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| − | {{Wageningen_UR/Menuv2}}
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| − | {{Wageningen_UR/StyleCSSv2}}
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| − | {{Wageningen_UR/MobileMenu}}
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| − | <html>
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| − | <div class="container-fluid OurContent">
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| − | <div class="row">
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| − | <div class="col-md-2 hidden-xs Main-Left-Column">
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| − | </div>
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| − | <div class="col-lg-8">
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| − | <div class="Main-Border">
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| − | <!--SideMenu-Wrapper-Start-->
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| − | <div id="SideMenu-Wrapper">
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| − | <div id="nav-anchor"></div>
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| − | <sidenav id="SideMenu">
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| − | <div class="menu-head">
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| − | <h4>Collaborations</h4>
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| − | </div>
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| − | <ul class="sidebar-nav">
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| − | <li class="menu-item">
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| − | <a href="Antigens">Antigens</a>
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| − | </li>
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| − | <li>
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| − | <a href="#HP">Human Practices</a>
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| − | </li>
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| − | </ul>
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| − | </sidenav>
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| − | </div>
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| − | <!-- SideMenu-Wrapper -->
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| − | <div class="Main-Center-Content-Column Overview">
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| − | <div id="breadcrumb-wrapper">
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| − | <ul class="breadcrumb">
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| − | <li><a href="https://2017.igem.org/Team:Wageningen_UR">Home</a></li>
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| − | <li><a href="https://2017.igem.org/Team:Wageningen_UR/Wet_Lab">Wet-lab</a></li>
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| − | <li>Results</li>
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| − | </ul>
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| − | </div>
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| − | <!--breadcrumb-wrapper -->
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| − | <section id="Antigens">
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| − | <section class="resultsoverview">
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| − | <div class="Title">
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| − | <h1>Results</h1> </div>
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| − | <p>
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| − | </p>
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| − | <div class="clearer"></div>
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| − | <p style="float: right;">
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| − | <img src="https://static.igem.org/mediawiki/2017/0/0f/T--Wageningen_UR--Results_Antigens.png" height="300" width="300" border="0px"></p>
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| − | <p>In order to develop the selectivity of Mantis against several diseases, we need proteins from the infectious agents that will be present in the blood of patients. In order to show the modularity of Mantis, selectivity against several diseases was developed. As placeholders, we selected the Zika virus, chikungunya virus and mayaro virus due to the availability of expertise in our university. Virus-like Particles (VLP), the structural proteins in the outer layer of the viruses, were produced in insect cells cultures and purified for their further use. To prove that Mantis not only applies to viral diseases, we also developed selectivity against <i>Trypanosoma brucei gambiense</i>, the causing agent of the African Sleeping Sickness (HAT). Surface antigens from this parasite were expressed in <i>E. coli</i> and purified for further use. </p>
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| − | <a href="https://2017.igem.org/Team:Wageningen_UR/Results/Viral_Antigens"><img src="https://static.igem.org/mediawiki/2017/4/40/T--Wageningen_UR--Results_Button_Virus.png" style="float: center; width: 20%; margin-left: 10%; margin-bottom: 0.5em;"></a>
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| − | <a href="https://2017.igem.org/Team:Wageningen_UR/Results/Tryps"><img src="https://static.igem.org/mediawiki/2017/0/09/T--Wageningen_UR--Results_Button_Trypanosoma.png" style="float: center; width: 20%; margin-left: 10%; margin-bottom: 0.5em;"></a>
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| − | <div class="figure-center">
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| − | <div class="figure-center-imagebox">
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| − | <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/c/c2/T--Wageningen_UR--Results_RL.png" />
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| − | </div>
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| − | </div>
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| − | </section>
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| − | <div class="clearer"></div>
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| − | <p style="float: left;">
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| − |
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| − | <img src="https://static.igem.org/mediawiki/2017/c/c8/T--Wageningen_UR--Results_Affibody.png" height="300" width="300" border="0px"></p>
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| − | <p>The biological tool that allows Mantis to be selective against a particular infectious is the affinity body, based on a domain from the Protein A from Staphylococcus aureus. This polypeptide is able to bind other proteins. The selectivity of the affinity body is determined by only 13 residues on 2 of the 3 helices.This makes it possible to engineer affinity molecules specific for new targets. In order to engineer an affinity body selective for the VLPs and the antigen from <i>T.b. gambiense</i>, oligonucleotides with a randomized sequence were used to amplify the sequence of the affinity body in order to obtain a library of affinities bodies with different sequences in the selectivity region. From this library, selective proteins were identified through several rounds of phage display. Affinities bodies with selectivity towards Zika virus/chikungunya virus/mayaro virus/Trypanosoma brucei were successfully engineered. </p>
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| − | <a href="https://2017.igem.org/Team:Wageningen_UR/Results/affinitybody"><img src="https://static.igem.org/mediawiki/2017/5/5f/T--Wageningen_UR--Results_Button_Affinitybody.png" style="float: center; width: 20%; margin-left: 10%; margin-bottom: 0.5em;"></a>
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| − | <a href="https://2017.igem.org/Team:Wageningen_UR/Results/Phage_Display"><img src="https://static.igem.org/mediawiki/2017/3/32/T--Wageningen_UR--Results_Button_Phagedisplay.png" style="float: center; width: 20%; margin-left: 10%; margin-bottom: 0.5em;"></a>
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| − | <div class="figure-center">
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| − | <div class="figure-center-imagebox">
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| − | <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/1/16/T--Wageningen_UR--Results_LR.png" />
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| − | </div>
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| − | </div>
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| − | <div class="clearer"></div>
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| − | <p style="float: right;">
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| − | <img src="https://static.igem.org/mediawiki/2017/c/c1/T--Wageningen_UR--Results_signal.png" height="300" width="300" border="0px"></p>
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| − | <p>In order to transduce the binding between the antigen and the affinity body into an intracellular signal, the Cpx system was used. The protein CpxP, which binds and inhibits CpxA, was fused to a placeholder affinity body selective for IgG. This system is expressed in the inner membrane of the bacteria, so spheroplasts were produced to allow the proteins the medium to get in contact with the affinity body fused to CpxP. To prevent the spontaneous diffusion of CpxP, it was tethered to the inner membrane through a linker connected to a transmembrane MBP protein. Once the IgG is added in the medium, it binds the affinity body which makes CpxP separate from CpxA. As a result, CpxA is activated, gaining kinase activity in its intracellular domain. This domain will phosphorylate CpxR, which in its phosphorylated form, it binds to other phosphorylated CpxR. The homodimer formed will act as a transcriptional factor, activating a reporter gene. </p>
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| − | <a href="https://2017.igem.org/Team:Wageningen_UR/Results/Cpx"><img src="https://static.igem.org/mediawiki/2017/d/da/T--Wageningen_UR--Results_Button_Signal.png" style="float: center; width: 20%; margin-left: 20%; margin-bottom: 0.5em;"></a>
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| − | <div class="figure-center">
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| − | <div class="figure-center-imagebox">
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| − | <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/c/c2/T--Wageningen_UR--Results_RL.png" />
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| − | </div>
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| − | </div>
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| − | <div class="clearer"></div>
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| − | <p style="float: left;">
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| − | <img src="https://static.igem.org/mediawiki/2017/b/b1/T--Wageningen_UR--Results_fluor.png" height="300" width="300" border="0px"></p>
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| − | <p>In order to create a fast response, we decoupled the Cpx system from the transcription and the translation of the reporter gene. In order to do this, we used bimolecular fluorescent complementation (BifC). This method uses fluorescent proteins split in two halves, which are fused to interacting proteins. When the two fusion proteins interact, the two halves of the fluorescent proteins will reassemble, giving place to a functional fluorescent protein. The bimolecular fluorescent complementation was succesfully integrated with the Cpx system, which allowed as to observe directly the interaction between the two phosphorylated CpxR proteins. Furthermore, we tested several fluorescent proteins whose implementation will improve Mantis' performance by increasing the intensity of the signal and reducing the maturation time of the reassembled protein. eYFP, mRFP, mCerulean, mVenus and sfGFP were tested as possible reporter proteins. </p>
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| − | <a href="URL"><img src="https://static.igem.org/mediawiki/2017/9/92/T--Wageningen_UR--Results_Button_Visualisation.png" style="float: center; width: 20%; margin-left: 10%; margin-bottom: 0.5em;"></a>
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| − | <a href="https://2017.igem.org/Team:Wageningen_UR/Results/Fluorescent"><img src="https://static.igem.org/mediawiki/2017/9/93/T--Wageningen_UR--Results_Button_FluorescentProteins.png" style="float: center; width: 20%; margin-left: 10%; margin-bottom: 0.5em;"></a>
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| − | <div class="figure-center">
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| − | <div class="figure-center-imagebox">
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| − | <img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/1/16/T--Wageningen_UR--Results_LR.png" />
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| − | </div>
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| − | </div>
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| − | <div class="clearer"></div>
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| − | <p style="float: right;">
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| − | <img src="https://static.igem.org/mediawiki/2017/5/5f/T--Wageningen_UR--Results_Amplify.png" height="300" width="300" border="0px"></p>
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| − | <p>We also have developed an alternative system based quorum sensing signalling. This system, while slower than the BifC system, allows for more robustness. The activation of the CpxR system leads to the activation of the quorum sensing system. This way, the cell that detects the antigen starts producing homocysteine lactone (AHL), which can diffuse to other cells. AHL has two effects on the cells. On one hand, it creates a positive feedback through the induction of the synthesis of more AHL. On the other hand, it induce the expression of COLE7 which leads to the lysis of the cell. The level of AHL necessary to activate the systems are regulated with a protein that degrades AHL. If enough antigen is detected, the quorum sensing system leads to the lysis of all the cells in the medium. In order to generate a visual signal linked to this lysis, two populations of cells were used. Both populations of cells are the same except that one of them express a TEV protease gene, while the other express a gene encoding a fluorescent protein fused to a quenching peptide. Once both populations are lysed, the two expressed proteins interact in the medium. The TEV protease cleaves the quenching peptide, activating the fluorescent protein and generating a fluorescent signal. </p>
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| − | <a href="URL"><img src="https://static.igem.org/mediawiki/2017/0/05/T--Wageningen_UR--Results_Button_QS.png" style="float: center; width: 20%; margin-left: 10%; margin-bottom: 0.5em;"></a>
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| − | <a href="URL"><img src="https://static.igem.org/mediawiki/2017/c/c7/T--Wageningen_UR--Results_Button_FluorescentQuenching.png" style="float: center; width: 20%; margin-left: 10%; margin-bottom: 0.5em;"></a>
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| − | </section>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="col-md-2 hidden-xs Main-Right-Column">
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </html>
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| − | {{Wageningen_UR/PageEnd}}
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| − | {{Wageningen_UR/MainJSv2}}
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