Difference between revisions of "Team:DTU-Denmark/Description"

 
(64 intermediate revisions by 7 users not shown)
Line 8: Line 8:
 
     <meta charset="utf-8">
 
     <meta charset="utf-8">
 
     <meta name="viewport" content="width=device-width, initial-scale=1">
 
     <meta name="viewport" content="width=device-width, initial-scale=1">
−
    <style>
 
−
        .check {
 
−
            color: #990000;
 
−
        }
 
−
    </style>
 
 
</head>
 
</head>
  
Line 27: Line 22:
 
                 <div class="caption">
 
                 <div class="caption">
 
                     <div class="col-md-5 col-sm-5 col-xs-12 title"> <!-- the approximate max number of characters ~ 400 --> <!-- EDIT -->
 
                     <div class="col-md-5 col-sm-5 col-xs-12 title"> <!-- the approximate max number of characters ~ 400 --> <!-- EDIT -->
−
                         <h1>Title<p class="lead">leader under the title, short introduction. Ubique moderatius efficiantur eum et, dico oporteat recusabo ius cu, pro id modus sadipscing. Maluisset patrioque eum ad, mel eius doctus accommodare eu, minimum deleniti repudiandae mel ea. Noster nostrud diceret sea no. Eos an nullam molestiae signiferumque, vel ne laudem ignota oblique. Duo te luptatum percipitur signiferumque, at dicunt iriure dolorem his.</p></h1>
+
                         <h1>Parts<p class="lead">We have constructed four new basic parts, 4 composite parts, a plasmid backbone and improved two parts made by a previous team. The parts are all a part of our molecular toolbox for protein expression in <i>Yarrowia lipolytica</i>. </p></h1>
 
                     </div>
 
                     </div>
 
                     <div class="col-md-2 col-sm-2 hidden-xs space"></div>
 
                     <div class="col-md-2 col-sm-2 hidden-xs space"></div>
 
                     <div class="col-md-5 col-sm-5 hidden-xs intro"> <!-- will be hidden on phones, duplicate the text to blockquote down below first section header, to show it there, when it dissapear-->
 
                     <div class="col-md-5 col-sm-5 hidden-xs intro"> <!-- will be hidden on phones, duplicate the text to blockquote down below first section header, to show it there, when it dissapear-->
 
                         <blockquote class="blockquote-reverse"> <!-- EDIT -->
 
                         <blockquote class="blockquote-reverse"> <!-- EDIT -->
−
                             <p>Quote Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer posuere erat a ante.</p>
+
                             <p>"A great building will never stand if you neglect the small bricks"</p>
−
                             <small>Someone famous in <cite title="Source Title">Source Title</cite></small>
+
                             <small>Ifeanyi Enoch Onuoha<cite title="Source Title"></cite></small>
 
                         </blockquote>       
 
                         </blockquote>       
 
                     </div>
 
                     </div>
Line 49: Line 44:
 
     <div class="col-md-9 col-sm-10 colLeft"> <!-- LEFT -->
 
     <div class="col-md-9 col-sm-10 colLeft"> <!-- LEFT -->
 
         <div><a class="anchor" id="section-1"></a>
 
         <div><a class="anchor" id="section-1"></a>
−
        <h2 class="h2">Section 1</h2>
+
            <h2 class="h2">Parts list</h2>
 
              
 
              
 
             <blockquote class="visible-xs"> <!-- quote from masterhead duplicate -->
 
             <blockquote class="visible-xs"> <!-- quote from masterhead duplicate -->
−
                 <p>Quote Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer posuere erat a ante.</p>
+
                 <p>"A great building will never stand if you neglect the small bricks"</p>
−
                 <small>Someone famous in <cite title="Source Title">Source Title</cite></small>
+
                 <small>Ifeanyi Enoch Onuoha<cite title="Source Title"></cite></small>
 
             </blockquote>
 
             </blockquote>
−
           
+
         
−
            <p>
+
−
                Has ut facer debitis, quo eu agam purto. In eum justo aeterno. Sea ut atqui efficiantur, mandamus deseruisse at est, erat natum cum eu. Quot numquam in vel. Salutatus euripidis moderatius qui ex, eu tempor volumus vituperatoribus has, ius ea ullum facer corrumpit.
+
−
            </p>
+
−
        </div> <!-- /overview-->
+
−
       
+
−
       
+
−
        <div><a class="anchor" id="section-2"></a>
+
−
        <h2 class="h2">Medal Requirements</h2>
+
−
           
+
−
            <h3 class="h3">Bronze</h3>
+
−
           
+
 
             <div class="grid-row">
 
             <div class="grid-row">
−
                 <div class="col-md-1 col-sm-1 col-xs-1">
+
                 <div class="col-md-3 col-sm-3 col-xs-12">
−
                   
+
                     <a href="http://parts.igem.org/Part:BBa_K2117000">BBa_K2117000</a>
−
                     <p><p class="check">&#10003;</p> Tryout blblblblblb</p>
+
 
                 </div>
 
                 </div>
−
                 <div class="col-md-9 col-sm-10 col-xs-12">
+
                 <div class="col-md-9 col-sm-9 col-xs-12">
−
                     <p>We <a href="https://igem.org/Team.cgi?team_id=2117">registered</a> for iGEM, have a great summer, and attend the Giant Jamboree.</p>
+
                     <p>Constitutive TEF1 promoter</p>
 
                 </div>
 
                 </div>
 
             </div> <!-- /grid-row -->
 
             </div> <!-- /grid-row -->
−
           
 
 
              
 
              
 
             <div class="grid-row">
 
             <div class="grid-row">
−
                 <div class="col-md-3 col-sm-2 col-xs-12">
+
                 <div class="col-md-3 col-sm-3 col-xs-12">
−
                     <p>&#10003; Register and attend</p>
+
                     <a href="http://parts.igem.org/Part:BBa_K2117001">BBa_K2117001</a>
 
                 </div>
 
                 </div>
−
                 <div class="col-md-9 col-sm-10 col-xs-12">
+
                 <div class="col-md-9 col-sm-9 col-xs-12">
−
                     <p>We <a href="https://igem.org/Team.cgi?team_id=2117">registered</a> for iGEM, have a great summer, and attend the Giant Jamboree.</p>
+
                     <p>hrGFP codon-optimized for <i>Yarrowia lipolytica</i></p>
 
                 </div>
 
                 </div>
 
             </div> <!-- /grid-row -->
 
             </div> <!-- /grid-row -->
 +
           
 
             <div class="grid-row">
 
             <div class="grid-row">
−
                 <div class="col-md-3 col-sm-2 col-xs-12">
+
                 <div class="col-md-3 col-sm-3 col-xs-12">
−
                     <p>&#10003; Deliverables</p>
+
                     <a href="http://parts.igem.org/Part:BBa_K2117002">BBa_K2117002</a>
 
                 </div>
 
                 </div>
−
                 <div class="col-md-9 col-sm-10 col-xs-12">
+
                 <div class="col-md-9 col-sm-9 col-xs-12">
−
                     <p>Meet all deliverables on the Requirements page (section 3).</p>
+
                     <p>Composite part consisting of the TEF1 promoter and the human proinsulin gene codon-optimized for <i>Y. lipolytica</i></p>
 
                 </div>
 
                 </div>
 
             </div> <!-- /grid-row -->
 
             </div> <!-- /grid-row -->
 +
           
 
             <div class="grid-row">
 
             <div class="grid-row">
−
                 <div class="col-md-3 col-sm-2 col-xs-12">
+
                 <div class="col-md-3 col-sm-3 col-xs-12">
−
                     <p>&#10003; Attribution</p>
+
                     <a href="http://parts.igem.org/Part:BBa_K2117003">BBa_K2117003</a>
 
                 </div>
 
                 </div>
−
                 <div class="col-md-9 col-sm-10 col-xs-12">
+
                 <div class="col-md-9 col-sm-9 col-xs-12">
−
                     <p>Create a page on your team wiki with clear attribution of each aspect of your project. This page must clearly attribute work done by the students and distinguish it from work done by others, including host labs, advisors, instructors, sponsors, professional website designers, artists, and commercial services.</p>
+
                     <p>Human proinsulin codon-optimized for <i>Y. lipolytica</i></p>
 
                 </div>
 
                 </div>
 
             </div> <!-- /grid-row -->
 
             </div> <!-- /grid-row -->
−
            <div class="grid-row">
 
−
                <div class="col-md-3 col-sm-2 col-xs-12">
 
−
                    <p>&#10003; Part</p>
 
−
                </div>
 
−
                <div class="col-md-9 col-sm-10 col-xs-12">
 
−
                    <p>Document at least one new standard BioBrick Part or Device central to your project and submit this part to the iGEM Registry (submissions must adhere to the iGEM Registry guidelines). You may also document a new application of a BioBrick part from a previous iGEM year, adding that documentation to the part main page.</p>
 
−
                </div>
 
−
            </div> <!-- /grid-row -->
 
−
           
 
−
           
 
 
              
 
              
−
            <h3 class="h3">Silver</h3>
 
 
             <div class="grid-row">
 
             <div class="grid-row">
−
                 <div class="col-md-3 col-sm-2 col-xs-12">
+
                 <div class="col-md-3 col-sm-3 col-xs-12">
−
                     <p>&#10003; Validated Part</p>
+
                     <a href="http://parts.igem.org/Part:BBa_K2117004">BBa_K2117004</a>
 
                 </div>
 
                 </div>
−
                 <div class="col-md-9 col-sm-10 col-xs-12">
+
                 <div class="col-md-9 col-sm-9 col-xs-12">
−
                     <p>Experimentally validate that at least one new BioBrick Part or Device of your own design and construction works as expected. Document the characterization of this part in the Main Page section of that Part’s/Device’s Registry entry. Submit this new part to the iGEM Parts Registry. This working part must be different from the part documented in bronze medal criterion #4.</p>
+
                     <p>SCR1'-tRNA promoter expressing sgRNA</p>
 
                 </div>
 
                 </div>
 
             </div> <!-- /grid-row -->
 
             </div> <!-- /grid-row -->
 +
         
 
             <div class="grid-row">
 
             <div class="grid-row">
−
                 <div class="col-md-3 col-sm-2 col-xs-12">
+
                 <div class="col-md-3 col-sm-3 col-xs-12">
−
                     <p>&#10003; Collaboration</p>
+
                     <a href="http://parts.igem.org/Part:BBa_K2117005">BBa_K2117005</a>
 
                 </div>
 
                 </div>
−
                 <div class="col-md-9 col-sm-10 col-xs-12">
+
                 <div class="col-md-9 col-sm-9 col-xs-12">
−
                     <p>Convince the judges you have helped any registered iGEM team from high school, a different track, another university, or another institution in a significant way by, for example, mentoring a new team, characterizing a part, debugging a construct, modeling/simulating their system or helping validate a software/hardware solution to a synbio problem.</p>
+
                     <p>hrGFP expressed by the constitutive TEF1 promoter</p>
 
                 </div>
 
                 </div>
 
             </div> <!-- /grid-row -->
 
             </div> <!-- /grid-row -->
 +
                       
 
             <div class="grid-row">
 
             <div class="grid-row">
−
                 <div class="col-md-3 col-sm-2 col-xs-12">
+
                 <div class="col-md-3 col-sm-3 col-xs-12">
−
                     <p>&#10003; Human Practices</p>
+
                     <a href="http://parts.igem.org/Part:BBa_K2117009">BBa_K2117009</a>
 
                 </div>
 
                 </div>
−
                 <div class="col-md-9 col-sm-10 col-xs-12">
+
                 <div class="col-md-9 col-sm-9 col-xs-12">
−
                     <p>iGEM projects involve important questions beyond the lab bench, for example relating to (but not limited to) ethics, sustainability, social justice, safety, security, and intellectual property rights. Demonstrate how your team has identified, investigated, and addressed one or more of these issues in the context of your project. Your activity could center around education, public engagement, public policy issues, public perception, or other activities (see the human practices hub for more information and examples of previous teams' exemplary work).</p>
+
                     <p>Semi-synthetic shuttle vector pSB1A8YL</p>
 
                 </div>
 
                 </div>
 
             </div> <!-- /grid-row -->
 
             </div> <!-- /grid-row -->
 
              
 
              
−
           
 
−
           
 
−
            <h3 class="h3">Gold</h3>
 
 
             <div class="grid-row">
 
             <div class="grid-row">
−
                 <div class="col-md-3 col-sm-2 col-xs-12">
+
                 <div class="col-md-3 col-sm-3 col-xs-12">
−
                     <p>&#10003; Integrated Human Practices</p>
+
                     <a href="http://parts.igem.org/Part:BBa_K2117010">BBa_K2117010</a>
 
                 </div>
 
                 </div>
−
                 <div class="col-md-9 col-sm-10 col-xs-12">
+
                 <div class="col-md-9 col-sm-9 col-xs-12">
−
                     <p>Expand on your silver medal activity by demonstrating how you have integrated the investigated issues into the design and/or execution of your project.</p>
+
                     <p>Composite part of TEF1 and enhanced YFP </p>
 
                 </div>
 
                 </div>
 
             </div> <!-- /grid-row -->
 
             </div> <!-- /grid-row -->
 +
           
 
             <div class="grid-row">
 
             <div class="grid-row">
−
                 <div class="col-md-3 col-sm-2 col-xs-12">
+
                 <div class="col-md-3 col-sm-3 col-xs-12">
−
                     <p>&#10003; Improve a previous part</p>
+
                     <a href="http://parts.igem.org/Part:BBa_K2117011">BBa_K2117011</a>
 
                 </div>
 
                 </div>
−
                 <div class="col-md-9 col-sm-10 col-xs-12">
+
                 <div class="col-md-9 col-sm-9 col-xs-12">
−
                     <p>Improve the function OR characterization of an existing BioBrick Part or Device and enter this information in the Registry. Please see the Registry help page on how to document a contribution to an existing part. This part must NOT be from your 2016 part number range.</p>
+
                     <p>Composite part of TEF1 and enhanced CFP</p>
 
                 </div>
 
                 </div>
 
             </div> <!-- /grid-row -->
 
             </div> <!-- /grid-row -->
 +
           
 
             <div class="grid-row">
 
             <div class="grid-row">
−
                 <div class="col-md-3 col-sm-2 col-xs-12">
+
                 <div class="col-md-3 col-sm-3 col-xs-12">
−
                     <p>&#10003; Proof of concept</p>
+
                     <a href="http://parts.igem.org/Part:BBa_K2117012">BBa_K2117012</a>
 
                 </div>
 
                 </div>
−
                 <div class="col-md-9 col-sm-10 col-xs-12">
+
                 <div class="col-md-9 col-sm-9 col-xs-12">
−
                     <p>Demonstrate a functional proof of concept of your project. Your proof of concept must consist of a BioBrick device; a single BioBrick part cannot constitute a proof of concept. (biological materials may not be taken outside the lab).</p>
+
                     <p>Improved <a href="http://parts.igem.org/Part:BBa_K530001">BBa_K530001</a> (<em>crtE</em>)</p>
 
                 </div>
 
                 </div>
 
             </div> <!-- /grid-row -->
 
             </div> <!-- /grid-row -->
 +
           
 
             <div class="grid-row">
 
             <div class="grid-row">
−
                 <div class="col-md-3 col-sm-2  col-xs-12">
+
                 <div class="col-md-3 col-sm-3 col-xs-12">
−
                     <p>&#10007; Demonstrate your work</p>
+
                     <a href="http://parts.igem.org/Part:BBa_K2117013">BBa_K2117013</a>
 
                 </div>
 
                 </div>
−
                 <div class="col-md-9 col-sm-10 col-xs-12">
+
                 <div class="col-md-9 col-sm-9 col-xs-12">
−
                     <p>Show your project working under real-world conditions. To achieve this criterion, you should demonstrate your whole system, or a functional proof of concept working under simulated conditions in the lab (biological materials may not be taken outside the lab).</p>
+
                     <p>Improved <a href="http://parts.igem.org/Part:BBa_K530002">BBa_K530002</a>  (<em>crtI</em>)</p>
 
                 </div>
 
                 </div>
 
             </div> <!-- /grid-row -->
 
             </div> <!-- /grid-row -->
−
         </div>
+
                     
−
       
+
         </div> <!-- /overview-->
−
       
+
−
       
+
−
       
+
−
       
+
−
       
+
 
          
 
          
−
       
+
         <div><a class="anchor" id="section-2"></a>
−
       
+
            <h2 class="h2">Improvement and Characterization of Two Existing Parts</h2>
−
       
+
           
−
       
+
−
       
+
−
         <div><a class="anchor" id="section-3"></a>
+
−
        <h2 class="h2">Section 3</h2>
+
 
             <p>
 
             <p>
−
                Has ut facer debitis, quo eu agam purto. In eum justo aeterno. Sea ut atqui efficiantur, mandamus deseruisse at est, erat natum cum eu. Quot numquam in vel. Salutatus euripidis moderatius qui ex, eu tempor volumus vituperatoribus has, ius ea ullum facer corrumpit.
+
            In our laboratory work, we wanted to work with the two BioBricks; <a href="http://parts.igem.org/Part:BBa_K530001">BBa_K530001</a> (<em>crtE</em> gene) and <a href="http://parts.igem.org/Part:BBa_K530002">BBa_K530002</a> (<em>crtI</em> gene) created by the John Hopkins iGEM team 2011. The genes encode the two enzymes Geranylgeranyl Diphosphate Synthase and Phytoene Desaturase, respectively, both from the wildtype strain of <i>xanthophyllomyces dendrorhous</i> used in the biosynthesis of beta-Carotene.
 
             </p>
 
             </p>
−
        </div>
 
  
−
        <div><a class="anchor" id="section-4"></a>
 
−
        <h2 class="h2">Section 4</h2>
 
 
             <p>
 
             <p>
−
                Has ut facer debitis, quo eu agam purto. In eum justo aeterno. Sea ut atqui efficiantur, mandamus deseruisse at est, erat natum cum eu. Quot numquam in vel. Salutatus euripidis moderatius qui ex, eu tempor volumus vituperatoribus has, ius ea ullum facer corrumpit.
+
            When looking into the specifications of these BioBricks, we realized that both genes contained illegal restriction sites.  
 +
            </p>
 +
            <p>
 +
            <a href="http://parts.igem.org/Part:BBa_K530001">BBa_K530001</a> contained an AgeI restriction site making the part incompatible with the RFC25 Freiburg Standard. The RFC25 standard allows for in-frame assembly of protein domains.
 +
            <a href="http://parts.igem.org/Part:BBa_K530002">BBa_K530002</a> contained the illegal restriction site BglII making the part incompatible with the RFC21 Berkely Standard, which enables in-frame assembly of proteins.  
 
             </p>
 
             </p>
−
        </div>
 
  
−
        <div><a class="anchor" id="section-5"></a>
 
−
        <h2 class="h2">Section 5</h2>
 
 
             <p>
 
             <p>
−
                Has ut facer debitis, quo eu agam purto. In eum justo aeterno. Sea ut atqui efficiantur, mandamus deseruisse at est, erat natum cum eu. Quot numquam in vel. Salutatus euripidis moderatius qui ex, eu tempor volumus vituperatoribus has, ius ea ullum facer corrumpit.
+
            We wanted to overcome these obstacle by removing the illegal restriction sites from the above described Biobricks.  
 
             </p>
 
             </p>
−
        </div>
 
  
−
        <div><a class="anchor" id="section-6"></a>
+
            <h3 class="h3">How did we do it?</h3>
−
        <h2 class="h2">Section 6</h2>
+
           
 
             <p>
 
             <p>
−
                Has ut facer debitis, quo eu agam purto. In eum justo aeterno. Sea ut atqui efficiantur, mandamus deseruisse at est, erat natum cum eu. Quot numquam in vel. Salutatus euripidis moderatius qui ex, eu tempor volumus vituperatoribus has, ius ea ullum facer corrumpit.
+
            The illegal restriction sites were removed using site directed mutagenesis with primers containing nucleotide substitutions in the two restriction sites (see Figure 1).
 
             </p>
 
             </p>
−
        </div>
 
  
−
        <div><a class="anchor" id="section-7"></a>
+
 
−
        <h2 class="h2">Section 7</h2>
+
            <figure class="figure">
 +
                <img id="img1" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/9/97/T--DTU-Denmark--sitedirected.png" alt="DESCRIPTION">
 +
                <figcaption class="figure-caption"><strong>Figure 1:</strong> Primers overlapping the restriction sites are designed with a single nucleotide change to disrupt the restriction site. The primers anneal to the template plasmid and replicate while introducing the point mutation. The elongated plasmids are digested with the enzyme DpnI, which cleaves at the methylated sites breaking down the circular template, resulting in a higher transformation efficiency of the linear PCR product. </figcaption>
 +
            </figure>
 +
           
 +
            <!-- The Modal with same picture-->
 +
            <div id="img1Modal" class="modal">
 +
                <span class="close img1">×</span>
 +
                <img class="modal-content" id="img1Img">
 +
                <div class="caption">DESCRIPTION</div>
 +
            </div>
 +
 
 +
 
 +
            <figure class="figure">
 +
                <img id="img2" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/c/cc/T--DTU-Denmark--primers.jpg" alt="DESCRIPTION">
 +
                <figcaption class="figure-caption"><strong>Table 1:</strong> Primers designed for removal of illegal restriction sites. Bold marks the nucleotide  substitution.</figcaption>
 +
            </figure>
 +
           
 +
            <!-- The Modal with same picture-->
 +
            <div id="img2Modal" class="modal">
 +
                <span class="close img2">×</span>
 +
                <img class="modal-content" id="img2Img">
 +
                <div class="caption">DESCRIPTION</div>
 +
            </div>
 +
 
 +
            <p>
 +
            The new plasmid, <a href="http://parts.igem.org/Part:BBa_K2117012">BBa_K2117012</a>, with the removed AgeI restriction site was double digested with the enzyme and SpeI to test if we had successfully removed the AgeI restriction site.
 +
            </p>
 +
 
 +
            <div class="row">
 +
                <div class="col-md-6 col-sm-6">
 +
                    <figure class="figure">
 +
                        <img width="400" id="img3" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/5/55/T--DTU-Denmark--digest1.jpg" alt="DESCRIPTION">
 +
                        <figcaption class="figure-caption"><strong>Figure 2:</strong> AgeI + SpeI digestion. Electrophoresis on a 1% agarose gel showing the digestion of  <a href="http://parts.igem.org/Part:BBa_K2117012">BBa_K2117012</a> (lane 1-3). <a href="http://parts.igem.org/Part:BBa_K530001">BBa_K530001</a>  digested with AgeI + SpeI and undigested <a href="http://parts.igem.org/Part:BBa_K530001">BBa_K530001</a> were used as controls (lane 4+5). </figcaption>
 +
                    </figure>
 +
                               
 +
                        <!-- The Modal with same picture-->
 +
                    <div id="img3Modal" class="modal">
 +
                        <span class="close img3">×</span>
 +
                        <img class="modal-content" id="img3Img">
 +
                        <div class="caption">DESCRIPTION</div>
 +
                    </div>
 +
                </div>
 +
                <div class="col-md-6 col-sm-6"><p style="color:transparent;">    h</p></div>
 +
            </div>
 +
 
 +
 
 
             <p>
 
             <p>
−
                Has ut facer debitis, quo eu agam purto. In eum justo aeterno. Sea ut atqui efficiantur, mandamus deseruisse at est, erat natum cum eu. Quot numquam in vel. Salutatus euripidis moderatius qui ex, eu tempor volumus vituperatoribus has, ius ea ullum facer corrumpit.
+
            The digestion showed the removal of the AgeI restriction site, shown on the gel picture by only one band on the <a href="http://parts.igem.org/Part:BBa_K2117012">BBa_K2117012</a> compared to two bands on the control <a href="http://parts.igem.org/Part:BBa_K530001">BBa_K530001</a>.
 +
            </p>
 +
 
 +
            <p>
 +
            <a href="http://parts.igem.org/Part:BBa_K2117012">BBa_K2117012</a> and <a href="http://parts.igem.org/Part:BBa_K2117013">BBa_K2117013</a>  were sent for sequencing with the verification primers VR and VF2 primers to further verify the removal of the restriction sites.
 +
            </p>
 +
 
 +
            <div class="row">
 +
                <div class="col-md-12">
 +
                    <figure class="figure">
 +
                        <img id="img4" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/9/9c/T--DTU-Denmark--AgeI.JPG" alt="DESCRIPTION">
 +
                        <figcaption class="figure-caption"><strong>Figure 3:</strong> Sequencing alignment of BBa_530001 (top sequence) and  <a href="http://parts.igem.org/Part:BBa_K2117012">BBa_K2117012</a> (bottom sequence). The alignment shows a nucleotide substitution in the AgeI restriction site. </figcaption>
 +
                    </figure>
 +
                   
 +
                    <!-- The Modal with same picture-->
 +
                    <div id="img4Modal" class="modal">
 +
                        <span class="close img4">×</span>
 +
                        <img class="modal-content" id="img4Img">
 +
                        <div class="caption">DESCRIPTION</div>
 +
                    </div>
 +
                </div>           
 +
                           
 +
                <div class="col-md-12">
 +
                    <figure class="figure">
 +
                        <img id="img5" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/a/ad/T--DTU-Denmark--BglII.jpg" alt="DESCRIPTION">
 +
                        <figcaption class="figure-caption"><strong>Figure 4:</strong> Sequencing alignment of BBa_530002 (top sequence) and  <a href="http://parts.igem.org/Part:BBa_K2117013">BBa_K2117013</a> (bottom sequence). The alignment shows a nucleotide substitution in the BglII restriction site. </figcaption>
 +
                    </figure>
 +
                                <!-- The Modal with same picture-->
 +
                    <div id="img5Modal" class="modal">
 +
                        <span class="close img5">×</span>
 +
                        <img class="modal-content" id="img5Img">
 +
                        <div class="caption">DESCRIPTION</div>
 +
                    </div>
 +
                </div>
 +
               
 +
            </div>
 +
 
 +
 
 +
            <p>
 +
                The sequencing results show a substitution in the restriction sites corresponding to the nucleotide substitution designed in the primers (Table 1).
 +
            </p>
 +
 
 +
            <p>
 +
                By deleting the two restriction sites in the <a href="http://parts.igem.org/Part:BBa_K2117012">BBa_K2117012</a> and <a href="http://parts.igem.org/Part:BBa_K2117013">BBa_K2117013</a> we have made the two BioBricks compatible with RFC25 and RFC21 standards, respectively.
 +
            </p>
 +
 
 +
            <h3 class="h3">Improvement of Characterization</h3>
 +
 
 +
            <p>
 +
                While working with <a href="http://parts.igem.org/Part:BBa_K530001">BBa_K530001</a>  and <a href="http://parts.igem.org/Part:BBa_K530002">BBa_K530002</a> we were struggling with assembly of the genes. Therefore, we sequenced the plasmids received in the distribution kit.
 +
            </p>
 +
 
 +
            <p>
 +
                Sequencing results of <a href="http://parts.igem.org/Part:BBa_K530002">BBa_K530002</a> showed nucleotide substitutions in the prefix and a large deletion in the suffix.
 +
            </p>
 +
 
 +
            <figure class="figure">
 +
                <img id="img6" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/b/b5/T--DTU-Denmark--prefix.png" alt="DESCRIPTION">
 +
                <figcaption class="figure-caption"><strong>Figure 5:</strong> Alignment of <a href="http://parts.igem.org/Part:BBa_K530002">BBa_K530002</a> sequence received from the parts page (top) and sequencing results of <a href="http://parts.igem.org/Part:BBa_K530002">BBa_K530002</a> from the distribution kit. Alignment shows many nucleotide substitutions in the prefix seuqence. </figcaption>
 +
            </figure>
 +
           
 +
            <div id="img6Modal" class="modal">
 +
                <span class="close img6">×</span>
 +
                <img class="modal-content" id="img6Img">
 +
                <div class="caption">DESCRIPTION</div>
 +
            </div>
 +
 
 +
            <figure class="figure">
 +
                <img id="img7" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/e/e2/T--DTU-Denmark--suffix.png" alt="DESCRIPTION">
 +
                <figcaption class="figure-caption"><strong>Figure 6:</strong> Alignment of <a href="http://parts.igem.org/Part:BBa_K530002">BBa_K530002</a> sequence received from the parts page (top) and sequencing results of <a href="http://parts.igem.org/Part:BBa_K530002">BBa_K530002</a> from the distribution kit. Alignment shows a deletion in the suffix sequence. </figcaption>
 +
            </figure>
 +
           
 +
            <div id="img7Modal" class="modal">
 +
                <span class="close img7">×</span>
 +
                <img class="modal-content" id="img7Img">
 +
                <div class="caption">DESCRIPTION</div>
 +
            </div>
 +
 
 +
            <p>
 +
                The alterations in prefix and suffix make the <a href="http://parts.igem.org/Part:BBa_K530002">BBa_K530002</a> incompatible with the BioBrick standard
 
             </p>
 
             </p>
 
         </div>
 
         </div>
 +
 +
 +
 
     </div> <!-- /LEFT -->
 
     </div> <!-- /LEFT -->
 
      
 
      
Line 226: Line 316:
 
     <div class="col-md-3 col-sm-2 colRight" id="scrollspy">  
 
     <div class="col-md-3 col-sm-2 colRight" id="scrollspy">  
 
         <ul class="nav" id="sidebar">
 
         <ul class="nav" id="sidebar">
−
             <li><a href="#section-1">Section 1</a></li>
+
             <li><a href="#section-1">Parts list</a></li>
−
             <li><a href="#section-2">Section 2</a></li>
+
             <li><a href="#section-2">Improvement and Characterization</a></li>
−
            <li><a href="#section-3">Section 3</a></li>
+
−
            <li><a href="#section-4">Section 4</a></li>
+
−
            <li><a href="#section-5">Section 5</a></li>
+
−
            <li><a href="#section-6">Section 6</a></li>
+
−
            <li><a href="#section-7">Section 7</a></li>
+
 
         </ul>
 
         </ul>
 
     </div> <!-- /RIGHT -->
 
     </div> <!-- /RIGHT -->

Latest revision as of 02:05, 20 October 2016

New HTML template for the wiki




Bootstrap Example

Parts

We have constructed four new basic parts, 4 composite parts, a plasmid backbone and improved two parts made by a previous team. The parts are all a part of our molecular toolbox for protein expression in Yarrowia lipolytica.


Parts list

"A great building will never stand if you neglect the small bricks"

Ifeanyi Enoch Onuoha
BBa_K2117000

Constitutive TEF1 promoter

BBa_K2117001

hrGFP codon-optimized for Yarrowia lipolytica

BBa_K2117002

Composite part consisting of the TEF1 promoter and the human proinsulin gene codon-optimized for Y. lipolytica

BBa_K2117003

Human proinsulin codon-optimized for Y. lipolytica

BBa_K2117004

SCR1'-tRNA promoter expressing sgRNA

BBa_K2117005

hrGFP expressed by the constitutive TEF1 promoter

BBa_K2117009

Semi-synthetic shuttle vector pSB1A8YL

BBa_K2117010

Composite part of TEF1 and enhanced YFP

BBa_K2117011

Composite part of TEF1 and enhanced CFP

BBa_K2117012

Improved BBa_K530001 (crtE)

BBa_K2117013

Improved BBa_K530002 (crtI)

Improvement and Characterization of Two Existing Parts

In our laboratory work, we wanted to work with the two BioBricks; BBa_K530001 (crtE gene) and BBa_K530002 (crtI gene) created by the John Hopkins iGEM team 2011. The genes encode the two enzymes Geranylgeranyl Diphosphate Synthase and Phytoene Desaturase, respectively, both from the wildtype strain of xanthophyllomyces dendrorhous used in the biosynthesis of beta-Carotene.

When looking into the specifications of these BioBricks, we realized that both genes contained illegal restriction sites.

BBa_K530001 contained an AgeI restriction site making the part incompatible with the RFC25 Freiburg Standard. The RFC25 standard allows for in-frame assembly of protein domains. BBa_K530002 contained the illegal restriction site BglII making the part incompatible with the RFC21 Berkely Standard, which enables in-frame assembly of proteins.

We wanted to overcome these obstacle by removing the illegal restriction sites from the above described Biobricks.

How did we do it?

The illegal restriction sites were removed using site directed mutagenesis with primers containing nucleotide substitutions in the two restriction sites (see Figure 1).

DESCRIPTION
Figure 1: Primers overlapping the restriction sites are designed with a single nucleotide change to disrupt the restriction site. The primers anneal to the template plasmid and replicate while introducing the point mutation. The elongated plasmids are digested with the enzyme DpnI, which cleaves at the methylated sites breaking down the circular template, resulting in a higher transformation efficiency of the linear PCR product.
DESCRIPTION
Table 1: Primers designed for removal of illegal restriction sites. Bold marks the nucleotide substitution.

The new plasmid, BBa_K2117012, with the removed AgeI restriction site was double digested with the enzyme and SpeI to test if we had successfully removed the AgeI restriction site.

DESCRIPTION
Figure 2: AgeI + SpeI digestion. Electrophoresis on a 1% agarose gel showing the digestion of BBa_K2117012 (lane 1-3). BBa_K530001 digested with AgeI + SpeI and undigested BBa_K530001 were used as controls (lane 4+5).

h

The digestion showed the removal of the AgeI restriction site, shown on the gel picture by only one band on the BBa_K2117012 compared to two bands on the control BBa_K530001.

BBa_K2117012 and BBa_K2117013 were sent for sequencing with the verification primers VR and VF2 primers to further verify the removal of the restriction sites.

DESCRIPTION
Figure 3: Sequencing alignment of BBa_530001 (top sequence) and BBa_K2117012 (bottom sequence). The alignment shows a nucleotide substitution in the AgeI restriction site.
DESCRIPTION
Figure 4: Sequencing alignment of BBa_530002 (top sequence) and BBa_K2117013 (bottom sequence). The alignment shows a nucleotide substitution in the BglII restriction site.

The sequencing results show a substitution in the restriction sites corresponding to the nucleotide substitution designed in the primers (Table 1).

By deleting the two restriction sites in the BBa_K2117012 and BBa_K2117013 we have made the two BioBricks compatible with RFC25 and RFC21 standards, respectively.

Improvement of Characterization

While working with BBa_K530001 and BBa_K530002 we were struggling with assembly of the genes. Therefore, we sequenced the plasmids received in the distribution kit.

Sequencing results of BBa_K530002 showed nucleotide substitutions in the prefix and a large deletion in the suffix.

DESCRIPTION
Figure 5: Alignment of BBa_K530002 sequence received from the parts page (top) and sequencing results of BBa_K530002 from the distribution kit. Alignment shows many nucleotide substitutions in the prefix seuqence.
DESCRIPTION
Figure 6: Alignment of BBa_K530002 sequence received from the parts page (top) and sequencing results of BBa_K530002 from the distribution kit. Alignment shows a deletion in the suffix sequence.

The alterations in prefix and suffix make the BBa_K530002 incompatible with the BioBrick standard

  • FIND US AT:
Facebook Twitter
  • DTU BIOBUILDERS
  • DENMARK
  • DTU - SØLTOFTS PLADS, BYGN. 221/002
  • 2800 KGS. LYNGBY

  • E-mail:
  • dtu-biobuilders-2016@googlegroups.com
  • MAIN SPONSORS:
Lundbeck fundation DTU blue dot Lundbeck fundation Lundbeck fundation