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<h2><span>INTERLAB STUDY:<span class='spacer'></span></h2>
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<p><span style="color: #ffffff;">&nbsp;</span></p>
 
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<h1 style=>Interlab</h1>
 
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<h1 style=>Introduction</h1>
 
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<h4><center>Our team decided to take part in the iGEM interlab study this year. This study<br>
 
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is organised by iGEM and is aimed at tackling a prominent issue regarding experimental  <br>
 
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fluorescent reporter data. At present, experiments using fluorescent reporters are almost <br>
 
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incomparable; separate groups often interpret fluorescence measurements in largely different <br>
 
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ways, making it hard to properly compare results. To combat this, the interlab study is <br>
 
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performed each year, iteratively refining an optimal protocol for fluorescence measurement. <br>
 
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Theoretically, this would be followed by anyone and yield comparably robust units of <br>
 
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fluorescence, facilitating more meaningful comparisons of data between researchers. <br>
 
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The study required us to measure a number of different fluorescent test devices sent to us by iGEM HQ, we followed their protocol and then shared the results we obtain. Teams around the world do the same; collectively, we provide a large set of data illustrating how similar the results were, potentially highlighting any aspects in which the protocol could be improved and, hopefully, showing that this is possible. This is quite an important issue, we felt we should participate and help in any way we could!
 
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</span></p>
 
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<h1 style=>Methods</h1>
 
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<h4><center>We were given 8 specimens to measure, termed “test devices”. These included a positive (BBa_I20270) and negative(BBa_R0040)  control, and 6 different test devices (BBa_J36400, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, BBa_J364005) .
 
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First we transformed these into DH5α-E. coli cells, then selected two resulting colonies to be prepared for measurement. The two colonies were grown overnight.
 
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But before the experiment could be undertaken, we needed to create calibrate our equipment by performing standard reference measurements, enabling the conversion of our data to absolute values. Firstly, we measured a solution of half LUDOX-S40, half water; fluorescein was then serially diluted by half  10 times and measured to give a standard curve.
 
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After 18 hours of growth, their optical density was measured and a new batch of LB/Chloramphenicol was inoculated to produce an OD of 0.02. These were then grown at 37°C, OD600nm and fluorescence were measured at 0, 2, 4, and 6 hours.
 
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For a full description of the methods, please visit the interlab official study page: </span></p>
 
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<h1 style=>Results</h1>
 
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<h4><center>Coming soon..</span></p>
 
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</center>
 
  
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<center><h1>Introduction</h1></center>
 +
<p>Our team decided to take part in the iGEM interlab study this year. This study
 +
is organised by iGEM and is aimed at tackling a prominent issue regarding experimental fluorescent reporter data. At present, experiments using fluorescent reporters are almost incomparable; separate groups often interpret fluorescence measurements in largely different ways, making it hard to properly compare results. To combat this, the interlab study is
 +
performed each year, iteratively refining an optimal protocol for fluorescence measurement.
 +
Theoretically, this would be followed by anyone and yield comparably robust units of fluorescence, facilitating more meaningful comparisons of data between researchers.
 +
<br><br>The study required us to measure a number of different fluorescent test devices sent to us by iGEM HQ, we followed their protocol and then shared the results we obtain. Teams around the world do the same; collectively, we provide a large set of data illustrating how similar the results were, potentially highlighting any aspects in which the protocol could be improved and, hopefully, showing that this is possible. This is quite an important issue, we felt we should participate and help in any way we could!</p>
 +
 +
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<center><h1>Methods</h1></center>
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<p>
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<img src="https://static.igem.org/mediawiki/2017/8/80/Unott_interlab_plate.jpeg"; style="float:right;height:auto;width:350px;padding:20px;">
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We were given 8 specimens to measure, termed “test devices”. These included a positive (BBa_I20270) and negative(BBa_R0040)  control, and 6 different test devices (BBa_J36400, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, BBa_J364005).
 +
<br><br>First we transformed these into DH5α-E. coli cells, then selected two resulting colonies to be prepared for measurement. The two colonies were grown overnight.
 +
<br><br>But before the experiment could be undertaken, we needed to calibrate our equipment by performing standard reference measurements, enabling the conversion of our data to absolute values. Firstly, we measured a solution of half LUDOX-S40, half water; fluorescein was then serially diluted by half  10 times and measured to give a standard curve.
 +
<br><br>After 18 hours of growth, the test device sample's optical density was measured, a new batch of LB/Chloramphenicol was inoculated to produce an OD of 0.02. These were then grown at 37°C, OD600nm and fluorescence were measured at 0, 2, 4, and 6 hours.
 +
<br><br>For a full description of the methods, please visit the <a href="https://2017.igem.org/Competition/InterLab_Study">official interlab study page</a>
 +
</p>
 +
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<center><h1>Results</h1></center>
  
  
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   <h3 class="box_header">LUDOX-S40 and Fluoroscein Reference Measurements</h3>
 
   <h3 class="box_header">LUDOX-S40 and Fluoroscein Reference Measurements</h3>
 
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   <div class="box_content">
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<p><span style="color: #000000;"><br /></span></p>
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<p><span style="color: #000000;">
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<table >
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  <tr>
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    <th> </th>
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    <th>LUDOX-HS40</th>
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    <th>H20</th>
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  </tr>
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  <tr>
 +
    <td>Replicate 1</td>
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    <td>S0.041</td>
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    <td>0.029</td>
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  </tr>
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  <tr>
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    <td>Replicate 2</td>
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    <td>0.045</td>
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    <td>0.03</td>
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  <tr>
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    <td>Replicate 3</td>
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    <td>0.043</td>
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    <td>0.032</td>
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  </tr>
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  <tr>
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    <td>Replicate 4</td>
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    <td>0.041</td>
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    <td>0.032</td>
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  <tr>
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    <td>Arith. Mean</td>
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    <td>0.0425</td>
 +
    <td>0.03075</td>
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  </tr>
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  <tr>
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    <td>Corrected Abs600</td>
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    <td>0.01175</td>
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 +
  </tr>
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  <tr>
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    <td>Reference OD600</td>
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    <td>0.0425</td>
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  </tr>
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  <tr>
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    <td>OD600/Abs600</td>
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    <td>3.617</td>
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  </tr>
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</table>
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<table >
 +
  <tr>
 +
  <th> uM Fluoroscein/a.u</th>
 +
    <th>50.00</th><th>25</th><th>12.5</th><th>6.25</th><th>3.13</th><th>1.56</th><th>0.78</th><th>0.39</th><th>0.2</th><th>0.1</th><th>0.05</th><th>0</th>
 +
  </tr>
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  <tr>
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    <td>Replicate 1</td>
 +
    <td>26172</td><td>17413</td><td>1031</td><td>5606</td><td>2988</td><td>1534</td><td>795</td><td>407</td><td>223</td><td>124</td><td>77</td><td>29</td>
 +
  </tr>
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  <tr>
 +
    <td>Replicate 2</td>
 +
    <td>26021</td><td>17369</td><td>10299</td><td>5641</td><td>2997</td><td>1529</td><td>788</td><td>410</td><td>224</td><td>124</td><td>78</td><td>30</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Replicate 3</td>
 +
    <td>26141</td><td>17333</td><td>10321</td><td>5602</td><td>2979</td><td>1527</td><td>787</td><td>409</td><td>225</td><td>126</td><td>78</td><td>30</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Replicate 4</td>
 +
    <td>26243</td><td>17204</td><td>10273</td><td>5570</td><td>2966</td><td>1523</td><td>789</td><td>417</td><td>216</td><td>126</td><td>76</td><td>28</td>
 +
  </tr>
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  <tr>
 +
    <td>Arith. Mean</td>
 +
    <td>26144</td><td>17330</td><td>10306</td><td>5605</td><td>2983</td><td>1528</td><td>7890</td><td>411</td><td>222</td><td>125</td><td>77</td><td>29</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Arith. Std. Dev.</td>
 +
<td>92.59</td><td>89.99</td><td>25.74</td><td>29.044</td><td>13.22</td><td>4.57</td><td>3.59</td><td>4.34</td><td>4.08</td><td>1.15</td><td>0.96</td><td>0.96</td>
 +
</tr></table><table>
 +
<tr>
 +
  <th> uM Fluoroscein/a.u</th>
 +
    <th>50.00</th><th>25</th><th>12.5</th><th>6.25</th><th>3.13</th><th>1.56</th><th>0.78</th><th>0.39</th><th>0.2</th><th>0.1</th><th>0.05</th>
 +
    </tr>
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    <tr>
 +
    <td>Mean uM Fluorescein/a.u.(×10^-3)</td>
 +
  <td>1.9</td><td>1.4</td><td>1.2</td><td>1.1</td><td>1</td><td>1</td><td>0.99</td><td>0.95</td><td>0.88</td><td>0.78</td><td>0.63</td>
 +
</tr>
 +
    <td>Mean of med-high levels</td>
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    <td>0.001168</td>
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</table>
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<br /></span></p>
 
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<tr><td><img src="https://static.igem.org/mediawiki/2017/3/3e/Fluorocein_stdcurve.png";></td>
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<td><img src="https://static.igem.org/mediawiki/2017/2/25/Fluorocein.png";></td></tr>
  
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<tr><td>Serial dilution of fluorescein intesnity plotted on linear scale</td><td>Fluorescein dilution of fluorescein intensity plotted on log (scale Graph)</td><tr></table>
  
  
  
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  <h3 class="box_header">Standard Results</h3>
 
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<p><span style="color: #000000;">Discussing options for CRISPRi and transposons to give assortment of levels for products. We spent a lot of time in meetings and planning to make sure that we were sure about our project. We went bowling as a team this week and discovered that Natalia is an incredibly precise bowler... hopefully her precision transfers to her pipetting skills - all will be revealed later.<br /></span></p>
 
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<center><img src="https://static.igem.org/mediawiki/2017/4/46/T--UNOTT--brainstormunott.jpeg"></center>
 
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   <h3 class="box_header"></h3>
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   <h3 class="box_header">OD600nm and Fluorescence Results</h3>
 
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<p><span style="color: #000000;">This week we really got our heads down and the biochemists planned our constructs and designed primers. In the meantime the rest of the team worked on outreach (i.e. sending hundreds of e-mails!). Vikram, the computer scientist, worked on lots of complex computer stuff and worked endlessly on our game, a Portal mod. Jake made us a new logo and designed a front page for our wiki which looks super professional now.</span></p>
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<table>
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<tr><th>Fluorescence Data <br>(colonies averaged and minus background)</th><th colspan="4">Timepoints (Hours)</th></tr>
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<tr><th>Test Device</th><th>0H</th><th>2H</th><th>4H</th><th>6H</th></tr>
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<tr><td>Negative Control</td><td>24.25</td><td>21</td><td>19.38</td><td>18.75</td></tr>
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<tr><td>Positive</td><td>27.8</td><td>38.6</td><td>81.9</td><td>107.5</td></tr>
 +
<tr><td>1</td><td>44.25</td><td>62.4</td><td>103.5</td><td>174.8</td></tr>
 +
<tr><td>2</td><td>28</td><td>40.1</td><td>93.5</td><td>137.4</td></tr>
 +
<tr><td>3</td><td>24</td><td>21.4</td><td>20.5</td><td>21.6</td></tr>
 +
<tr><td>4</td><td>28</td><td>36.5</td><td>79.6</td><td>124</td></tr>
 +
<tr><td>5</td><td>25</td><td>28.4</td><td>40.5</td><td>45.1</td></tr>
 +
<tr><td>6</td><td>23.1</td><td>21</td><td>9.75</td><td>19.4</td></tr></table>
 +
</table><table>
 +
<tr><th>OD600nm Data (colonies averaged and minus background)</th></tr>
 +
<tr><th>Test Device</th><th>0H</th><th>2H</th><th>4H</th><th>6H</th></tr>
 +
<tr><td>Negative Control</td><td>0.01</td><td>0.07</td><td>0.3</td><td>0.55</td></tr>
 +
<tr><td>Positive control</td><td>0.01</td><td>0.06</td><td>0.26</td><td>0.55</td></tr>
 +
<tr><td>1</td><td>0.01</td><td>0.02</td><td>0.03</td><td>0.07</td></tr>
 +
<tr><td>2</td><td>0.001</td><td>0.05</td><td>0.22</td><td>0.5</td></tr>
 +
<tr><td>3</td><td>0.07</td><td>0.06</td><td>0.25</td><td>0.48</td></tr>
 +
<tr><td>4</td><td>0.01</td><td>0.03</td><td>0.13</td><td>0.4</td></tr>
 +
<tr><td>5</td><td>0.08</td><td>0.05</td><td>0.23</td><td>0.54</td></tr>
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<tr><td>6</td><td>0.08</td><td>0.61</td><td>0.25</td><td>0.5</td></tr></table>
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  <h3 class="box_header">WEEK 3</h3>
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<p><span style="color: #000000;">This week we started lab work! 5 of us (biotechnologists and biochemists) were in the lab learning the techniques needed for iGEM.</span></p>
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<td><img src="https://static.igem.org/mediawiki/2017/1/17/Od600_graph_UNOTT.png"; style:"float:left;width:50px;height:150px;padding:20px;"></td>
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<p><span style="color: #000000;"><span style="text-decoration: underline;">Monday</span> - Primers and enzymes were ordered for one of our two constructs today so we started on the InterLab study until they arrive! We transformed the controls and devices into DH5a competent cells that were provided for us. We set up overnight cultures of TOP10 cells for making electrocompetent cells, and we also made O/N cultures of E.coli carrying the backbone we need for our pReporter which was donated by a lab member. This is called pSTLS.&nbsp; Vikram released a teaser trailer for the game we have made (INSERT LINK HERE). Vik's computer then died</span></p>
+
<td><img src="https://static.igem.org/mediawiki/2017/0/06/Interlab_graph_fluo.png"; style:"float:right;width:50px;height:150px;padding:20px;"><t/d>
−
<p><span style="color: #000000;"><span style="text-decoration: underline;">Tuesday</span> - Vik's computer is alive again. Unfortunately our transformations only gave very small colonies on some plates, and none on others. We re-streaked those colonies that we did have. We extracted plasmid DNA from the O/N cultures of pSTLS and others for the lab to get some practice. We learnt to use the NanoDrop to quantify DNA. Outside of the lab, Georgette was busy creating our very first vlog which will be shared on the social media pages. Vik is looking at building hardware. Today we also skyped Edinburgh UG team to discuss possible collaborations</span></p>
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</tr>
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<p><span style="color: #000000;"><span style="text-decoration: underline;">Wednesday</span> - Waiting for primers to arrive. NEB sent us some enzymes and competent cells so thanks to NEB!</span></p>
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<tr><td>OD600nm measurements of all test devices (minus background), biological replicates were averaged.</td><td>Fluoresence measurements of all test devices (minus background), biological replicates were averaged.</td></tr></table>
−
<p><span style="color: #000000;"><span style="text-decoration: underline;">Thursday</span> - Today we learnt to electroporate DH5a and TOP10 cells. We transformed them with either a plasmid containing dCas9 or our low copy backbone, pSTLS. We also set up overnight cultures for the InterLab Study.</span></p>
+
<br>
−
<p><span style="color: #000000;"><span style="text-decoration: underline;">Friday</span> - DISASTER! Two of our overnight colonies didn't grow so we have to restart the overnights on Monday. Ah well, we are learning what science is like in reality. We went for a social today - Laser quest! Georgette won by far, Ellie lost miserably (-8300 points). Georgette was appointed new team leader due to Ellie's shocking performance... just kidding!</span></p>
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<tr><td><img src="https://static.igem.org/mediawiki/2017/b/b2/Full_fluoro_UNOTT.jpeg";></td></tr><tr><td>Graph depicting each test device's averaged fluorescence intensity minus background</td></tr>
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<tr><td><img src="https://static.igem.org/mediawiki/2017/a/ac/OD600_full_graph_unott.jpeg";></td></tr><tr><td>Graph depicting each test device's averaged OD600nm minus background</td></tr>
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Latest revision as of 03:57, 2 November 2017





INTERLAB STUDY:

Introduction

Our team decided to take part in the iGEM interlab study this year. This study is organised by iGEM and is aimed at tackling a prominent issue regarding experimental fluorescent reporter data. At present, experiments using fluorescent reporters are almost incomparable; separate groups often interpret fluorescence measurements in largely different ways, making it hard to properly compare results. To combat this, the interlab study is performed each year, iteratively refining an optimal protocol for fluorescence measurement. Theoretically, this would be followed by anyone and yield comparably robust units of fluorescence, facilitating more meaningful comparisons of data between researchers.

The study required us to measure a number of different fluorescent test devices sent to us by iGEM HQ, we followed their protocol and then shared the results we obtain. Teams around the world do the same; collectively, we provide a large set of data illustrating how similar the results were, potentially highlighting any aspects in which the protocol could be improved and, hopefully, showing that this is possible. This is quite an important issue, we felt we should participate and help in any way we could!

Methods

We were given 8 specimens to measure, termed “test devices”. These included a positive (BBa_I20270) and negative(BBa_R0040) control, and 6 different test devices (BBa_J36400, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, BBa_J364005).

First we transformed these into DH5α-E. coli cells, then selected two resulting colonies to be prepared for measurement. The two colonies were grown overnight.

But before the experiment could be undertaken, we needed to calibrate our equipment by performing standard reference measurements, enabling the conversion of our data to absolute values. Firstly, we measured a solution of half LUDOX-S40, half water; fluorescein was then serially diluted by half 10 times and measured to give a standard curve.

After 18 hours of growth, the test device sample's optical density was measured, a new batch of LB/Chloramphenicol was inoculated to produce an OD of 0.02. These were then grown at 37°C, OD600nm and fluorescence were measured at 0, 2, 4, and 6 hours.

For a full description of the methods, please visit the official interlab study page

Results

LUDOX-S40 and Fluoroscein Reference Measurements

LUDOX-HS40 H20
Replicate 1 S0.041 0.029
Replicate 2 0.045 0.03
Replicate 3 0.043 0.032
Replicate 4 0.041 0.032
Arith. Mean 0.0425 0.03075
Corrected Abs600 0.01175
Reference OD600 0.0425
OD600/Abs600 3.617
uM Fluoroscein/a.u 50.002512.56.253.131.560.780.390.20.10.050
Replicate 1 261721741310315606298815347954072231247729
Replicate 2 2602117369102995641299715297884102241247830
Replicate 3 2614117333103215602297915277874092251267830
Replicate 4 2624317204102735570296615237894172161267628
Arith. Mean 26144173301030656052983152878904112221257729
Arith. Std. Dev. 92.5989.9925.7429.04413.224.573.594.344.081.150.960.96
uM Fluoroscein/a.u 50.002512.56.253.131.560.780.390.20.10.05
Mean uM Fluorescein/a.u.(×10^-3) 1.91.41.21.1110.990.950.880.780.63
Mean of med-high levels 0.001168

Serial dilution of fluorescein intesnity plotted on linear scaleFluorescein dilution of fluorescein intensity plotted on log (scale Graph)

OD600nm and Fluorescence Results

Fluorescence Data
(colonies averaged and minus background)		Timepoints (Hours)
Test Device0H2H4H6H
Negative Control24.252119.3818.75
Positive27.838.681.9107.5
144.2562.4103.5174.8
22840.193.5137.4
32421.420.521.6
42836.579.6124
52528.440.545.1
623.1219.7519.4
OD600nm Data (colonies averaged and minus background)
Test Device0H2H4H6H
Negative Control0.010.070.30.55
Positive control0.010.060.260.55
10.010.020.030.07
20.0010.050.220.5
30.070.060.250.48
40.010.030.130.4
50.080.050.230.54
60.080.610.250.5

OD600nm measurements of all test devices (minus background), biological replicates were averaged.Fluoresence measurements of all test devices (minus background), biological replicates were averaged.

Graph depicting each test device's averaged fluorescence intensity minus background
Graph depicting each test device's averaged OD600nm minus background