Difference between revisions of "Team:Cork Ireland/Collaborations"

 
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          <li><a href="project.html">Project</a></li>
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}
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          <li><a href="Human Practice.html">Human Practice</a></li>
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          <li><a href="Results.html">Results</a></li>
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          <li><a href="Attributions.html">Attributions</a></li>
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          <li><a href="Parts.html">Parts</a></li>
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          <li><a href="team.html">Team</a></li>
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          <li><a href="Collaborations.html">Collaborations</a>
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  <h1>Interlab Study</h1>
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<div class="question">
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Provenance and releases:</div><br>
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<div class="question">Who did the work for the interlab study?</div>
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}
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<p class="IL">The work was carried out by Brandon Malone.</p><br>
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<div class="question">When was the work carried out for the study?<br></div>
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<p class="IL">The devices were constructed over a period from June 26th to July 17th. The measurements were done at the start of the following week and the data was analysed and graphed shortly afterwards.</p><br>
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<div class="question">Do you consent to your data being included in subsequent publications?<br></div>
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<p class="IL">All persons involved consent to the data being published in subsequent studies.</p><br>
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<div class="question">What is model of measuring tool & how is it configured?</div>
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<p class="IL">Short intro:<br>
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nav ul a:hover {
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The purpose of the interlab study is to measure fluorescence data from three devices. Igem teams from around the world are invited to take part in this study and analyse the difference in fluorescence from three devices i.e diferent promotors from the Anderson series of promotors combined with a GFP part as outlined in the interlab study overview page.<br><br>
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Assembly:<br>
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color: white;
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The promotor’s respective names are J23101, J23106 & J23117. The GFP part is I13504. Each part is present in the distribution kit, the first step of the project was therefore to assemble the devices (promotor plus GFP generator). Due to difficulties encountered experimentally more than one assembly method was trialled. 3A assembly was carried out and so too was the traditional restrict & ligation method.<br><br>
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}
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Timeline:<br>
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22/06/15 – individual promotors and GFP taken from the distribution kit and transformed into dh5 alpha.  17 F -4th plate= J23101<br>
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19 p-4th plate- J23117 4th plate<br>
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J23106- 17 P 4th plate<br>
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I13504-17 C 3rd plate-pSB1C3<br>
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Transformations were repeated on 25/06/15 as no colonies were present.<br>
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Plate 1 – 20K –K823005- J23101 –Psb1C3<br>
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Plate 1 – 22A J23106<br>
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Plate 1- 22kJ23117-Psb1c3<br><br>
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Here J23101 & J23117 had colonies, overnights were grown up with 3 liquid cultures prepared for each one.<br>
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Transformation repeated for J23106 with more DNA being used on 26/06/15.<br><br>
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27/06/15 Plasmid preps were made of Promotors and GFP generating biobrick. The plasmid DNA concentrations were then recorded using nanodrop.<br><br> Plasmids were restricted using biobrick standard restriction digests and then ligated togethed following gel extraction.<br><br>
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Eco + Spe used for promotors & Xba 1 + PSt1 used for GFP generator.<br><br>
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6/7/15. Following successful ligations for devices 1 and 2, overnights were prepared from the plates where some of the colonies that grew showed green fluorescence.<br><br>
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However the ligations involving promotor J23117 were unsuccessful in that colonies formed but none of which showed green fluorescence. Colony PCR was done to investigate and results showed that some of the correct size plasmids were there but there was no green fluorescence.<br> <br>
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The primers used were the VF2 & VR2 primers which are complementary to the regions of the prefix and suffix. Commercially available solis biodyne was used as a ready to go mastermix of Taq polymerase, buffer and dNTPS.<br><br>
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Now both 3A assembly and traditional restriction/ ligation were trialled simultaneously to ligate J23117 and I13504.Each method of assembly was done in triplicate to increase the likelihood of success. <br><br>
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Following this on the 9th July, one of the colonies that formed on the plates showed green fluorescence and was taken and grew overnight. <br><br>
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Plasmid preps were made of all overnights that were successful and these were subsequently diluted 1:1000 and retransformed whereby a greater proportion of the colonies showed green flourecenece. 3 biological replicates were taken from these and by the 14th July, fluorescence was ready to be measures using the plate reader. As controls, colonies without promotors but containing the GFP generator I13594 were grown overnight and also LB media inoculated with antibiotic was also left overnight at 37 with the rest of the samples.<br><br>
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Each of the biological replicates was tested three different times using the plate reader. The average and standard deviation of both technical replicates and biological replicates was obtained. This average was then divided by the OD at 600nm.<br><br>
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Digests were performed on all the devices to show that they were the correct size by restriction mapping. <br><br></p>
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<div class="question">Cloning Protocol-traditional restriction & ligation:</div>
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.content {
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<p class="IL">The following applies to each of the three devices. The promotor parts shall be from here on known as the “promotor” and the part BBa_I13504 shall be known as GFP generator. <br>
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clear: both;
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After plasmid prep, restrict both parts for the device. Digest 1 microgram approx. of Promotor DNA with EcoRI and SpeI and roughly 1 microgram of GFP generator DNA with XbaI and PstI. <br>
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height: 100%;
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A typical protocol is as follows:
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}
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Pipette the following into a PCR tube and incubate for the required amount of time (normally between 1 hr and 6 hrs depending on enzyme activity, amount of enzyme used and DNA concentration)<br>
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X uL  dH2O
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.col {
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X uL  Plasmid DNA
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2 uL  10x buffer
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width: 12%;
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1 uL (each) enzyme
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margin: 0;
 +
}
  
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Following this the digests should be run on an agarose gel. The bands containing the required parts should be cut and using a commercially available Gel extraction kit or the correct reagents, the DNA should be extracted from the gel. DNA concentration should be obtained using a nanodrop.
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#main_col {
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Following this the parts can be cloned together using DNA ligase. The result of the cloning should be transformed into LB media with the correct antibiotic resistance.</p><br>
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<figure>
 
−
  <img style="height:265px;" src="https://static.igem.org/mediawiki/2015/f/f5/Rsz_1interlab3.jpg"><figcaption>Restriction mapping<br>
 
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Lane 1 =  ladder<br>
 
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Lane 2=undigested plasmid of J23101 + I13504 ligation<br>
 
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Lane 3= device 1 digested with ECoRI and PstI<br>
 
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Lane 4= device 2 digested with EcoRI & PstI<br>
 
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Lane 5= device 3 digested with EcoRI & PstI<br>
 
−
Run using an 0.8 % gel
 
−
</figcaption>
 
−
</figure>
 
  
−
<div class="question">
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Plate reader protocol:</div>
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<p class="IL">
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In order to measure the fluorescence, liquid cultures were grown of all the biological replicates. 100uL of these cultures was then pipetted into a 96 well plate and the OD 600 was measured. Dilutions were made of the absorbance at 600nm to give an OD 600 close to 0.5 in order to normalise the results across all the replicates. 100uL of the overnight was pipetted out 2 more times and diluted in order to provide technical replicates. Technical replicates all were derived from the same colony as the initial biological replicate.
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The samples on the 96 well plate were then analysed. The OD600 was measured and the fluorescence was measured using 488 nm wavelength to excite the sample and 532 nm wavelength for emission.<br>
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        color: #E02121;
 +
}
  
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  <figure>
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#team h2, #collabs h2 {
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<img src="https://static.igem.org/mediawiki/2015/0/07/Rsz_1interlab2.jpg"><figcaption>Plate reader</figcaption>
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  </figure><br>
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<div class="question">Discussion:</div>
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<p class="IL">
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The results obtained showed for the most part what would have been expected, the promotor J23101 and GFP generator had the highest fluorescence. The understanding from previous work is that J23101 is the strongest promotor, J23106 the next one after that and the weakest being J23117. The results of the experimentation somewhat supported this view although the relative strengths of the promotors were slightly different from what was previously recorded. All of the devices were in the PSB1C3 high copy number plasmid backbone in order to reduce the number of variables in the experiment.</p><br>
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<div class="question">Devices:</div>
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<p class="IL">
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padding: 10px;
−
The three devices that are required to be studied are:<br>
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}
−
1)  Device 1 is a composite of promotor J23101 and a RBS, GFP generator and a terminator I13504<br>
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−
2)  Device 2 is a composite of promotor J23106 and the part I13504<br>
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3)  Device 3 is a composite of promotor J23117 and the part I13504<br>
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−
All three devices were cloned in to a PSB1C3 vector.
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</p>
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−
<br>
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  <img src="https://static.igem.org/mediawiki/2015/a/ad/Rsz_11interlab1.jpg"><p>Plate reader manufacturer</p>
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<script type="text/javascript">
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</script>
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−
</body>
+
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 +
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 +
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 +
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<footer>
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#notebook div {
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 +
background-color: white;
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margin: 15px;
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−
      <h6>Cork iGEM 2015</h6>
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#interlab div {
−
+
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−
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+
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 +
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    <div class="col-sm-4">
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#interlab div h3 {
 +
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 +
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−
      <h6>About Us</h6>
+
#interlab div p, #interlab div li {
−
      <p style="color: #eee;">
+
font-size: 1.2em;
−
Our project aims to develop a bacterial-based diagnostic system. This builds on our foundational work last year, where we showed that it was possible to use bacteria to detect specific target DNA sequences. This has the potential to be applied in a variety of settings in which simple, low-tech and cost-effective DNA detection is required. These areas include veterinary diagnostics and medical diagnostics in regional hospitals or resource-poor developing countries.</p>
+
}
−
   
+
−
    </div><!-- col-sm-4 end -->
+
  
 +
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 +
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−
    <div class="col-sm-2">
+
.question {
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−
      <h6>Navagation</h6>
+
.answer {
−
      <ul class="unstyled">
+
padding-bottom: 15px;
−
        <li><a href="#">Home</a></li>
+
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−
        <li><a href="#">Project</a></li>
+
}
−
        <li><a href="#">Human Practice</a></li>
+
−
        <li><a href="#">Results</a></li>
+
−
      </ul>
+
  
−
    </div><!-- col-sm-2 end -->
+
#interlab .result_pic {
 +
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 +
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 +
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 +
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 +
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 +
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 +
}
  
−
    <div class="col-sm-2">
+
#achieve h2 {
 +
text-align: center;
 +
}
  
−
      <h6>Contact Us</h6>
+
#collabs figure {
 +
        text-align: center;
 +
}
  
−
    </div><!-- col-sm-2 end -->
+
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margin: 10px;
 +
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 +
}
  
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      <h6><span class="glyphicon glyphicon-heart"></span> by Aoife</h6>
+
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                    <h2>Trinity College Dublin & University College London</h2>
 +
 +
<p>This year, Cork iGEM collaborated with both the Trinity College Dublin and the University College London iGEM teams. The Basehunter system was put together in an anonymous way so that the participating teams could not see what reagents were present or absent in order to avoid biased results. Cork iGEM put together tubes containing mastermixes and labelled them using unbiased characters such as A,B and C that were then sent via post. The participating iGEM teams agreed to test the systems and give feedback regarding the protocol. We were unfortunately unable to obtain test results from the UCL team due to time constraints. They did however successfully receive the kit. Mariola Sebastián gave us feedback regarding out Basehunter System,"I really liked that the tubes were labeled with numbers and letters. I think its great because you don't need to know what each one contains in order to follow the protocol, which makes it more accessible, and the simple labeling makes them easier to find and work with. In the final version of the kit. I think it would be great if you could add more of the needed things, like sterile plates and sterile LB Media, to speed and simplify the process".</p>
 +
 +
<figure id="Lab_pic">
 +
<img src="https://static.igem.org/mediawiki/2015/8/8d/UCLCorkCollab.jpg"/>
 +
<figcaption>
 +
Mariola from UCL iGEM working on our protocol
 +
</figcaption>
 +
</figure>
 +
 +
<p>The aim of this collaboration was to investigate if the Basehunter system could be both easily transported and explained with a protocol. A written protocol and a video protocol were prepared. The video protocol can be seen below and the written protocol can be seen <a href="https://static.igem.org/mediawiki/2015/4/45/Basehunter-Protocols-amy-xoxo.pdf">here</a>.</p>
 +
 +
<p>Trinity tested the system and these were the results obtained in the blind testing; Sent to the team were 6 tubes containing the HPV 55bp Detector. Tubes 1-3 were to be positive controls, while tubes 4-6 were to be negative. This was not disclosed to the experimenting team at TCD.</p>
 +
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2015/c/c7/CollabGraphsCork.JPG"/>
 +
</figure>
 +
 +
<p>Feedback from the team was positive and they reported - “The Protocols were very clear indeed and easy to follow, and it helped greatly that there was an alternative to using the PCR machine when incubating the coloured tubes.”</p>
 +
 +
<p>These results proved that the detector could be posted to other labs and that the protocols were simply followed. In addition, results achieved were consistent with those obtained in our lab at UCC. See figures 1 and 2 for results reported by TCD.</p>
 +
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2015/d/df/PlatesTCDCorkCollab.JPG"/>
 +
</figure>
 +
 +
<p>Also, to assist TCD, we researched into the possibility of constructing a detector to detect artemisinin resistance in species of plasmodium falciparum, the parasite that causes malaria. TCD informed us that this is a significant problem in areas where malaria is endemic, as usually the most widely used treatment for malaria is artemisinin. Between the two teams, we found that the most common mutations that confer resistance are SNP’s on the Kelch 13 propellor (Haldar et al., 2015) . The mutations are point mutations, single base changes. Our detector was not sensitive or specific enough to pick up these small changes.</p>
 +
 +
<video width="100%" height="100%" controls>
 +
<source src="https://static.igem.org/mediawiki/2015/3/3f/Igem_protocol_video_fixed_2.mp4">
 +
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 +
</div>
 +
</div>
 +
</figure>
 +
 +
<h4>References:</h4>
 +
<p>Haldar, K. et. al, April 2015, “A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria”, Nature, 520, 683–687</p>
 +
                    </div>
 +
 +
<div id="team2">
 +
<h2>Meetups</h3>
 +
 +
<p>In July Cork iGEM, along with Trinity College Dublin (TCD) iGEM, organised a meetup that was posted on iGEM.org. Cork iGEM travelled to Dublin where we met up with TCD for the first time. TCD met us at the front gate with a very welcoming sign and brought us to a hang out space called the Global Room.</p>
 +
 +
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2015/0/0f/MeetupCork1.JPG" alt="" />
 +
<figcaption>
 +
</figcaption>
 +
</figure>
 +
 +
<p>We provided feedback on their project work and told them what to expect at the Jamboree. We were in contact with the team during its early days before the team members were selected and stressed the need for a diverse team from many backgrounds. This was something we learned at iGEM in our previous year and knew it was important to relay this advice. We also suggested the team not focus entirely on lab work and engage with the community throughout their project.</p>
 +
 +
<p>Over the course of working on the projects, we kept in contact with the team and aided them in their efforts to create a Youtube Channel “iGEM Academy” with videos of common lab protocols by providing a video tutorial of our “Detector Reaction” for use with our BaseHunter parts.</p>
 +
<p>We gave a presentation of our project and our results up to the beginning of July. </p>
 +
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2015/8/86/MeetupCork2.JPG" alt="" />
 +
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 +
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 +
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<figure>
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<figure>
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<figure>
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 +
<figcaption>
 +
</figcaption>
 +
</figure>
 +
 +
<p>TCD brought us to their lab and talked us through their plans for the project over the summer months. They then kindly gave us a campus tour and brought us to see the beautiful Book of Kells and the Long Room. We had a social that evening! </p>
 +
 +
<p>In August this year we had the opportunity to meet up with members of the UCL iGEM Team at the IndieBio Demo Dinner & Summer Party. We also met up with them the following day at Forma labs. We spoke about our projects and planning for the Jamboree. We spoke about London Hackspace and the hacker community in London and the future of Cork's hacker community.</p>
 +
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2015/4/49/MeetupCork6.JPG" alt="" />
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Latest revision as of 15:29, 9 November 2015

Collaborations

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Trinity College Dublin & University College London

This year, Cork iGEM collaborated with both the Trinity College Dublin and the University College London iGEM teams. The Basehunter system was put together in an anonymous way so that the participating teams could not see what reagents were present or absent in order to avoid biased results. Cork iGEM put together tubes containing mastermixes and labelled them using unbiased characters such as A,B and C that were then sent via post. The participating iGEM teams agreed to test the systems and give feedback regarding the protocol. We were unfortunately unable to obtain test results from the UCL team due to time constraints. They did however successfully receive the kit. Mariola Sebastián gave us feedback regarding out Basehunter System,"I really liked that the tubes were labeled with numbers and letters. I think its great because you don't need to know what each one contains in order to follow the protocol, which makes it more accessible, and the simple labeling makes them easier to find and work with. In the final version of the kit. I think it would be great if you could add more of the needed things, like sterile plates and sterile LB Media, to speed and simplify the process".

Mariola from UCL iGEM working on our protocol

The aim of this collaboration was to investigate if the Basehunter system could be both easily transported and explained with a protocol. A written protocol and a video protocol were prepared. The video protocol can be seen below and the written protocol can be seen here.

Trinity tested the system and these were the results obtained in the blind testing; Sent to the team were 6 tubes containing the HPV 55bp Detector. Tubes 1-3 were to be positive controls, while tubes 4-6 were to be negative. This was not disclosed to the experimenting team at TCD.

Feedback from the team was positive and they reported - “The Protocols were very clear indeed and easy to follow, and it helped greatly that there was an alternative to using the PCR machine when incubating the coloured tubes.”

These results proved that the detector could be posted to other labs and that the protocols were simply followed. In addition, results achieved were consistent with those obtained in our lab at UCC. See figures 1 and 2 for results reported by TCD.

Also, to assist TCD, we researched into the possibility of constructing a detector to detect artemisinin resistance in species of plasmodium falciparum, the parasite that causes malaria. TCD informed us that this is a significant problem in areas where malaria is endemic, as usually the most widely used treatment for malaria is artemisinin. Between the two teams, we found that the most common mutations that confer resistance are SNP’s on the Kelch 13 propellor (Haldar et al., 2015) . The mutations are point mutations, single base changes. Our detector was not sensitive or specific enough to pick up these small changes.

iGEM Paris-Saclay Collaboration Survey Badge

References:

Haldar, K. et. al, April 2015, “A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria”, Nature, 520, 683–687

Meetups

In July Cork iGEM, along with Trinity College Dublin (TCD) iGEM, organised a meetup that was posted on iGEM.org. Cork iGEM travelled to Dublin where we met up with TCD for the first time. TCD met us at the front gate with a very welcoming sign and brought us to a hang out space called the Global Room.

We provided feedback on their project work and told them what to expect at the Jamboree. We were in contact with the team during its early days before the team members were selected and stressed the need for a diverse team from many backgrounds. This was something we learned at iGEM in our previous year and knew it was important to relay this advice. We also suggested the team not focus entirely on lab work and engage with the community throughout their project.

Over the course of working on the projects, we kept in contact with the team and aided them in their efforts to create a Youtube Channel “iGEM Academy” with videos of common lab protocols by providing a video tutorial of our “Detector Reaction” for use with our BaseHunter parts.

We gave a presentation of our project and our results up to the beginning of July.

TCD brought us to their lab and talked us through their plans for the project over the summer months. They then kindly gave us a campus tour and brought us to see the beautiful Book of Kells and the Long Room. We had a social that evening!

In August this year we had the opportunity to meet up with members of the UCL iGEM Team at the IndieBio Demo Dinner & Summer Party. We also met up with them the following day at Forma labs. We spoke about our projects and planning for the Jamboree. We spoke about London Hackspace and the hacker community in London and the future of Cork's hacker community.