom - Continued on website design and wrote several pieces concerning UCL to be used on the website when it goes live.<br />
</p><br />
<p class="body_text"><br />
Robin - Continued on website coding.<br />
</p><br />
<p class="body_text"><br />
Weiling & Catrin - Researched for project sponsors and potential contacts.<br />
</p><br />
<p class="body_text"><br />
Alex, Ruxi, StJohn & Andy - Continued research into the project ideas.<br />
</p><br />
<p class="minor_title">18th June</p><br />
<p class="body_text"><br />
The group met with advisors Darren Nesbeth and Philipp Boeing to discuss the three project suggestions. The 'Neural Network' proposal was effectively ruled out due to the high risk and low probablility of project success in terms of medals.<br />
</p><br />
<p class="body_text"><br />
The anti-cancer project was previously the favoured idea, but after extensive review ,the Alzheimers project gained favour due to being relatively new (and hence exciting) to iGEM compared to a cancer project, which has been done several times already at iGEM. No final decision has been made however, work has continued on researching both projects. The wiki is also still being worked on.<br />
</p><br />
<p class="body_text"><br />
The team also had a social gathering: pizza for lunch.<br />
</p><br />
<p class="minor_title">19th June</p><br />
<p class="body_text"><br />
The group continued work on all three projects in order to send improved proposals to Darren Nesbeth by the end of the day. Many professors and experts were also emailed to seek guidance, in particular for the Alzheimer's project which seems to be particularly difficult.<br />
</p><br />
<p class="minor_title">20th June</p><br />
<p class="body_text"><br />
Tom - Prepared a presentation to be given next week about iGEM to prospective UCL students to raise interest in the engineering faculty and also the iGEM competition. After this was complete, joined the rest of the group in research. Also performed wiki coding for the team page and notebook page.<br />
</p><br />
<p class="body_text"><br />
The group continued what was started yesterday: Rectifying the proposals, with both sent off at the end of the day once they were complete. A group meeting was held at the end of the day to gauge interest and vote for the most popular idea, followed by a social gathering.<br />
</p><br />
<p class="minor_title">21st June</p><br />
<p class="body_text"><br />
Tom - Continued wiki design, coding and content uploads.<br />
Alex - Continued to redraft the proposal for Alzheimer's<br />
StJohn - Continued to redraft the proposal for Cancer<br />
</p><br />
<p class="body_text"><br />
KC - Researched into other iGEM teams to colloborate with and initiated correspondence via email<br />
</p><br />
<p class="body_text"><br />
The team then discusses which project was favoured. It was fairly even but Alzheimer's was slightly more popular.<br />
</p><br />
<p class="minor_title">24th June</p><br />
<p class="body_text"><br />
Tom continued wiki design whilst the rest of the group performed research.<br />
</p><br />
<p class="body_text"><br />
Once this was complete, the group had a meeting with Yanika Borg and Philipp Boeing concerning the two project ideas. Philipp favoured the Alzheimer's project whilst Yanika was somewhat undecided. <br />
</p><br />
<p class="body_text"><br />
A vote was taken with Alzheimer's being the prefered project by the group as a whole once more, although consensus was not fully reached. The group agreed to decide on the project on Wednesday proceeding a meeting with Prof. Lazaros Lukas.<br />
</p><br />
<br />
<p class="minor_title">25th June</p><br />
<p class="body_text"><br />
The group continued with general research, and also went to the Wellcome trust to seek any extra information, although this was unfruitful.<br />
</p><br />
<br />
<p class="minor_title">27th June</p><br />
<p class="body_text"><br />
The group voted 29 -11 in favour of Alzheimer's after a meeting with Prof. Lazaro Lukas, who was helpful and seemed excited about the project. The group also met advisor Yanika Borg and she agreed with the choice. The group also scheduled lab safety training for next thursday.<br />
</p><br />
<br />
<p class="minor_title">28th June</p><br />
<p class="body_text"><br />
Tom presented to prospective students about the iGEM project for the day.<br />
</p><br />
<p class="body_text"><br />
Weiling, Alex, Andy & Catrin began to produce a 'stop motion' explanation of the Alzheimer's project.<br />
</p><br />
<p class="body_text"><br />
KC, Robin and StJohn discussed lab protocols and also modelling ideas.<br />
</p><br />
<p class="minor_title">29th June</p><br />
<p class="body_text"><br />
Tom, Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi & StJohn – Continued work on the proposals for the meeting with Dr. Nesbeth on Thursday.<br />
</p><br />
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Exam period - iGEM work to commence full time after the slog through exams.<br />
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Exam period - iGEM work to commence full time after the slog through exams.<br />
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