Difference between revisions of "Template:Team:Utrecht/MainBody"

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<li data-key="home" class="active" data-url="home">Home</li>

<li data-key="exp">Experimental</li>

<li data-key="hp">Human practices</li>

<li data-key="team">Team & Sponsors</li>

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<img id="figure-1" style="position: absolute; top: 25px; left: 128px; " src="https://static.igem.org/mediawiki/2017/b/b4/Uututorial_1.png">

<img id="figure-2" style="position: absolute; top: 25px; left: 128px; display: none;" src="https://static.igem.org/mediawiki/2017/f/f5/Uututorial_2.png">

<img id="figure-3" style="position: absolute; top: 25px; left: 128px; display: none;" src="https://static.igem.org/mediawiki/2017/3/3b/Uututorial_3.png">

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The OUTCASST two-component system consists of two proteins, expressed to the membrane of a dryable cell. One of the proteins is a Cas9-fusion and the other contains Cpf1. Both proteins can be given a guide RNA that makes it bind to a specific, user-chosen, complementary sequence. When both proteins bind a DNA fragment from a sample, they co-localize, so that a transcription factor is released intracellularly which then induces an intracellular reporter mechanism such as a dye or fluorescent signal.

It is very important that measurements in all engineering disciplines are reliable and reproducible. However, the compatibility of measurements in different labs around the world has always been difficult. This is why there is a need for robust measurement procedures. The iGEM Measurement Committee has chosen Green Fluorescent Protein as the measurement marker for this study, as it is one of the most commonly used markers in the lifesciences. For this fourth interlab study, fluorescence measurements were performed with E. coli. Six different GFP expression plasmids and additional positive and negative control plasmids were used. The protocols for this study were provided by the iGEM organisation to ensure method uniformity between participating labs.

<li />E. coli DH5-alpha cells

<li />Protocol for the transformation of competent E. coli: single tube transformation protocol (http://parts.igem.org/Help:Protocols/Transformation)

All eight plasmids were transformed in competent E. coli DH5-alpha cells with heat shock. After incubating overnight at 37°C, single colonies were picked and grown in 5mL LB medium with chloramphenicol. These cultures were grown at 37°C while being shaken at 200 rpm (instead of the recommended 220 rpm).

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           <h2>Westerdijk</h2>

         <p>The first person we talked to came from the Westerdijk Fungal Biodiversity Institute in Utrecht. Here, they perform mycological research, wherein they focus on the determination and classification of fungi&hellip;</p>

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           <h2>UMCU</h2>

         <p>We used this information and went to two DNA genome diagnostic labs, one in Utrecht and one in Amsterdam. At the University Medical Center of Utrecht we talked about the non-invasive prenatal testing and the use of our system to test for genetic conditions&hellip;</p>

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<b>1.1 Lab Safety</b><br><br>

<b>1.2 User Safety</b><br><br>

<b>1.3 Patient Safety</b><br><br>

<b>1.4 Public Safety</b><br><br>

<b>1.5 Environment</b><br><br>

We have also heard that the tool should have a low incidence of false positive and negative results and that our device should distinguish DNA strands with one different base pair. We want to take this information into account to decide the target DNA. There are two possibilities from which we can choose. The first option would be to permit certain mutations in the target DNA, to prevent getting a false negative result in some cases. The second option would be to use a very conserved domain as target DNA and don’t allow any mismatches. From our perspective, we think the second option would be more suitable, since the specificity in our system is a very valuable aspect of the design. We have chosen to use the satellite DNA, which is present in the T. cruzi parasite as a 195 base pair repeat with about 100,000 copies (Aldert Bart, Academical Medical Center Amsterdam: Clinical molecular parasitologist).

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<h2 class="subhead" id="subhead-2">Movie: Meet the team</h2>

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<h2 class="subhead" id="subhead-2">Movie: The problem; the detection of infectious disease has to be improved</h2>

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<h2 class="subhead" id="subhead-2">Movie: Why Chagas Disease?</h2>

           <h2>Quincy Holzaphfel</h2>

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