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

 
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<h2>Project description</h2>
 
  
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<h3>Background</h3>
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In Denmark today, less than half the waste produced is recycled, which means that more than 3.5 million tons get burned off each year. We have abundant waste streams from the industry such as glycerol from biodiesel production, byproducts from rapeseed production, used cooking oil and ordinary household waste.
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Cell factories are becoming an increasing factor in the industry today, where different microorganisms are utilized to produce various compounds from therapeutics, organic acids, food additives etc. Currently however, the sustainability of these industrial processes is limited by the narrow substrate range of the organisms used. The most common feeds in use are simple carbohydrates such as glucose produced by enzymatic hydrolysis from edible plants such as maize, rice and wheat.
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It is widely established that the dimorphic yeast <i>Yarrowia lipolytica</i> grows well on a broad range of substrates such as alcohols, fatty acids, glycerol as well as on simple sugars in complex mixtures, whereas the conventional and widely used yeast <i>Saccharomyces cerevisiae</i> only grows well on a very limited amount of substrates such as glucose. Furthermore, the protein modification and secretion systems of <i>Y. lipolytica</i> gives rise to a higher potential as a cell factory for production of a variety of therapeutics, food additives etc. Both of the species are fast growing, which contributes to the final efficiency as cell factories.
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Nonetheless, <i>Y. lipolytica</i> has not been applied in industry as widely as <i>S. cerevisiae</i> due to lack of tools for genetic engineering and as genetic manipulation has been tedious and time consuming.
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                        <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>
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                    <div class="col-md-2 col-sm-2 hidden-xs space"></div>
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                    <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-->
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                        <blockquote class="blockquote-reverse"> <!-- EDIT -->
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                            <p>"A great building will never stand if you neglect the small bricks"</p>
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                            <small>Ifeanyi Enoch Onuoha<cite title="Source Title"></cite></small>
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                        </blockquote>    
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                    </div>
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                </div>
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            </div>
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    </div>
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<br>
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<div class="container">
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<div class="row"> <!--Must sorround both content and sidebar-->
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    <div class="col-md-9 col-sm-10 colLeft"> <!-- LEFT -->
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        <div><a class="anchor" id="section-1"></a>
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            <h2 class="h2">Parts list</h2>
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            <blockquote class="visible-xs"> <!-- quote from masterhead duplicate -->
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                <p>"A great building will never stand if you neglect the small bricks"</p>
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                <small>Ifeanyi Enoch Onuoha<cite title="Source Title"></cite></small>
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            </blockquote>
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            <div class="grid-row">
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                <div class="col-md-3 col-sm-3 col-xs-12">
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                    <a href="http://parts.igem.org/Part:BBa_K2117000">BBa_K2117000</a>
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                </div>
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                <div class="col-md-9 col-sm-9 col-xs-12">
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                    <p>Constitutive TEF1 promoter</p>
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                </div>
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            </div> <!-- /grid-row -->
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            <div class="grid-row">
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                <div class="col-md-3 col-sm-3 col-xs-12">
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                    <a href="http://parts.igem.org/Part:BBa_K2117001">BBa_K2117001</a>
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                </div>
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                <div class="col-md-9 col-sm-9 col-xs-12">
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                    <p>hrGFP codon-optimized for <i>Yarrowia lipolytica</i></p>
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                </div>
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            </div> <!-- /grid-row -->
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            <div class="grid-row">
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                <div class="col-md-3 col-sm-3 col-xs-12">
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                    <a href="http://parts.igem.org/Part:BBa_K2117002">BBa_K2117002</a>
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                </div>
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                <div class="col-md-9 col-sm-9 col-xs-12">
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                    <p>Composite part consisting of the TEF1 promoter and the human proinsulin gene codon-optimized for <i>Y. lipolytica</i></p>
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                </div>
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            </div> <!-- /grid-row -->
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            <div class="grid-row">
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                <div class="col-md-3 col-sm-3 col-xs-12">
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                    <a href="http://parts.igem.org/Part:BBa_K2117003">BBa_K2117003</a>
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                </div>
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                <div class="col-md-9 col-sm-9 col-xs-12">
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                    <p>Human proinsulin codon-optimized for <i>Y. lipolytica</i></p>
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                </div>
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            </div> <!-- /grid-row -->
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            <div class="grid-row">
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                <div class="col-md-3 col-sm-3 col-xs-12">
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                    <a href="http://parts.igem.org/Part:BBa_K2117004">BBa_K2117004</a>
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                </div>
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                <div class="col-md-9 col-sm-9 col-xs-12">
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                    <p>SCR1'-tRNA promoter expressing sgRNA</p>
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                </div>
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            </div> <!-- /grid-row -->
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            <div class="grid-row">
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                <div class="col-md-3 col-sm-3 col-xs-12">
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                    <a href="http://parts.igem.org/Part:BBa_K2117005">BBa_K2117005</a>
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                </div>
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                <div class="col-md-9 col-sm-9 col-xs-12">
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                    <p>hrGFP expressed by the constitutive TEF1 promoter</p>
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                </div>
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            </div> <!-- /grid-row -->
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            <div class="grid-row">
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                <div class="col-md-3 col-sm-3 col-xs-12">
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                    <a href="http://parts.igem.org/Part:BBa_K2117009">BBa_K2117009</a>
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                </div>
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                <div class="col-md-9 col-sm-9 col-xs-12">
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                    <p>Semi-synthetic shuttle vector pSB1A8YL</p>
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                </div>
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            </div> <!-- /grid-row -->
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            <div class="grid-row">
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                <div class="col-md-3 col-sm-3 col-xs-12">
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                    <a href="http://parts.igem.org/Part:BBa_K2117010">BBa_K2117010</a>
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                </div>
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                <div class="col-md-9 col-sm-9 col-xs-12">
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                    <p>Composite part of TEF1 and enhanced YFP </p>
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                </div>
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            </div> <!-- /grid-row -->
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            <div class="grid-row">
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                <div class="col-md-3 col-sm-3 col-xs-12">
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                    <a href="http://parts.igem.org/Part:BBa_K2117011">BBa_K2117011</a>
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                </div>
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                <div class="col-md-9 col-sm-9 col-xs-12">
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                    <p>Composite part of TEF1 and enhanced CFP</p>
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                </div>
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            </div> <!-- /grid-row -->
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            <div class="grid-row">
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                <div class="col-md-3 col-sm-3 col-xs-12">
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                    <a href="http://parts.igem.org/Part:BBa_K2117012">BBa_K2117012</a>
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                </div>
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                <div class="col-md-9 col-sm-9 col-xs-12">
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                    <p>Improved <a href="http://parts.igem.org/Part:BBa_K530001">BBa_K530001</a> (<em>crtE</em>)</p>
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                </div>
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            </div> <!-- /grid-row -->
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 +
            <div class="grid-row">
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                <div class="col-md-3 col-sm-3 col-xs-12">
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                    <a href="http://parts.igem.org/Part:BBa_K2117013">BBa_K2117013</a>
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                </div>
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                <div class="col-md-9 col-sm-9 col-xs-12">
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                    <p>Improved <a href="http://parts.igem.org/Part:BBa_K530002">BBa_K530002</a>  (<em>crtI</em>)</p>
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                </div>
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            </div> <!-- /grid-row -->
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        </div> <!-- /overview-->
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        <div><a class="anchor" id="section-2"></a>
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            <h2 class="h2">Improvement and Characterization of Two Existing Parts</h2>
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            <p>
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            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.
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            </p>
  
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<h3>Aim</h3>
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            <p>
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<p>
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            When looking into the specifications of these BioBricks, we realized that both genes contained illegal restriction sites.
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This project aims to develop the chassis for a versatile and efficient cell factory that can transform abundant waste streams into valuable products.
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            </p>
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</p>
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            <p>
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            <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.
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            <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.  
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            </p>
  
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            <p>
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            We wanted to overcome these obstacle by removing the illegal restriction sites from the above described Biobricks.
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            </p>
  
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<h3>Methods</h3>
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            <h3 class="h3">How did we do it?</h3>
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<ol>
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<li>Substrate screening</li>
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            <p>
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<p>
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            The illegal restriction sites were removed using site directed mutagenesis with primers containing nucleotide substitutions in the two restriction sites (see Figure 1).
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To confirm the ability of <i>Y. lipolytica</i> for efficient utilization of an impure mixture of compounds, various waste streams will be investigated as a substrate. We chose mixtures of fats, present in biodiesel waste or vegetation waters from rapeseed oil production, as well as sugars, which are present in molasses or brewery waste. In order to demonstrate the versatility of  <i>Y. lipolytica</i>, we are going even further and ferment homogenized household waste.
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            </p>
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</p>
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<li>Product</li>
 
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<p>
 
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As a proof of concept we aim to demonstrate the production of both an extracellular heterologous protein and an engineered metabolite, and show how a valuable product can be produced by our cell factory utilizing waste streams.
 
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We will implement a codon optimized version of the human proinsulin gene along with a native <i>Y. lipolytica</i> promoter and secretion signal into <i>Y. lipolytica</i>.
 
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Using an already constructed plasmid with <i>S. cerevisiae</i> optimized genes from the bacterium <i>Erwinia uredovora</i> encoding four enzymes, we will implement the biosynthesis pathway of beta-carotene in <i>Y. lipolytica</i> by using the <a href="http://parts.igem.org/Part:BBa_K152005"> K152005 biobrick</a>.
 
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</p>
 
  
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<li>Molecular toolbox</li>
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            <figure class="figure">
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<p>
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                <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">
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This project tries to solve the lack of the well-proven tools for <i>Y. lipolytica</i>. We will develop a standardized genetic toolbox, including CRISPR/Cas9-mediated genome editing. The molecular toolbox will bring new opportunities such as an introduction of new pathways, adjusting waste utilization and targeting genetic manipulations.
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                <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>
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            </figure>
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                <div class="caption">DESCRIPTION</div>
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            </div>
  
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<p>Tell us about your project, describe what moves you and why this is something important for your team.</p>
 
  
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            <figure class="figure">
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                <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">
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                <figcaption class="figure-caption"><strong>Table 1:</strong> Primers designed for removal of illegal restriction sites. Bold marks the nucleotide  substitution.</figcaption>
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                <div class="caption">DESCRIPTION</div>
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            </div>
  
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<h5>What should this page contain?</h5>
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            <p>  
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<ul>
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            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.  
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<li> A clear and concise description of your project.</li>
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            </p>
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<li>A detailed explanation of why your team chose to work on this particular project.</li>
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<li>References and sources to document your research.</li>
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<li>Use illustrations and other visual resources to explain your project.</li>
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                        <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">
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                        <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>
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                    </figure>
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                        <!-- The Modal with same picture-->
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                        <div class="caption">DESCRIPTION</div>
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                <div class="col-md-6 col-sm-6"><p style="color:transparent;">    h</p></div>
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<h5>Advice on writing your Project Description</h5>
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            <p>
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            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>.
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            </p>
  
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<p>
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            <p>
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We encourage you to put up a lot of information and content on your wiki, but we also encourage you to include summaries as much as possible. If you think of the sections in your project description as the sections in a publication, you should try to be consist, accurate and unambiguous in your achievements.  
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            <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.  
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</p>
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            </p>
  
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<p>
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            <div class="row">
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Judges like to read your wiki and know exactly what you have achieved. This is how you should think about these sections; from the point of view of the judge evaluating you at the end of the year.
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                    <figure class="figure">
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                        <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">
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                        <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>
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                        <div class="caption">DESCRIPTION</div>
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                        <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">
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                        <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>
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                    </figure>
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                                <!-- The Modal with same picture-->
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                        <img class="modal-content" id="img5Img">
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                        <div class="caption">DESCRIPTION</div>
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                    </div>
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                </div>
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            </div>
  
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            <p>
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                The sequencing results show a substitution in the restriction sites corresponding to the nucleotide substitution designed in the primers (Table 1).
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            </p>
  
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<div class="column half_size" >
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            <p>
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                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.
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            </p>
  
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<h5>References</h5>
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            <h3 class="h3">Improvement of Characterization</h3>
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<p>iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you thought about your project and what works inspired you.</p>
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</div>
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            <p>
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                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.
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            </p>
  
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            <p>
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                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.
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            </p>
  
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<h5>Inspiration</h5>
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<p>See how other teams have described and presented their projects: </p>
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                <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>
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                <div class="caption">DESCRIPTION</div>
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            </div>
  
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            <figure class="figure">
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<li><a href="https://2014.igem.org/Team:Imperial/Project"> Imperial</a></li>
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                <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">
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<li><a href="https://2014.igem.org/Team:UC_Davis/Project_Overview"> UC Davis</a></li>
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                <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>
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<li><a href="https://2014.igem.org/Team:SYSU-Software/Overview">SYSU Software</a></li>
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                <div class="caption">DESCRIPTION</div>
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            </div>
  
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            <p>
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                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
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            <li><a href="#section-1">Parts list</a></li>
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            <li><a href="#section-2">Improvement and Characterization</a></li>
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Latest revision as of 02:05, 20 October 2016

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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

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