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| | <h2 class="section-heading">Experiments</h2> | | <h2 class="section-heading">Experiments</h2> |
| | <p class="lead"> | | <p class="lead"> |
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| − | <h4>Overview</h4>
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| − | <br>
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| − | <p class="lead">
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| − | General verification</p>
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| − | <p class="lead">
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| − | Vector creation
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| − | <ul style="text-align:left; color:white;">
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| − | <li>Biobrick compatible (failed) </li>
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| − | <li>Vector design </li>
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| − | <li>CPP tag insertion </li>
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| − | </ul>
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| | </p> | | </p> |
| − | <br>
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| − | <p class="lead">
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| − | Evaluating protein import:
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| − | <ul style="text-align:left; color:white;">
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| − | <li>Expression of fluorescent proteins</li>
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| − | <li>Purification and import of the expressed proteins</li>
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| − | </ul>
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| − | </p>
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| − | <br>
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| − | <h4>Verifications and biobrick creation</h4>
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| − | <p class="lead">
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| − | In all three sub projects, we have used gel electrophoresis and sequencing to verify our stepwise experiments. Read more about our general verifications and biobrick creation under <a href="https://2017.igem.org/Team:UCopenhagen/Interdependency">interdependency</a>.
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| − | </p>
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| − | <h4>Vector creation</h4>
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| − | <p class="lead">
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| − | Our vector is a modified version of the <a href="https://www.addgene.org/vector-database/2623/">pET102 vector</a>, which contains a USER cassette, and a his tag after the USER cassette. The USER cassette is used for cloning genes into. We build the vector design on prSET102; an existing vector in our lab, containing the USER cassette and some restriction sites. <br><br>
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| − | We have made two versions of the vector: one for expression of untagged YFP and BFP, that was also used in the interdependency subproject. The other has a CPP tag before the USER cassette, which will add CPP to the proteins.
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| − |
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| − | </p>
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| − |
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| − | <figure>
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| − | <br>
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| − | <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/5/5d/CPPvector.png" alt="" width="250" height="200">
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| − | <br>
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| − | <figcaption><b>Figure 1 </b>Overview of final vectors. First level: The two vectors, unlinearized. Second level: linearized by opening in USER casette. Third level: Insertion of YFP. 4: Produced YFP protein with and without CPP tag. YFP used as example; the same method is used for expression of BFP with and without CPP tag.
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| − | </figcaption>
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| − | </figure>
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| − | <br>
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| − |
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| | </div> | | </div> |
| | </div> | | </div> |
