/* The grid: Four equal columns that floats next to each other */

.column {

    padding: 10px;

}

.column img:hover {

    opacity: 1;

}

/* Clear floats after the columns */

.row:after {

    content: "";

    display: table;

    clear: both;

}

.container {

    display: none;

}

/* Expanding image text */

#imgtext {

    left: 15px;

    color: white;

    font-size: 20px;

}

/* Closable button inside the expanded image */

.closebtn {

    position: absolute;

    top: 10px;

      }

.blue-letter  {

    color:blue;

}

.red-letter{

  color:red;

}  

          /*  THIS LINE WILL CHANGE THE BACKGROUND IMAGE FOR PARALLAX*/  

          .parallax {

          }

        <div class="igem-icon"><a href="https://2018.igem.org/Main_Page">

              <!------------ CHANGE THIS FOR ANOTHER ANIMATED TEXT -------------------->

      <!-- ONLY IF YOU REALLY REALLY NEED TO: change the class from "svg-wrapper" to "sv" to have the text at the bottom of the image -->

      <div class="svg-wrapper">

<svg version="1.1" id="Layer_1" xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink" x="0px" y="0px"

viewBox="0 0 2412 244" style="enable-background:new 0 0 2412 244;" xml:space="preserve">

<style type="text/css">

c-6.2-8.8-11-14.8-14.4-18c-3.4-3.2-6.1-5-7.9-5.6C2222.2,36.1,2217.2,35.8,2209.1,35.8z"/>

</div>

.btn-group span {

  display: inline-block;

                             <h2>General Concept</h2>

                              </p>

                             <img src="https://static.igem.org/mediawiki/2018/1/1a/T--Uppsala--phageflowchart.svg" alt="Flowchart Phage Display" class="center" height="50%" width="50%">  

                             <p><b>Figure 1:</b> Flowchart over the workflow of a typical phage display screening</p>  

<p>For the purpose of our project we need a way to detect the parasites. A peptide binding specifically to the exterior of the strongyle would fulfill this purpose.  Since we are working with an under-researched organism, starting with a random peptide library seemed reasonable. The only similar organisms that have been used in this method were <i>C. elegens</i>, but as it is a free living nematode whereas strongyles are endoparasites we had reason to believe that the exterior of the membranes would differ greatly due to the different environments the different species inhabit.<br><br>

</p>

<h1>Experiment</h1>

<div>

<h2>New Application</h2>

<h3>Unknown genome/proteome </h3>

<p>Due to our target strongyle having an unknown genome/proteome, selection of a single target protein is not feasible. Efforts could have been made to separate surface proteins from the organism, but such an approach does not provide any guarantees of the extracted proteins displaying correct conformations. To preserve each of the possible protein targets in a physiological state, the entire organism was used for the experiment. </p> <br></br>

<h3>Immobilization hard for whole worms</h3>

<p>To be able to wash away unbound phages, while retaining the bound phages, existing protocols employ various methods for immobilizing the target. Since the target of our experiment is the surface of an entire organism rather than a single protein, the common methods of fixing the target were ineffective, and a new method had to be developed.  

</p> <br></br>

<h3>Experiment procedure</h3>

<p>

With the experiment necessitating the use of phage display on an entire organism, no existing protocol was entirely applicable. This meant that we had to adapt a protocol as much as it was possible and introduce completely new solutions to problems unique to our application.  

</p>

<p>

For a detailed description of our protocol click <a href="https://static.igem.org/mediawiki/2018/9/97/T--Uppsala--phageprotocol.pdf">here</a>. Our whole organism phage display experiments started with preparation of the containers that the whole procedure were performed in microcentrifuge tubes with filter inserts. The preparation consisted of blocking the tubes with blocking buffer to prevent non-specific interactions. 

<br>

To avoid selecting for phage with affinity for the plastic and not the organism, an affinity screening for the tubes was performed before introducing the target. After collecting the phage elute that doesn’t bind to the tubes, we performed our first affinity screening against the small strongyle. This was done by introducing the 12-mer peptides expressing phages to the strongyle placed in the filter tubes. Unbound phages were then washed away followed by elution and collection of the bound phages with an acidic buffer. All washing and elution steps were performed in the filter tubes, where liquid during centrifugation could pass through the filters, leaving the strongyle still on the top of the filter.

</p>

<!-- Begining of 3 card template -->

                        <div class="inner-card left-card">

                            <img class="content-card-img" src="https://static.igem.org/mediawiki/2018/0/0f/T--Uppsala--filter_only%282%29.svg">

                            <div class="inner-card-text">  

                                <p><strong>Figure 1:</strong> Filter tube blocked with Blocking Buffer.</p>

                            </div>

                        <div class="inner-card middle-card">

                            <img class="content-card-img"  src="https://static.igem.org/mediawiki/2018/3/32/T--Uppsala--worm_in_2.svg">

<div class="inner-card-text">  

<p><strong>Figure 2:</strong> The strongyles are added, excess liquid spun down and discarded.</p>

                            </div>

</div>

 

                        </div>

<p>

</p>

</div>

<div>

<h2>Phage Titering</h2>

<p>

Phage titering is done after every affinity screening to assess the amount of phages that bind to the target. By following the titering protocol consisting of plating phages together with mid-log phase bacteria visible blue plaques are formed on Xgal/IPTGl plates. The goal with plating is to achieve plates with around 100 plaques, which is fulfilled by doing several dilution series of the infected bacteria. The reason for this is that plaques will only increase linearly with added phages when the multiplicity of infection (MOI) is much less than 1. Also low MOI result in one DNA sequence per plaque. To asses the titre, plaque forming unit (pfu) can be calculated by multiple numbers of plaques with the bacteria dilution.  

</p>

<p>

Phage titering is also carried out after every phage amplification in order to assess the success of the amplification and to make sure a sufficient amount of phages for the subsequente affinity screening are present.

</p>

<p>

We did three rounds of panning to select for phages having affinity peptides for the target. The last phage titering could then be used to select single clones to be prepared for sequencing.  

</p>

</div>

<h2>Verifying the Affinity and Analysis of DNA</h2>

<p>

So how do we prove that the selected phages clones really binds to our target? With the help of ELISA (enzyme-linked immunosorbent assay) single phage clones affinity to the target could be tested by screening against plastic binders. This allowed us to determine which samples are viable. The ELISA was performed in the centrifugal filter-tubes as to expose the phages to the same environment as the panning stage of the process.  

</p>

 

 

 

<!-- Beginning of 3 card template -->

<h2>Sample titers from Series 1</h2>

                             <img class="content-card-img" style="border:0px" src="https://static.igem.org/mediawiki/2018/d/d6/T--Uppsala--p2e.png">                            <div class="inner-card-text">  

                                <p><strong>Figure 5</strong> Titered Eluate: Panning 2</p>

                             <img class="content-card-img" id="adjusted-img" style="border:0px" src="https://static.igem.org/mediawiki/2018/2/25/T--Uppsala--p3e.png">

<div class="inner-card-text">  

<p> <!-- Paste your content --> <strong>Figure 6:</strong> Titered Eluate: Panning 3 </p>

</div>

<div>

<p><strong>Table 1:</strong> Titering results. *Negative Panning for Interactions with the Tube were Performed in Conjugate with Regular Panning.<img src="https://static.igem.org/mediawiki/2018/4/40/T--Uppsala--titertable.png" alt="Titering table" class="center" height="50%" width="50%">  

                         <div class="side-img"">

                          <!-- Here goes the big image to the right -->  

                          <img src=https://static.igem.org/mediawiki/2018/3/39/T--Uppsala--phageelisa_small.jpg>

<p><strong>Figure 7:</strong>Negative control of phage ELISA, containing ONLY monoclonal M13 antibodies and NO phage.</p>   

                  

                     <div><h2>Sequenced Samples and Computational Analysis</h2></div>

    <tbody>

        <tr>

            <td>Series 1

            </td>

            <td>EF01122224

            </td>

            <td>TP<span class="blue-letter">I</span>FL<span class="red-letter">P</span>TPA<span class="blue-letter">Q</span>EH---

            </td>

            <td> Yes

            </td>

            <td> No

            </td>

            <td> No

            </td>

        </tr>

            <td>Series 3

            </td>

            <td>EF01122218

            </td>

            <td>---FS<span class="red-letter">P</span>TQA<span class="blue-letter">N</span>T<span class="blue-letter">IH</span>RW

            </td>

            <td>No

            </td>

            <td>No

            </td>  

            <td>No

            </td>

    </tr>

</table>

<!-- The four columns -->

<div class="row">

    <img class="galleryimg" src="https://static.igem.org/mediawiki/2018/4/43/T--Uppsala--peptide_upload_17.png" alt="EF01122217" style="width:100%" onclick="myFunction(this);">

    <img class="galleryimg" src="https://static.igem.org/mediawiki/2018/5/55/T--Uppsala--peptide_18.png" alt="EF01122218" style="width:100%" onclick="myFunction(this);">

<div class="container">

  <span onclick="this.parentElement.style.display='none'" class="closebtn">&times;</span>

  <img id="expandedImg" style="width:100%">

  <div id="imgtext"></div>

</div>

<script>

    var expandImg = document.getElementById("expandedImg");

    var imgText = document.getElementById("imgtext");

    expandImg.src = imgs.src;

    imgText.innerHTML = imgs.alt;

    expandImg.parentElement.style.display = "block";

</script>

<div>

<h1>Conclusions</h1>

<p>

Due to the fact that our ELISA-test was inconclusive we can not be sure that our peptides are specific binders. However, only one was predicted as a Polysterene binder and none contain any known TUP-sequences. A blastp on Biopanning Databank showed that EF01122224 showed some similarities with an antibody for a pork tapeworm epithelial proteins and EF01122218 for an antibody for rabbit epithelia. The E-values were high, 185  and 10 respectively, but those two would be promising candidates for future studies.  

</p>

</div>

</div>

<div>

<h1>Links</h1>

</div>

<!-------BUTTONS ------->

<div>

<h2>References</h2>

<p>

<b>[1]</b>Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science. 1985;228:1315–1317.

<br>

<b>[2]</b> Arap, Marco Antonio. (2005). Phage display technology: applications and innovations.<a href="https://dx.doi.org/10.1590/S1415-47572005000100001"><i>Genetics and Molecular Biology, 28(1)</i>, 1-9.</a>

<br>

<b>[3]</b>  Qiang, Xu, Keyong Sun, Lijun Xing, Yifeng Xu, Hong Wang, Zhengpin Zhou, Juan Zhang, m.fl. ”Discovery of a Polystyrene Binding Peptide Isolated from Phage Display Library and Its Application in Peptide Immobilization”. Scientific Reports 7, nr 1 (december 2017). <a href="https://doi.org/10.1038/s41598-017-02891-x">https://doi.org/10.1038/s41598-017-02891-x</a>.

<br>