Team:Warwick/Parts

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             <li> <a href = "/Team:Warwick/Project"> PROJECT </a> </li>
             <li> <a href = "/Team:Warwick/Project"> PROJECT </a> </li>
             <li> <a href = "/Team:Warwick/Team"> TEAM </a> </li>
             <li> <a href = "/Team:Warwick/Team"> TEAM </a> </li>
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             <li> <a href = "/Team:Warwick/Parts"> PARTS </a> </li>
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             <li> <a href = "/Team:Warwick/Parts"> <span> PARTS </span> </a> </li>
             <li> <a href = "/Team:Warwick/Modelling"> MODELLING </a> </li>
             <li> <a href = "/Team:Warwick/Modelling"> MODELLING </a> </li>
             <li> <a href = "/Team:Warwick/Notebook"> NOTEBOOK </a> </li>
             <li> <a href = "/Team:Warwick/Notebook"> NOTEBOOK </a> </li>
             <li> <a href = "/Team:Warwick/Human"> POLICY & PRACTICES </a> </li>
             <li> <a href = "/Team:Warwick/Human"> POLICY & PRACTICES </a> </li>
             <!--<li> <a href = "/Team:Warwick/Measurements"> MEASUREMENTS </a> </li>-->
             <!--<li> <a href = "/Team:Warwick/Measurements"> MEASUREMENTS </a> </li>-->
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             <li> <a href = "/Team:Warwick/Interlab"> <span> INTERLAB </span> </a> </li>
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             <li> <a href = "/Team:Warwick/Interlab"> INTERLAB </a> </li>
             <li> <a href = "/Team:Warwick/Attributions"> ATTRIBUTIONS </a> </li>
             <li> <a href = "/Team:Warwick/Attributions"> ATTRIBUTIONS </a> </li>
             <li> <a href = "/Team:Warwick/Judging"> JUDGING </a> </li>
             <li> <a href = "/Team:Warwick/Judging"> JUDGING </a> </li>
         </div>
         </div>
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<div id="secondaryMenu">
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<li> <a href = "/Team:Warwick/Parts/Aptazyme"> APTAZYME </a> </li>
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<li> <a href = "/Team:Warwick/Parts/IRES"> IRES </a> </li>
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<li> <a href = "/Team:Warwick/Parts/sirna"> siRNA </a> </li>
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<li> <a href = "/Team:Warwick/Parts/Neomycin"> NEOMYCIN </a> </li>
 +
<li> <a href = "/Team:Warwick/Parts/T7"> T7 </a> </li>
 +
<li> <a href = "/Team:Warwick/Parts/RdRp"> RdRp </a> </li>
 +
<li> <a href = "/Team:Warwick/Parts/P2a"> P2A </a> </li>
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<li> <a href = "/Team:Warwick/Parts/MS2"> MS2 </a> </li>
 +
<li> <a href = "/Team:Warwick/Parts/3promoter"> PROMOTERS </a> </li>
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<li> <a href = "/Team:Warwick/Parts/Testing"> TESTING MODULES </a> </li>
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<li> <a href = "/Team:Warwick/Parts/bb"> EXISTING BIOBRICK </a> </li>
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</div>
         <div id = "pageContentWrapper">
         <div id = "pageContentWrapper">
             <div id = "pageContent">
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             <!-- THIS IS WHERE YOUR MAIN BODY GOES -->
             <!-- THIS IS WHERE YOUR MAIN BODY GOES -->
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<p> Below is an interactive schematic of our system, hover your mouse over any part to see a brief description, and click to follow through to the Registry and see a more in depth description, and information regarding sequences and results. </p>
 +
<div id="pop1" class="popbox">
 +
    <h2>5' Promoter</h2>
 +
    <p>This is derived from the 5i UTR of the HCV virus strain 1b isolate Con1. The secondary structure of this sequence acts as a binding site and initiates replication of the system by RdRp. It also includes the first 16 amino acids of the first gene of HCV as this has been shown to increase the efficacy of binding of the RdRp to the 5' promoter.</p>
 +
</div>
 +
<div id="pop2" class="popbox">
 +
    <h2>IRES</h2>
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    <p>This
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<div id="toc-wrapper" style='width:16%; float:left; position:fixed; text-indent:-21px;'><table id="toc" class="toc" style='width:100%; font-size:12px; color:green;'><tr><td>
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acts as an initiation for eukaryotic
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<ul>
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ribosomes and begins translation of the
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<li style="list-style-type: none;"><a href="#Introduction"><span class="tocnumber"> 1. </span><span class="toctext"> Introduction </span></a></li>
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<li style="list-style-type: none;"><a href="#Timeline"><span class="tocnumber"> 2. </span><span class="toctext"> The experimental timeline </span></a></li>
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-
<li style="list-style-type: none;"><a href="#Protocols"><span class="tocnumber"> 3. </span><span class="toctext"> Protocols and methodology </span></a></li>
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-
<li style="list-style-type: none;"><a href="#Measurements"><span class="tocnumber"> 4. </span><span class="toctext"> Measurements and results </span></a></li>
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-
</ul>
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-
</td></tr></table><script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script></div>
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following protein sequence. We
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<div id="main_content" style='min-height:100%; width: 80%; float: right; border-left:2px black solid; padding-left:10px'>
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compared two different IRESs: the
 +
classical EMCV IRES used in many papers,which has been shown to be compatible
-
<h1 id="Introduction"> Introduction </h1><br><br>
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with replicons and the NKRF IRES derived from the 3’UTR of
-
<p>
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the mammalian NF-kappaB repressing
-
The interlab study is an attempt by iGEM HQ to conduct a comparative analysis of the different methods employed by the varied and diverse teams internationally to arrive at useful data. A key problem in science is ascertaining absolute measurements; there is no point in one measuring the fluorescence of some given part, only to arrive at arbitrary units whose meaning to other scientists is near zero. Standards must be set, in order to embue our results with any useful meaning. The interlab study is a step towards that ultimate goal of blanket standardisation in the synthetic biological context.
+
-
</p><br><br>
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-
<p>
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factor, which we also chose to test because during
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It is suggested that a team conduct the interlab study as a preamble to the main event. However, we are the first example of an iGEM team at Warwick, and coalesced rather late in the day. Hence we decided just to dedicate three members of the team to pursuing it in parallel to our other endeavours, as a side quest. It was primarily undertaken by <b> Dan Goss </b>, <b> Waqar Yousaf </b> and <b> Chelsey Tye </b>, with support from advisers <b> Will Rostain </b> and <b> Sian Davies </b>. All consent is given in accordance with the <a href="http://www.wtfpl.net/"> WTFPL </a> license!
+
-
</p><br><br>
+
-
<h1 id="Timeline"> The experimental timeline </h1><br><br>
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investigation regarding the efficacy and
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<p> The remit of the interlab study boils down to constructing and characterising, albeit minimally, three devices. They share a lot of similarities, and the objective is obviously not to create some wacky new form of life, but to measure well characterised and well understood parts in order to measure the measuring equipment, as it were. </p>
+
strength of the EMCV IRES, the NKRF derived
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<p> Therefore, over a period of about 1 month, we engineered the three devices from the brief via transformation, miniprep, digestion, ligation, and all the protocols you would expect (more on that below). What follows is a timetable of our experimental work in the wet lab: </p>
+
IRES was shown to be 30-fold more  
-
<table style="width:100%">
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efficient however had never been previously used in Huh7.</p>
 +
</div>
 +
<div id="pop3" class="popbox">
 +
    <h2>MS2 Box</h2>
 +
    <p>The MS2 box acts as a binding
-
<tr>
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site for the MS2 coat protein, formed
-
<th> Date </th>
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-
<th> Protocols and measurements </th>
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-
</tr>
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-
<tr>
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downstream in the replicon, which
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<td> 05/08/2014 </td>
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-
<td> <b> Transformed </b> all parts from kit plates, including an <a href="http://parts.igem.org/Part:BBa_J04450"> RFP-producing control </a> </td>
+
-
</tr>
+
-
<tr>
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represses translation hence preventing
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<td> 06/08/2014 </td>
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-
<td> <b> Isolated/inoculated </b> one colony from each and and grew them up overnight </td>
+
-
</tr>
+
-
<tr>
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exponential growth of the replicon.</p>
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<td> 07/08/2014 </td>
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</div>
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<td> <b> Miniprepped </b> the overnights to secure sufficient plasmid DNA for digest </td>
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<div id="pop4" class="popbox">
-
</tr>
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    <h2>Neomycin Resistance</h2>
 +
    <p>This was included in order to select
-
<tr>
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for the cells which had been
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<td> 08/08/2014 </td>
+
-
<td> <b> Restriction digested </b> parts and linearised plasmid backbones for assembly </td>
+
-
</tr>
+
-
<tr>
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successfully transfected with the
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<td> 08/08/2014 </td>
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-
<td> <b> Ligated </b> digested parts to produce devices 2 and 3 </td>
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-
</tr>
+
-
<tr>
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replicon. This is commonly used in
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<td> 08/08/2014 </td>
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-
<td> <b> Transformed </b> ligation products over weekend </td>
+
-
</tr>
+
-
<tr>
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human cell studies and allows survival
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<td> 11/08/2014 </td>
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-
<td> <b> Inoculated </b> colonies of ligation products </td>
+
-
</tr>
+
-
<tr>
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of eukaryotic cells in the prescence of
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<td> 12/08/2014 </td>
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-
<td> <b> Miniprepped </b> ligation products to ascertain plasmid DNA of devices 2/3 </td>
+
-
</tr>
+
-
<tr>
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geneticin.</p>
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<td> 12/08/2014 </td>
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</div>
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<td> <b> Gel electrophoresis </b> assay of these devices, with positive results </td>
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<div id="pop5" class="popbox">
-
</tr>
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    <h2>Aptazyme</h2>
 +
    <p>This is an RNA enzyme which self-
-
<tr>
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cleaves in the presence of
-
<td> ... </td>
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-
<td> Hiatus period (focusing on other work!) </td>
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-
</tr>
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-
<tr>
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theophylline. Theophylline is not
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<td> 20/08/2014 </td>
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-
<td> <b> Transformation </b> of all devices from plasmid DNA for measurement </td>
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-
</tr>
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-
<tr>
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endogenous to mammalian cells
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<td> 21/08/2014 </td>
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-
<td> <b> Inoculated </b> colonies of each device (three biological replicates for each) </td>
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-
</tr>
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-
<tr>
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hence acts as a selective “off-switch”
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<td> 22/08/2014 </td>
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-
<td> <b> Refreshed </b> cultures in M9 minimal media in the morning </td>
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-
</tr>
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-
<tr>
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in an instance such as, Hepatitis C
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<td> 22/08/2014 </td>
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-
<td> <b> Measured optical density and fluorescence </b> with plate reader overnight </td>
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-
</tr>
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-
<tr>
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infection or tumour formation which
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<td> 25/08/2014 </td>
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<td> <b> Collected </b> data from plate reader, but gain was set to 100 so had to repeat </td>
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-
</tr>
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-
<tr>
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is exacerbated by lack of DPP-IV.</p>
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<td> 26/08/2014 </td>
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</div>
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<td> <b> Inoculated </b> colonies of each device (three biological replicates for each) </td>
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<div id="pop6" class="popbox">
-
</tr>
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    <h2>siRNA</h2>
 +
    <p>This is the functional element of our replicon and shows a
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<tr>
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potential area for RNA experimentation. This sequence was
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<td> 27/08/2014 </td>
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-
<td> <b> Refreshed </b> cultures in M9 minimal media in the morning </td>
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-
</tr>
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-
<tr>
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specifically designed using RNA fold and investigating the minimum
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<td> 27/08/2014 </td>
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-
<td> <b> Measured optical density and fluorescence </b> with plate reader overnight </td>
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-
</tr>
+
-
<tr>
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free energy bond formation, in order to form a secondary structure
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<td> 28/08/2014 </td>
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<td> <b> Collected </b> data from plate reader and imported into Excel for analysis </td>
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-
</tr>
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-
<tr>
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in the positive sense that would not include a length of double
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<td> 29/08/2014 </td>
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-
<td> The end! </td>
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-
</tr>
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-
</table>
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stranded RNA longer than 16 bases and hence would not be
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<h1 id="Protocols"> Protocols and methodology </h1>
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recognised by Dicer and our replicon would remain intact for further
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<p> Many of the protocols mentioned above which we used were harvested straight from the iGEM website, but we also used content from previous iGEM teams and instructions packaged with kits. The specific materials and procedures can be accessed through clicking the relevant hyperlink: </p> <br>
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replication. However, in the negative sense the secondary structure
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<p>
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will form a hairpin recognised by Dicer which will result in cleavage
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<ul>
+
-
<li> <a href="http://parts.igem.org/Help:Protocols/Transformation">Transformation </a> </li>
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<li> <a href="http://parts.igem.org/Help:3A_Assembly_Kit/Growing">Growing (which I refer to mostly as inoculation)</a> </li>
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-
<li> <a href="http://www.qiagen.com/gb/resources/resourcedetail?id=331740ca-077f-4ddd-9e5a-2083f98eebd5&lang=en">Miniprep </a></li>
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-
<li> <a href="http://parts.igem.org/Help:Protocols/Restriction_Digest">Restriction digest </a></li>
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-
<li> <a href="http://parts.igem.org/Help:Protocols/Ligation">Ligation </a></li>
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-
<li> <a href="https://2010.igem.org/Team:Newcastle/Gel_electrophoresis">Gel electrophoresis </a></li>
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-
<li> 3A Assembly basically comprises digestion and ligation </li>
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-
</ul>
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</p>
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<p> To acquire the measurements necessary, we used a microplate reader, rather than a flow cytometer or something similar. To do this, we used the <a href="http://www.tecan.com/platform/apps/product/index.asp?MenuID=2575&ID=4890&Menu=1&Item=21.2.10.2"> Tecan Infinite F500 </a>, seen below. </p> <br>
+
and release of the siRNA from the RNA strand allowing it to bind in a  
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<img style = "width:70%;" align="middle" src="https://static.igem.org/mediawiki/2014/2/2f/Warwick_Interlab_Tecan.jpg"> <br>
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complimentary fashion to the 3’UTR of DPP-IV mRNA and cause RNA
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<p> This machine is very flexible, supporting various different plate sizes (from 6- to 1536-well plates!) and various modes of measurement (supporting absorbance wavelength from 230-1000nm and excitation wavelengths from 230-900nm). It is configured in to take the readings we were after using the following setup: </p> <br>
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silencing hence reducing the amount of DPP-IV protein present in the  
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<p>
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cell.  We used siRNA sequences from various papers to design this
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<ul style="float:none;">
+
-
<li> 96 well plate (see diagram below) </li>
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-
<li> Temperature: 37 degrees Celsius </li>
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-
<li> Absorbance measurement wavelength: 600nm (OD) </li>
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-
<li> Excitation wavelength: 465nm (GFP) </li>
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-
<li> Emission wavelength: 530nm (GFP) </li>
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-
<li> GFP gain: 35 </li>
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-
</ul>
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-
</p> <br>
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<p> We then ran the programme ‘GFPandOD_overnight’, for which one cycle runs like so: </p><br><br>
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hairpin.</p>
 +
</div>
 +
<div id="pop7" class="popbox">
 +
    <h2>MS2 Coat Protein</h2>
 +
    <p>This coding sequence forms
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<p>
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a protein which binds to the MS2 box, hairpin
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<ol style="float:none;">
+
-
<li> Orbital shaking (amplitude 2.5mm, frequency 246.7rpm) for 200s </li>
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<li> Wait for 20s </li>
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<li> Repeat shaking for 200s </li>
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<li> Wait for 2s </li>
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<li> Repeat shaking for 200s </li>
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<li> Take measurements (takes about four minutes)  </li>
+
-
</ol>
+
-
</p> <br>
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<p> This whole process takes 15 minutes per cycle, and would run 80 cycles, coming to 20 hours, unless interrupted. Generally the optical density (that is, the growth of cells) has maxed out earlier than that. I stopped my measurements after 14 hours because growth had reached a plateau. </p><br>
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type structure, and represses translation of the  
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<p> In terms of the protocol for preparing the samples and the plate for the reader, the first step was to concoct some M9 minimal media in which to refresh inoculated overnights before putting them into the plate reader. For 200ml of M9, combine the ingredients given, paying attention to amounts, concentrations and preparation advice given, and make up with H2O: </p><br>
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replicon.</p>
 +
</div>
 +
<div id="pop8" class="popbox">
 +
    <h2>P2A</h2>
 +
    <p>This is derived from the Porcine
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<p>
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Teschovirus-1 genome and cleaves with high
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<ul style="float:none;">
+
-
<li> 20μl, 1M CaCl2 (A) </li>
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<li> 400μl, 1M MgSO4 (A) </li>
+
-
<li> 200μl, 10mM FeSO4¬ (F) </li>
+
-
<li> 40ml, 5x M9 salts (A) </li>
+
-
<li> 3.2ml, 50% glycerol  (A) </li>
+
-
<li> 8ml, casamino acids 5% (A) </li>
+
-
<li> 20μl, 10mg/ml thiamine (F) </li>
+
-
<li> 2ml, 2mg/ml uracil (F) </li>
+
-
<li> 2ml 3mg/ml leucine (F) </li>
+
-
<li> 50μl, 8-10M pH 7.4 NaOH </li>
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-
</ul>
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-
</p> <br>
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<p> <b> NB. </b> Here <b> A </b> = autoclaved and <b> F </b> = filter sterilised. </p><br>
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efficiency and leaving no scar following the
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<p> The final solution should be 7.4pH, so usually best to make up with water, and then add NaOH carefully.
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formation of a polyprotein MS2 coat protein,  
-
Now that you have M9, the protocol is quite simple: </p><br>
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-
<p>
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P2A and RdRp. This is required as the HCV
-
<ol style="float:none;">
+
-
<li> Inoculate three colonies (biological replicates) from each device overnight in 5ml LB broth, as given in the above ‘growing’ protocol </li>
+
-
<li> We need to refresh the samples in M9. First transfer broth to falcon tubes and spin down </li>
+
-
<li> Pipette away or decant the supernatant and resuspend the pellet in M9 </li>
+
-
<li> Prepare 1:50 concentration of sample in 1ml of M9 </li>
+
-
<li> Put refreshed samples in a shaking incubator at 37 degrees Celsius for six hours </li>
+
-
<li> Retrieve samples, get a 96 well plate. Any given well should contain 10μl of sample solution and be made up to 200μl with M9. Prepare three wells per sample (technical replicates) </li>
+
-
<li> In the top row, put 200μl of M9 in every well; these act as the blanks </li>
+
-
<li> Where possible, avoid putting samples in wells along the boundary of the plate </li>
+
-
<li> Into all unused wells, pipette 200μl of distilled water </li>
+
-
<li> Put the plate into the machine and run the programme defined above </li>
+
-
</ol>
+
-
</p> <br>
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-
<p> Hence if my devices are D1, D2 and D3, biological replicates denoted by A, B and C, and technical replicates denoted by #1, #2 and #3, then the table below describes exactly my 96 well plate: </p><br>
+
genome which has one open reading frame
-
<img style = "width:70%;" align="middle" src="https://static.igem.org/mediawiki/2014/4/4a/Warwick_Interlab_Plate.jpg"><br>
+
from which one long polyprotein is produced.  
-
<p> The point of three technical replicates is that it is easy to spot anomalies – they are the odd ones out. With regards to optical density, most samples were ending up at around 1, so to eliminate any anomalous data I used a difference threshold of 20% of this. That is, if any member of a triplet of technical replicates was more than 0.2 away from both other values at end of play, that datum would be excluded. This test led to no exclusions. </p>
+
RdRp is the final protein in this polygenic
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<p> Then I considered instead the GFP measurements, and applied a similar protocol, using a 20% difference threshold specific to the datum being considered. If any overflowing was registered, as was the case for many incarnations of device 1, I considered the last full row of data, before any overflowing took place. In this fashion, I excluded from my data set D3 A #3, D3 B #1 and D3 C #1. Excluding these values prior to taking averages allowed me to then consider, by comparison of the final averages for each set of three technical replicates (unless one had been excluded), whether the inclusion of any biological replicates should be disputed. This analysis led me to discard all data related to device 3 (D3) since none of its three final averages were compatible with each other by my 20% test. I therefore held off calculating the standard deviation of these data. </p><br>
+
mRNA and hence does not have a start codon,  
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<p> Any excluded data has a red background on the <b> Excel databook </b>, which can be found <a href"https://static.igem.org/mediawiki/2014/e/ef/Warwick_Interlab_Measurements.xls"> here </a>. </p><br>
+
adding a start codon could be disastrous for
-
<p> In terms of controls, he M9 blanks provided a baseline of optical density of about 0.07. Anything over that was contributed by the cells in the sample. The blanks were not significantly different from one another, nor had any given blank changed significantly over time. This consistency and display of constant values proved the M9 to not be contaminated. </p>
+
the function of RdRp.</p>
 +
</div>
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<div id="pop9" class="popbox">
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    <h2>RdRp</h2>
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    <p>This is a polymerase derived from Hepatitis C Virus Strain
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<p> The two quantities we measures were the intensity of green light produced by the sample upon excitation by the plate reader (that is, green fluorescence), and the optical density (i.e. absorbance) of the sample. </p>
+
1b isolate Con1 which catalyses the replication of RNA from an RNA template, reffered to as NS5B in
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<p> Timewise, a full run with the above settings would be 20 hours, but I ran it only for 14 hours with good results. and costwise, this is mainly a question of capital outlay. A used Tecan machine alone sets you back $10,000. But no iGEM team would have to fork out for this. Their costs are primarily the M9 materials, several of which are commonplace in most labs or cheap otherwise, and the well plates. One 96-well microtitre plate can set you back $50 or so, so this is probably the most expensive cost associated with repeated plate reading. </p>
+
the contact of the HCV genome. Heterelogous expression of NS5B has been achieved in insect and  
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<p> There were practical limits to the possible measurements. Preparation of the samples to put in the machine takes a good 24 hours, and the machine itself needs to run for a minimum of 10 or 12 hours with this configuration. It can also only handle one plate at once, although of course you could have more wells (although generally this sends the preparation time soaring accordingly). So you can maximally test 96x5=480 samples a week, and it would take a big chunk of your time. Expertise with the software is also required. </p>
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bacterial hosts, with RNA-dependent RNA synthesis initiated de novo (Behrens et al., 1996; Lohmann
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<p> <b> Optical Density </b> </p>
+
et al., 1997). Structural studies indicate the hydrophobic C-terminal 21 amino acid residues cause
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<p> Units: The optical density or absorbance is the logarithmic ratio of light incident on a material to light which penetrates the material. It therefore has no units. </p>
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insertion into the membrane with other intracellular protein-protein interactions implicated (Moradpour
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<p> Precision: As a logarithmic ratio, OD is always greater than zero. The Tecan Infinite F500 specification says the full range is 0-4. Using the output data, we can determine that the Tecan is precise to 6 significant figures, although this seems to stretch to 7 s.f. when OD is in excess of 1. </p>
+
et al., 2004) making the final 63 bases essential for HCV RNA replication in eukaryotic cells
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<p> Accuracy: Containing as it does a monochromator rather than filters, the F500 does not require calibration. </p>
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(Moradpour et al., 2004). In prokaryotic cells the final 21 amino acids are expendable. RdRp initiates
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<p> <b> Fluorescence </b> </p>
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viral RNA synthesis with nucleotidyl transfer activity found within palm motifs A and C, with several
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<p> Units: The fluorescence measurements are relative rather than absolute, without units. The blanks, containing no GFP, serve as a reference from which to consider all other measurements. </p>
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amino acid residues implicated in nucleotide triphosphate contact (Bressanelli et al., 2002). NS5B
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<p> Precision: Again by observing the data gathered by the Tecan, it seems to be precise to 7 s.f, but across the whole range this time. All my data sat within the wide range 1400 – 65000. </p>
+
activity has been demonstrated ''in vitro'', with synthesis of full length HCV RNA (Lohmann et al.,  
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<p> Accuracy: As before, this machine does not require calibration. </p>
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1997; Ferrari et al., 1999). 5’ and 3’ untranslated regions (UTRs) of the HCV genome contains
-
<h1 id="Measurements"> Measurements and results </h1>
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ordered RNA structures, which are evolutionary conserved and contain crucial cis-acting elements for
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<p> Please see the accompanying <a href="https://static.igem.org/mediawiki/2014/e/ef/Warwick_Interlab_Measurements.xls">Excel spreadsheet </a> for all the measurements proper. It includes annotations, and identifiers for each column of data that follow the same naming scheme as in the well plate diagram above. Also included is the .asc file and equivalent text file containing the data in its original format. </p> <br>
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viral RNA replication. 150 nt in the 3’ termini of HCV RNA contains elements which are essential for  
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<p> The directly measured quantities are fluorescence and OD. We derived various quantities from the data, including OD/time, fluorescence/time, and fluorescence/OD. Much of the data for this is in the ‘Derived Quantities’ worksheet contained within the Excel document. And the relevant graphs are given below for devices 1 and 2, as well as in the Excel document. The exclusion of device 3 is explained in Section II. </p> <br>
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RdRp binding and replication of viral RNA (Cheng et al., 1999; Yi and Lemon, 2003).</p>
 +
</div>
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<div id="pop10" class="popbox">
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    <h2>3' Promoter</h2>
 +
    <p>Each unique RNA dependent RNA polymerase (RdRP) initiates de novo replication of a RNA strand by interacting with RdRP-specific RNA sequences, henceforth called RdRP/RNA promoters. The RdRP chosen for our project is taken from the Hepatitis C virus (HCV) and it recognizes a limited set of such sequences. All of them possess a few common characteristics: an initiation cytidylate at the 3’ end, where the replication starts; and a stable secondary structure – single stranded tail and a stem of various length. For our project in addition to the indigenous to HCV RdRP promoters, we designed alternative promoter sequences previously identified by Heinz et. Al. as templates for replication by the HCV RdRP.</p>
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<img style="width:70%; float:none;" align="middle" src="https://static.igem.org/mediawiki/2014/2/2b/Warwick_Interlab_ODTime.jpg"> <br>
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<a href="/Team:Warwick/Parts/3promoter" class="popper" data-popbox="pop1"><img src="https://static.igem.org/mediawiki/2014/7/7f/5%27_promoter-01-01.png" width="100%"></a>
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</td>
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<a href="/Team:Warwick/Parts/IRES" class="popper" data-popbox="pop2"><img src="https://static.igem.org/mediawiki/2014/0/01/IRES-01.png" width="100%"></a>
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<a href="/Team:Warwick/Parts/MS2" class="popper" data-popbox="pop3"><img src="https://static.igem.org/mediawiki/2014/5/50/MS2_box-01.png" width="100%"></a>
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<a href="/Team:Warwick/Parts/Neomycin" class="popper" data-popbox="pop4"><img src="https://static.igem.org/mediawiki/2014/6/6c/Neomycin_Resistance-01.png" width="100%"></a>
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<a href="/Team:Warwick/Parts/Aptazyme" class="popper" data-popbox="pop5"><img src="https://static.igem.org/mediawiki/2014/d/d7/Aptazyme-01.png" width="100%"></a>
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<a href="/Team:Warwick/Parts/sirna" class="popper" data-popbox="pop6"><img src="https://static.igem.org/mediawiki/2014/0/0c/SiRNA-01.png" width="100%"></a>
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<a href="/Team:Warwick/Parts/IRES" class="popper" data-popbox="pop2"><img src="https://static.igem.org/mediawiki/2014/f/fa/IRES_2-01.png" width="100%"></a>
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<a href="/Team:Warwick/Parts/MS2" class="popper" data-popbox="pop7"><img src="https://static.igem.org/mediawiki/2014/a/a5/MS2_coat_protein-01.png" width="100%"></a>
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<a href="/Team:Warwick/Parts/P2a" class="popper" data-popbox="pop8"><img src="https://static.igem.org/mediawiki/2014/c/c5/P2A-01.png" width="100%"></a>
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<a href="/Team:Warwick/Parts/RdRp" class="popper" data-popbox="pop9"><img src="https://static.igem.org/mediawiki/2014/3/38/RdRp-01.png" width="100%"></a>
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<a href="/Team:Warwick/Parts/3promoter" class="popper" data-popbox="pop10"><img src="https://static.igem.org/mediawiki/2014/c/c7/3%27_promoter-01.png" width="100%"></a>
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Revision as of 22:34, 17 October 2014

Below is an interactive schematic of our system, hover your mouse over any part to see a brief description, and click to follow through to the Registry and see a more in depth description, and information regarding sequences and results.

5' Promoter

This is derived from the 5i UTR of the HCV virus strain 1b isolate Con1. The secondary structure of this sequence acts as a binding site and initiates replication of the system by RdRp. It also includes the first 16 amino acids of the first gene of HCV as this has been shown to increase the efficacy of binding of the RdRp to the 5' promoter.

IRES

This acts as an initiation for eukaryotic ribosomes and begins translation of the following protein sequence. We compared two different IRESs: the classical EMCV IRES used in many papers,which has been shown to be compatible with replicons and the NKRF IRES derived from the 3’UTR of the mammalian NF-kappaB repressing factor, which we also chose to test because during investigation regarding the efficacy and strength of the EMCV IRES, the NKRF derived IRES was shown to be 30-fold more efficient however had never been previously used in Huh7.

MS2 Box

The MS2 box acts as a binding site for the MS2 coat protein, formed downstream in the replicon, which represses translation hence preventing exponential growth of the replicon.

Neomycin Resistance

This was included in order to select for the cells which had been successfully transfected with the replicon. This is commonly used in human cell studies and allows survival of eukaryotic cells in the prescence of geneticin.

Aptazyme

This is an RNA enzyme which self- cleaves in the presence of theophylline. Theophylline is not endogenous to mammalian cells hence acts as a selective “off-switch” in an instance such as, Hepatitis C infection or tumour formation which is exacerbated by lack of DPP-IV.

siRNA

This is the functional element of our replicon and shows a potential area for RNA experimentation. This sequence was specifically designed using RNA fold and investigating the minimum free energy bond formation, in order to form a secondary structure in the positive sense that would not include a length of double stranded RNA longer than 16 bases and hence would not be recognised by Dicer and our replicon would remain intact for further replication. However, in the negative sense the secondary structure will form a hairpin recognised by Dicer which will result in cleavage and release of the siRNA from the RNA strand allowing it to bind in a complimentary fashion to the 3’UTR of DPP-IV mRNA and cause RNA silencing hence reducing the amount of DPP-IV protein present in the cell. We used siRNA sequences from various papers to design this hairpin.

MS2 Coat Protein

This coding sequence forms a protein which binds to the MS2 box, hairpin type structure, and represses translation of the replicon.

P2A

This is derived from the Porcine Teschovirus-1 genome and cleaves with high efficiency and leaving no scar following the formation of a polyprotein MS2 coat protein, P2A and RdRp. This is required as the HCV genome which has one open reading frame from which one long polyprotein is produced. RdRp is the final protein in this polygenic mRNA and hence does not have a start codon, adding a start codon could be disastrous for the function of RdRp.

RdRp

This is a polymerase derived from Hepatitis C Virus Strain 1b isolate Con1 which catalyses the replication of RNA from an RNA template, reffered to as NS5B in the contact of the HCV genome. Heterelogous expression of NS5B has been achieved in insect and bacterial hosts, with RNA-dependent RNA synthesis initiated de novo (Behrens et al., 1996; Lohmann et al., 1997). Structural studies indicate the hydrophobic C-terminal 21 amino acid residues cause insertion into the membrane with other intracellular protein-protein interactions implicated (Moradpour et al., 2004) making the final 63 bases essential for HCV RNA replication in eukaryotic cells (Moradpour et al., 2004). In prokaryotic cells the final 21 amino acids are expendable. RdRp initiates viral RNA synthesis with nucleotidyl transfer activity found within palm motifs A and C, with several amino acid residues implicated in nucleotide triphosphate contact (Bressanelli et al., 2002). NS5B activity has been demonstrated ''in vitro'', with synthesis of full length HCV RNA (Lohmann et al., 1997; Ferrari et al., 1999). 5’ and 3’ untranslated regions (UTRs) of the HCV genome contains ordered RNA structures, which are evolutionary conserved and contain crucial cis-acting elements for viral RNA replication. 150 nt in the 3’ termini of HCV RNA contains elements which are essential for RdRp binding and replication of viral RNA (Cheng et al., 1999; Yi and Lemon, 2003).

3' Promoter

Each unique RNA dependent RNA polymerase (RdRP) initiates de novo replication of a RNA strand by interacting with RdRP-specific RNA sequences, henceforth called RdRP/RNA promoters. The RdRP chosen for our project is taken from the Hepatitis C virus (HCV) and it recognizes a limited set of such sequences. All of them possess a few common characteristics: an initiation cytidylate at the 3’ end, where the replication starts; and a stable secondary structure – single stranded tail and a stem of various length. For our project in addition to the indigenous to HCV RdRP promoters, we designed alternative promoter sequences previously identified by Heinz et. Al. as templates for replication by the HCV RdRP.

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