http://2014.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Xen+sm2014.igem.org - User contributions [en]2024-03-28T11:01:16ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:Imperial/PartsTeam:Imperial/Parts2014-10-18T03:59:29Z<p>Xen sm: </p>
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<h1>Parts</h1><br />
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<ul><br />
<li><a data-scroll href="#featured">Featured Parts</a><br />
</li><br />
<li><a data-scroll href="#table">Table of Parts</a><br />
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<h2>Featured Parts</h2><br />
<div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321340">BBa_K1321340</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>N-terminal linker + double CBD</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>Double cellulose binding domain (dCBD) using two cellulose binding domains from <i>Trichoderma reesei</i> cellobiohydrolases, with an N-terminal linker and internal linker sequence between the two domains which are derived from the endogenous cellobiohydrolase linker sequence. This part is in RFC(25) Freiberg fusion format to allow for easy use in protein fusions. dCBD binding affinity for cellulose was <a href="https://2014.igem.org/Team:Imperial/Functionalisation"> characterised </a> using sfGFP fusions.<br />
</p><br />
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<h3><a href="http://parts.igem.org/Part:BBa_K1321339">BBa_K1321339</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>CBDcenA + Linker</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>Cellulose binding domain (CBD) of Endoglucanase A (cenA) from <i>Cellulomonas fimi</i> with an endogenous C-terminal linker. The part is in Freiburg format (RFC 25) for ease of use in protein fusions.</p><br />
</div><br />
</div><br />
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</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321014"> BBa_K1321014</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>CBDcipA with N and C-terminal linker </p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>This CBD is from the Cellulosomal -scaffolding protein A (cipA) of <i>Clostridium thermocellum </i> including the endogenous linker sequences at the N and C-terminus. It is in RFC25 format to allow for easy use in protein fusions. At present the cloning for these constructs is still in progress to correct an illegal EcoRI site which was identified in the parts with this CBD. This can be achieved with a silent mutation via site-directed mutagenesis and we aim to send these parts to the registry once this is complete.<br />
</p><br />
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</div><br />
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<h3><a href="http://parts.igem.org/Part:BBa_K1321005">BBa_K1321005</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>Synthetic Phytochelatin (PC) EC20</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>A general heavy metal-binding peptide consisting of 20 Glu-Cys (EC) repeats in Freiburg format (RFC[25]) to allow for easy use in fusion proteins. We created a library of CBD fusions with this part.<br />
</p><br />
</div><br />
</div><br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p><i>Vitreoscilla</i> haemoglobin (VHb)</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>This part is a haemoglobin isolated from <i>Vitreoscilla</i> (VHb) which improves the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions. We used this part in <i> G. xylinus </i> to try and improve growth rate and cellulose production.<br />
</p><br />
</div><br />
</div><br />
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</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a>, <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>pSEVA331 and pSEVA321 backbones</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>These are two broad host range vector backbones for which we have demonstrated use in <i>E. coli</i> and <i>Gluconacetobacter xylinus </i>.<br />
</p><br />
</div><br />
</div> <br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321334"> BBa_K1321334</a>, <a href="http://parts.igem.org/Part:BBa_K1321335">BBa_K1321335</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>Cellulose synthase operon AcsAB and AcsCD</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>Designed and refactored from <i>G. xylinus</i>, this operon was inserted into <i>E.coli </i> for cellulose synthesis. <br />
</p><br />
</div><br />
</div> <br />
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</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a>,<a href="http://parts.igem.org/Part:BBa_K1321306"> BBa_K1321306</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p><i>Gluconacetobacter xylinus</i> strains ATCC53582 and Kombucha Isolate</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>We created and characterised a library of parts for this organism, with the aim to increase the accessibility the chassis for future work in iGEM. By sharing these strains and their genomes on the registry we hope this will contribute to their ease of use and characterisation.<br />
</p><br />
</div><br />
</div> <br />
</div><br />
</div><br />
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</section><br />
<section id="table"><br />
<h2>Table of Parts</h2><br />
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</section><br />
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<groupparts>iGEM014 Imperial</groupparts><br />
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{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/Mass_Production_and_ProcessingTeam:Imperial/Mass Production and Processing2014-10-18T03:21:37Z<p>Xen sm: </p>
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<div class="pure-u-1-1 main"><br />
<h1>Mass Production and Processing</h1><br />
<div id="navWrap"><br />
<div id="subNav"><br />
<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<br />
<li><a data-scroll href="#references">References</a><br />
</li><br />
</ul><br />
<br class="clearLeft" /><br />
</div><br />
</div><br />
<br />
<section id="introduction"><br />
<h2>Introduction</h2><br />
<br/> <br />
<br />
<figure class="content-image image-right"><br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/7/7b/IC14-Post-prod-fig-1-blue.png ><br />
<figcaption>Figure 1: Blue dyed bacterial cellulose</figcaption><br />
</figure><br />
<br />
<p> Bacterial cellulose (BC) exhibits a <a href="https://2014.igem.org/Team:Imperial/Project_Background">multitude of different properties</a> depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation steps. </p><br />
<br />
<p> By mass producing cellulose this enables a better understanding of what material properties can be realistically produced during the short duration of iGEM. More importantly, it improves the likelihood of finding suitable processing candidates for the project’s aim of making a customisable ultrafiltration membrane, at the same time as allowing room for creativity and exploration of the remarkable properties of cellulose. </p><br />
<br />
<h3>Minimum requirements</h3><br />
<ol><br />
<li>Treatment of BC requires killing the cells, particularly if the cells are genetically engineered, which is the aim for putting the customisable in ultrafiltration membranes.</li><br />
<li>Based on brainstorming with Central Saint Martins student Zuzana, removing the colour of BC is required as it looks displeasing to the eye otherwise, and seems counter-intuituve to filter clean water with cellulose coloured like turbid water. </li><br />
<li>Removal of the smell of BC has also been raised as a requirement, particularly by producers who work in close contact with the processing facilities.</li><br />
</ol><br />
<br />
<h3>Mass Production Methods</h3><br />
<br />
<figure class="content-image image-right image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/0/04/IC14-post-prod-fig-2-pellicle-comparison.png > <br />
<figcaption>Figure 2. left: A granular pellicle, right: even pellicle</figcaption> <br />
</figure> <br />
<br />
<p>Setting up the mass production of cellulose was done according to the <a href="https://2014.igem.org/Team:Imperial/Protocols#gluconacetobacter">Kombucha media protocol</a> , which involved setting up 61 trays with media and G. xylinus and yeast co-culture as shown in figure 3. The trays were left to grow up over 7 days, after which diminishing pellicle growth was detected. Upon harvesting, the pellicles were sorted according to granular pellicles (see figure 2 left) and even pellicles (figure 2 right). All pellicles were kept in distilled water in large plastic buckets or containers. </p><br />
<br />
<p>Below shows the general workflow we employed to mass produce our cellulose and illustrates the process of manufacturing biomaterials with significantly different properties despite originating from the same BC source.<br />
</p><br />
<br />
<img class="content-image image-full" src="https://static.igem.org/mediawiki/2014/b/bf/IC14-mass_production2.png"> <br />
<figure class="content-image image-full"><br />
<table class="tg image-full"><br />
<br />
<h3> Cost Analysis </h3><br />
<p> Producing this large amount of cellulose enabled us to analyse cost in some detail, as shown in Table 1. </p><br />
<tr><br />
<th class="tg-e3zv">Component</th><br />
<th class="tg-e3zv">Quantity</th><br />
<th class="tg-e3zv">Source</th><br />
<th class="tg-e3zv">Cost breakdown (£)</th><br />
<th class="tg-e3zv">Cost (£)</th><br />
</tr><br />
<tr><br />
<td class="tg-031e">Water</td><br />
<td class="tg-031e">4l</td><br />
<td class="tg-031e">London South West Water</td><br />
<td class="tg-031e">4 liters of £5.5195 per m3</td><br />
<td class="tg-031e">0.02</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">400 g granulated sugar</td><br />
<td class="tg-031e">400g</td><br />
<td class="tg-031e">Tesco's</td><br />
<td class="tg-031e">79p per 1 kg</td><br />
<td class="tg-031e">0.32</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Clipper green tea tea bags</td><br />
<td class="tg-031e">4</td><br />
<td class="tg-031e">Clipper tea</td><br />
<td class="tg-031e">300 teabags for £9.99</td><br />
<td class="tg-031e">0.13</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Aspall organic cider vinegar</td><br />
<td class="tg-031e">2</td><br />
<td class="tg-031e">Aspall Suffolk</td><br />
<td class="tg-031e">400 ml of a £2.25 500 ml bottle</td><br />
<td class="tg-031e">1.80</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Total</td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv">2.27</td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Product</td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Component</td><br />
<td class="tg-e3zv">Quantity</td><br />
<td class="tg-e3zv">Source</td><br />
<td class="tg-e3zv">Price breakdown (£)</td><br />
<td class="tg-e3zv">Price per g (£)</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Bacterial cellulose yield</td><br />
<td class="tg-031e">60 cm by 40 cm = 0.24 m2</td><br />
<td class="tg-031e">production from single tray</td><br />
<td class="tg-031e">110 g/m2 x 0.24 m2 = 26.4g</td><br />
<td class="tg-e3zv">0.09</td><br />
</tr><br />
</table><br />
<figcaption>Table 1: Cost analysis for production of bacterial cellulose</figcaption><br />
</figure><br />
</section><br />
<section id="references"><br />
<h2>References</h2><br />
<br />
</section><br />
</div><br />
<br />
<br />
<br />
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</body><br />
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{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/Mass_Production_and_ProcessingTeam:Imperial/Mass Production and Processing2014-10-18T03:16:31Z<p>Xen sm: </p>
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<div>{{:Team:Imperial/Templates:header}}<br />
<html><br />
<body><br />
<div class="content-wrapper"><br />
<br />
<div class="pure-g"><br />
<br />
<div class="pure-u-1-1 main"><br />
<h1>Mass Production and Processing</h1><br />
<div id="navWrap"><br />
<div id="subNav"><br />
<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<br />
<li><a data-scroll href="#references">References</a><br />
</li><br />
</ul><br />
<br class="clearLeft" /><br />
</div><br />
</div><br />
<br />
<section id="introduction"><br />
<h2>Introduction</h2><br />
<br/> <br />
<br />
<figure class="content-image image-right"><br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/7/7b/IC14-Post-prod-fig-1-blue.png ><br />
<figcaption>Figure 1: Blue dyed bacterial cellulose</figcaption><br />
</figure><br />
<br />
<p> Bacterial cellulose (BC) exhibits a <a href="https://2014.igem.org/Team:Imperial/Project_Background">multitude of different properties</a> depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation steps. </p><br />
<br />
<p> By mass producing cellulose this enables a better understanding of what material properties can be realistically produced during the short duration of iGEM. More importantly, it improves the likelihood of finding suitable processing candidates for the project’s aim of making a customisable ultrafiltration membrane, at the same time as allowing room for creativity and exploration of the remarkable properties of cellulose. </p><br />
<br />
<h3>Minimum requirements</h3><br />
<ol><br />
<li>Treatment of BC requires killing the cells, particularly if the cells are genetically engineered, which is the aim for putting the customisable in ultrafiltration membranes.</li><br />
<li>Based on brainstorming with Central Saint Martins student Zuzana, removing the colour of BC is required as it looks displeasing to the eye otherwise, and seems counter-intuituve to filter clean water with cellulose coloured like turbid water. </li><br />
<li>Removal of the smell of BC has also been raised as a requirement, particularly by producers who work in close contact with the processing facilities.</li><br />
</ol><br />
<br />
<h3>Mass Production Methods</h3><br />
<br />
<figure class="content-image image-right image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/0/04/IC14-post-prod-fig-2-pellicle-comparison.png > <br />
<figcaption>Figure 2. left: A granular pellicle, right: even pellicle</figcaption> <br />
</figure> <br />
<br />
<p>Setting up the mass production of cellulose was done according to the <a href="https://2014.igem.org/Team:Imperial/Protocols#gluconacetobacter">Kombucha media protocol</a> , which involved setting up 61 trays with media and G. xylinus and yeast co-culture as shown in figure 3. The trays were left to grow up over 7 days, after which diminishing pellicle growth was detected. Upon harvesting, the pellicles were sorted according to granular pellicles (see figure 2 left) and even pellicles (figure 2 right). All pellicles were kept in distilled water in large plastic buckets or containers. </p><br />
<br />
<p>Below shows the general workflow we employed to mass produce our cellulose </p><br />
<br />
<img class="content-image image-full" src="https://static.igem.org/mediawiki/2014/b/bf/IC14-mass_production2.png"> <br />
<figure class="content-image image-full"><br />
<table class="tg image-full"><br />
<br />
<h3> Cost Analysis </h3><br />
<p> Producing this large amount of cellulose enabled us to analyse cost in some detail, as shown in Table 1. </p><br />
<tr><br />
<th class="tg-e3zv">Component</th><br />
<th class="tg-e3zv">Quantity</th><br />
<th class="tg-e3zv">Source</th><br />
<th class="tg-e3zv">Cost breakdown (£)</th><br />
<th class="tg-e3zv">Cost (£)</th><br />
</tr><br />
<tr><br />
<td class="tg-031e">Water</td><br />
<td class="tg-031e">4l</td><br />
<td class="tg-031e">London South West Water</td><br />
<td class="tg-031e">4 liters of £5.5195 per m3</td><br />
<td class="tg-031e">0.02</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">400 g granulated sugar</td><br />
<td class="tg-031e">400g</td><br />
<td class="tg-031e">Tesco's</td><br />
<td class="tg-031e">79p per 1 kg</td><br />
<td class="tg-031e">0.32</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Clipper green tea tea bags</td><br />
<td class="tg-031e">4</td><br />
<td class="tg-031e">Clipper tea</td><br />
<td class="tg-031e">300 teabags for £9.99</td><br />
<td class="tg-031e">0.13</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Aspall organic cider vinegar</td><br />
<td class="tg-031e">2</td><br />
<td class="tg-031e">Aspall Suffolk</td><br />
<td class="tg-031e">400 ml of a £2.25 500 ml bottle</td><br />
<td class="tg-031e">1.80</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Total</td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv">2.27</td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Product</td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Component</td><br />
<td class="tg-e3zv">Quantity</td><br />
<td class="tg-e3zv">Source</td><br />
<td class="tg-e3zv">Price breakdown (£)</td><br />
<td class="tg-e3zv">Price per g (£)</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Bacterial cellulose yield</td><br />
<td class="tg-031e">60 cm by 40 cm = 0.24 m2</td><br />
<td class="tg-031e">production from single tray</td><br />
<td class="tg-031e">110 g/m2 x 0.24 m2 = 26.4g</td><br />
<td class="tg-e3zv">0.09</td><br />
</tr><br />
</table><br />
<figcaption>Table 1: Cost analysis for production of bacterial cellulose</figcaption><br />
</figure><br />
</section><br />
<section id="references"><br />
<h2>References</h2><br />
<br />
</section><br />
</div><br />
<br />
<br />
<br />
</div><br />
</body><br />
</html><br />
{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/Mass_Production_and_ProcessingTeam:Imperial/Mass Production and Processing2014-10-18T03:15:10Z<p>Xen sm: </p>
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<div>{{:Team:Imperial/Templates:header}}<br />
<html><br />
<body><br />
<div class="content-wrapper"><br />
<br />
<div class="pure-g"><br />
<br />
<div class="pure-u-1-1 main"><br />
<h1>Mass Production and Processing</h1><br />
<div id="navWrap"><br />
<div id="subNav"><br />
<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<br />
<li><a data-scroll href="#references">References</a><br />
</li><br />
</ul><br />
<br class="clearLeft" /><br />
</div><br />
</div><br />
<br />
<section id="introduction"><br />
<h2>Introduction</h2><br />
<br/> <br />
<br />
<figure class="content-image image-right"><br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/7/7b/IC14-Post-prod-fig-1-blue.png ><br />
<figcaption>Figure 1: Blue dyed bacterial cellulose</figcaption><br />
</figure><br />
<br />
<p> Bacterial cellulose (BC) exhibits a <a href="https://2014.igem.org/Team:Imperial/Project_Background">multitude of different properties</a> depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation steps. </p><br />
<br />
<p> By mass producing cellulose this enables a better understanding of what material properties can be realistically produced during the short duration of iGEM. More importantly, it improves the likelihood of finding suitable processing candidates for the project’s aim of making a customisable ultrafiltration membrane, at the same time as allowing room for creativity and exploration of the remarkable properties of cellulose. </p><br />
<br />
<h3>Minimum requirements</h3><br />
<ol><br />
<li>Treatment of BC requires killing the cells, particularly if the cells are genetically engineered, which is the aim for putting the customisable in ultrafiltration membranes.</li><br />
<li>Based on brainstorming with Central Saint Martins student Zuzana, removing the colour of BC is required as it looks displeasing to the eye otherwise, and seems counter-intuituve to filter clean water with cellulose coloured like turbid water. </li><br />
<li>Removal of the smell of BC has also been raised as a requirement, particularly by producers who work in close contact with the processing facilities.</li><br />
</ol><br />
<br />
<h3>Mass Production Methods</h3><br />
<br />
<figure class="content-image image-right image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/0/04/IC14-post-prod-fig-2-pellicle-comparison.png > <br />
<figcaption>Figure 2. left: A granular pellicle, right: even pellicle</figcaption> <br />
</figure> <br />
<br />
<p>Setting up the mass production of cellulose was done according to the <a href="https://2014.igem.org/Team:Imperial/Protocols#gluconacetobacter">Kombucha media protocol</a> , which involved setting up 61 trays with media and G. xylinus and yeast co-culture as shown in figure 3. The trays were left to grow up over 7 days, after which diminishing pellicle growth was detected. Upon harvesting, the pellicles were sorted according to granular pellicles (see figure 2 left) and even pellicles (figure 2 right). All pellicles were kept in distilled water in large plastic buckets or containers. </p><br />
<br />
<p>Below shows the general workflow we employed to mass produce our cellulose </p><br />
<br />
<img class="content-image image-full" src="https://static.igem.org/mediawiki/2014/b/bf/IC14-mass_production2.png"> <br />
<figure class="content-image image-full"><br />
<table class="tg image-full"><br />
<tr><br />
<th class="tg-e3zv">Component</th><br />
<th class="tg-e3zv">Quantity</th><br />
<th class="tg-e3zv">Source</th><br />
<th class="tg-e3zv">Cost breakdown (£)</th><br />
<th class="tg-e3zv">Cost (£)</th><br />
</tr><br />
<tr><br />
<td class="tg-031e">Water</td><br />
<td class="tg-031e">4l</td><br />
<td class="tg-031e">London South West Water</td><br />
<td class="tg-031e">4 liters of £5.5195 per m3</td><br />
<td class="tg-031e">0.02</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">400 g granulated sugar</td><br />
<td class="tg-031e">400g</td><br />
<td class="tg-031e">Tesco's</td><br />
<td class="tg-031e">79p per 1 kg</td><br />
<td class="tg-031e">0.32</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Clipper green tea tea bags</td><br />
<td class="tg-031e">4</td><br />
<td class="tg-031e">Clipper tea</td><br />
<td class="tg-031e">300 teabags for £9.99</td><br />
<td class="tg-031e">0.13</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Aspall organic cider vinegar</td><br />
<td class="tg-031e">2</td><br />
<td class="tg-031e">Aspall Suffolk</td><br />
<td class="tg-031e">400 ml of a £2.25 500 ml bottle</td><br />
<td class="tg-031e">1.80</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Total</td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv">2.27</td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Product</td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Component</td><br />
<td class="tg-e3zv">Quantity</td><br />
<td class="tg-e3zv">Source</td><br />
<td class="tg-e3zv">Price breakdown (£)</td><br />
<td class="tg-e3zv">Price per g (£)</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Bacterial cellulose yield</td><br />
<td class="tg-031e">60 cm by 40 cm = 0.24 m2</td><br />
<td class="tg-031e">production from single tray</td><br />
<td class="tg-031e">110 g/m2 x 0.24 m2 = 26.4g</td><br />
<td class="tg-e3zv">0.09</td><br />
</tr><br />
</table><br />
<figcaption>Table 1: Cost analysis for production of bacterial cellulose</figcaption><br />
</figure><br />
</section><br />
<section id="references"><br />
<h2>References</h2><br />
<br />
</section><br />
</div><br />
<br />
<br />
<br />
</div><br />
</body><br />
</html><br />
{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/Mass_Production_and_ProcessingTeam:Imperial/Mass Production and Processing2014-10-18T03:12:16Z<p>Xen sm: </p>
<hr />
<div>{{:Team:Imperial/Templates:header}}<br />
<html><br />
<body><br />
<div class="content-wrapper"><br />
<br />
<div class="pure-g"><br />
<br />
<div class="pure-u-1-1 main"><br />
<h1>Mass Production and Processing</h1><br />
<div id="navWrap"><br />
<div id="subNav"><br />
<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<br />
<li><a data-scroll href="#references">References</a><br />
</li><br />
</ul><br />
<br class="clearLeft" /><br />
</div><br />
</div><br />
<br />
<section id="introduction"><br />
<h2>Introduction</h2><br />
<br/> <br />
<br />
<figure class="content-image image-right"><br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/7/7b/IC14-Post-prod-fig-1-blue.png ><br />
<figcaption>Figure 1: Blue dyed bacterial cellulose</figcaption><br />
</figure><br />
<br />
<p> Bacterial cellulose (BC) exhibits a <a href="https://2014.igem.org/Team:Imperial/Project_Background">multitude of different properties</a> depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation steps. </p><br />
<br />
<p> By mass producing cellulose this enables a better understanding of what material properties can be realistically produced during the short duration of iGEM. More importantly, it improves the likelihood of finding suitable processing candidates for the project’s aim of making a customisable ultrafiltration membrane, at the same time as allowing room for creativity and exploration of the remarkable properties of cellulose. </p><br />
<br />
<h3>Minimum requirements</h3><br />
<ol><br />
<li>Treatment of BC requires killing the cells, particularly if the cells are genetically engineered, which is the aim for putting the customisable in ultrafiltration membranes.</li><br />
<li>Based on brainstorming with Central Saint Martins student Zuzana, removing the colour of BC is required as it looks displeasing to the eye otherwise, and seems counter-intuituve to filter clean water with cellulose coloured like turbid water. </li><br />
<li>Removal of the smell of BC has also been raised as a requirement, particularly by producers who work in close contact with the processing facilities.</li><br />
</ol><br />
<br />
<h3>Mass Production Methods</h3><br />
<br />
<figure class="content-image image-right image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/0/04/IC14-post-prod-fig-2-pellicle-comparison.png > <br />
<figcaption>Figure 2. left: A granular pellicle, right: even pellicle</figcaption> <br />
</figure> <br />
<br />
<p>Setting up the mass production of cellulose was done according to the <a href="https://2014.igem.org/Team:Imperial/Protocols#gluconacetobacter">Kombucha media protocol</a> , which involved setting up 61 trays with media and G. xylinus and yeast co-culture as shown in figure 3. The trays were left to grow up over 7 days, after which diminishing pellicle growth was detected. Upon harvesting, the pellicles were sorted according to granular pellicles (see figure 2 left) and even pellicles (figure 2 right). All pellicles were kept in distilled water in large plastic buckets or containers. </p><br />
<br />
<br />
<br />
<img class="content-image image-full" src="https://static.igem.org/mediawiki/2014/b/bf/IC14-mass_production2.png"> <br />
<figure class="content-image image-full"><br />
<table class="tg image-full"><br />
<tr><br />
<th class="tg-e3zv">Component</th><br />
<th class="tg-e3zv">Quantity</th><br />
<th class="tg-e3zv">Source</th><br />
<th class="tg-e3zv">Cost breakdown (£)</th><br />
<th class="tg-e3zv">Cost (£)</th><br />
</tr><br />
<tr><br />
<td class="tg-031e">Water</td><br />
<td class="tg-031e">4l</td><br />
<td class="tg-031e">London South West Water</td><br />
<td class="tg-031e">4 liters of £5.5195 per m3</td><br />
<td class="tg-031e">0.02</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">400 g granulated sugar</td><br />
<td class="tg-031e">400g</td><br />
<td class="tg-031e">Tesco's</td><br />
<td class="tg-031e">79p per 1 kg</td><br />
<td class="tg-031e">0.32</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Clipper green tea tea bags</td><br />
<td class="tg-031e">4</td><br />
<td class="tg-031e">Clipper tea</td><br />
<td class="tg-031e">300 teabags for £9.99</td><br />
<td class="tg-031e">0.13</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Aspall organic cider vinegar</td><br />
<td class="tg-031e">2</td><br />
<td class="tg-031e">Aspall Suffolk</td><br />
<td class="tg-031e">400 ml of a £2.25 500 ml bottle</td><br />
<td class="tg-031e">1.80</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Total</td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv">2.27</td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Product</td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Component</td><br />
<td class="tg-e3zv">Quantity</td><br />
<td class="tg-e3zv">Source</td><br />
<td class="tg-e3zv">Price breakdown (£)</td><br />
<td class="tg-e3zv">Price per g (£)</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Bacterial cellulose yield</td><br />
<td class="tg-031e">60 cm by 40 cm = 0.24 m2</td><br />
<td class="tg-031e">production from single tray</td><br />
<td class="tg-031e">110 g/m2 x 0.24 m2 = 26.4g</td><br />
<td class="tg-e3zv">0.09</td><br />
</tr><br />
</table><br />
<figcaption>Table 1: Cost analysis for production of bacterial cellulose</figcaption><br />
</figure><br />
</section><br />
<section id="references"><br />
<h2>References</h2><br />
<br />
</section><br />
</div><br />
<br />
<br />
<br />
</div><br />
</body><br />
</html><br />
{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/Mass_Production_and_ProcessingTeam:Imperial/Mass Production and Processing2014-10-18T03:09:50Z<p>Xen sm: </p>
<hr />
<div>{{:Team:Imperial/Templates:header}}<br />
<html><br />
<body><br />
<div class="content-wrapper"><br />
<br />
<div class="pure-g"><br />
<br />
<div class="pure-u-1-1 main"><br />
<h1>Mass Production and Processing</h1><br />
<div id="navWrap"><br />
<div id="subNav"><br />
<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<br />
<li><a data-scroll href="#references">References</a><br />
</li><br />
</ul><br />
<br class="clearLeft" /><br />
</div><br />
</div><br />
<br />
<section id="introduction"><br />
<h2>Introduction</h2><br />
<br/> <br />
<br />
<figure class="content-image image-right"><br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/7/7b/IC14-Post-prod-fig-1-blue.png ><br />
<figcaption>Figure 1: Blue dyed bacterial cellulose</figcaption><br />
</figure><br />
<br />
<p> Bacterial cellulose (BC) exhibits a <a href="https://2014.igem.org/Team:Imperial/Project_Background">multitude of different properties</a> depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation steps. </p><br />
<br />
<p> By mass producing cellulose this enables a better understanding of what material properties can be realistically produced during the short duration of iGEM. More importantly, it improves the likelihood of finding suitable processing candidates for the project’s aim of making a customisable ultrafiltration membrane, at the same time as allowing room for creativity and exploration of the remarkable properties of cellulose. </p><br />
<br />
<h3>Minimum requirements</h3><br />
<ol><br />
<li>Treatment of BC requires killing the cells, particularly if the cells are genetically engineered, which is the aim for putting the customisable in ultrafiltration membranes.</li><br />
<li>Based on brainstorming with Central Saint Martins student Zuzana, removing the colour of BC is required as it looks displeasing to the eye otherwise, and seems counter-intuituve to filter clean water with cellulose coloured like turbid water. </li><br />
<li>Removal of the smell of BC has also been raised as a requirement, particularly by producers who work in close contact with the processing facilities.</li><br />
</ol><br />
<br />
<h3>Mass Production Methods</h3><br />
<br />
<figure class="content-image image-right"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/0/04/IC14-post-prod-fig-2-pellicle-comparison.png > <br />
<figcaption>Figure 2. left: A granular pellicle, right: even pellicle</figcaption> <br />
</figure> <br />
<br />
<p>Setting up the mass production of cellulose was done according to the Kombucha media protocol (link), which involved setting up 61 trays with media and G. xylinus and yeast co-culture as shown in figure 3. The trays were left to grow up over 7 days, after which diminishing pellicle growth was detected. Upon harvesting, the pellicles were sorted according to granular pellicles (see figure 2 left) and even pellicles (figure 2 right). All pellicles were kept in distilled water in large plastic buckets or containers. </p><br />
<br />
<br />
<br />
<img class="content-image image-full" src="https://static.igem.org/mediawiki/2014/b/bf/IC14-mass_production2.png"> <br />
<figure class="content-image image-full"><br />
<table class="tg image-full"><br />
<tr><br />
<th class="tg-e3zv">Component</th><br />
<th class="tg-e3zv">Quantity</th><br />
<th class="tg-e3zv">Source</th><br />
<th class="tg-e3zv">Cost breakdown (£)</th><br />
<th class="tg-e3zv">Cost (£)</th><br />
</tr><br />
<tr><br />
<td class="tg-031e">Water</td><br />
<td class="tg-031e">4l</td><br />
<td class="tg-031e">London South West Water</td><br />
<td class="tg-031e">4 liters of £5.5195 per m3</td><br />
<td class="tg-031e">0.02</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">400 g granulated sugar</td><br />
<td class="tg-031e">400g</td><br />
<td class="tg-031e">Tesco's</td><br />
<td class="tg-031e">79p per 1 kg</td><br />
<td class="tg-031e">0.32</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Clipper green tea tea bags</td><br />
<td class="tg-031e">4</td><br />
<td class="tg-031e">Clipper tea</td><br />
<td class="tg-031e">300 teabags for £9.99</td><br />
<td class="tg-031e">0.13</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Aspall organic cider vinegar</td><br />
<td class="tg-031e">2</td><br />
<td class="tg-031e">Aspall Suffolk</td><br />
<td class="tg-031e">400 ml of a £2.25 500 ml bottle</td><br />
<td class="tg-031e">1.80</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Total</td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv">2.27</td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Product</td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Component</td><br />
<td class="tg-e3zv">Quantity</td><br />
<td class="tg-e3zv">Source</td><br />
<td class="tg-e3zv">Price breakdown (£)</td><br />
<td class="tg-e3zv">Price per g (£)</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Bacterial cellulose yield</td><br />
<td class="tg-031e">60 cm by 40 cm = 0.24 m2</td><br />
<td class="tg-031e">production from single tray</td><br />
<td class="tg-031e">110 g/m2 x 0.24 m2 = 26.4g</td><br />
<td class="tg-e3zv">0.09</td><br />
</tr><br />
</table><br />
<figcaption>Table 1: Cost analysis for production of bacterial cellulose</figcaption><br />
</figure><br />
</section><br />
<section id="references"><br />
<h2>References</h2><br />
<br />
</section><br />
</div><br />
<br />
<br />
<br />
</div><br />
</body><br />
</html><br />
{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/Mass_Production_and_ProcessingTeam:Imperial/Mass Production and Processing2014-10-18T03:09:15Z<p>Xen sm: </p>
<hr />
<div>{{:Team:Imperial/Templates:header}}<br />
<html><br />
<body><br />
<div class="content-wrapper"><br />
<br />
<div class="pure-g"><br />
<br />
<div class="pure-u-1-1 main"><br />
<h1>Mass Production and Processing</h1><br />
<div id="navWrap"><br />
<div id="subNav"><br />
<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<br />
<li><a data-scroll href="#references">References</a><br />
</li><br />
</ul><br />
<br class="clearLeft" /><br />
</div><br />
</div><br />
<br />
<section id="introduction"><br />
<h2>Introduction</h2><br />
<br/> <br />
<br />
<figure class="content-image image-right"><br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/7/7b/IC14-Post-prod-fig-1-blue.png ><br />
<figcaption>Figure 1: Blue dyed bacterial cellulose</figcaption><br />
</figure><br />
<br />
<p> Bacterial cellulose (BC) exhibits a <a href="https://2014.igem.org/Team:Imperial/Project_Background">multitude of different properties</a> depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation steps. </p><br />
<br />
<p> By mass producing cellulose this enables a better understanding of what material properties can be realistically produced during the short duration of iGEM. More importantly, it improves the likelihood of finding suitable processing candidates for the project’s aim of making a customisable ultrafiltration membrane, at the same time as allowing room for creativity and exploration of the remarkable properties of cellulose. </p><br />
<br />
<h3>Minimum requirements</h3><br />
<ol><br />
<li>Treatment of BC requires killing the cells, particularly if the cells are genetically engineered, which is the aim for putting the customisable in ultrafiltration membranes.</li><br />
<li>Based on brainstorming with Central Saint Martins student Zuzana, removing the colour of BC is required as it looks displeasing to the eye otherwise, and seems counter-intuituve to filter clean water with cellulose coloured like turbid water. </li><br />
<li>Removal of the smell of BC has also been raised as a requirement, particularly by producers who work in close contact with the processing facilities.</li><br />
<br />
<h3>Mass Production Methods</h3><br />
<br />
<p>Setting up the mass production of cellulose was done according to the Kombucha media protocol (link), which involved setting up 61 trays with media and G. xylinus and yeast co-culture as shown in figure 3. The trays were left to grow up over 7 days, after which diminishing pellicle growth was detected. Upon harvesting, the pellicles were sorted according to granular pellicles (see figure 2 left) and even pellicles (figure 2 right). All pellicles were kept in distilled water in large plastic buckets or containers. </p><br />
<br />
<figure class="content-image image-right"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/0/04/IC14-post-prod-fig-2-pellicle-comparison.png > <br />
<figcaption>Figure 2. left: A granular pellicle, right: even pellicle</figcaption> <br />
</figure> <br />
<br />
<br />
<img class="content-image image-full" src="https://static.igem.org/mediawiki/2014/b/bf/IC14-mass_production2.png"> <br />
<figure class="content-image image-full"><br />
<table class="tg image-full"><br />
<tr><br />
<th class="tg-e3zv">Component</th><br />
<th class="tg-e3zv">Quantity</th><br />
<th class="tg-e3zv">Source</th><br />
<th class="tg-e3zv">Cost breakdown (£)</th><br />
<th class="tg-e3zv">Cost (£)</th><br />
</tr><br />
<tr><br />
<td class="tg-031e">Water</td><br />
<td class="tg-031e">4l</td><br />
<td class="tg-031e">London South West Water</td><br />
<td class="tg-031e">4 liters of £5.5195 per m3</td><br />
<td class="tg-031e">0.02</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">400 g granulated sugar</td><br />
<td class="tg-031e">400g</td><br />
<td class="tg-031e">Tesco's</td><br />
<td class="tg-031e">79p per 1 kg</td><br />
<td class="tg-031e">0.32</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Clipper green tea tea bags</td><br />
<td class="tg-031e">4</td><br />
<td class="tg-031e">Clipper tea</td><br />
<td class="tg-031e">300 teabags for £9.99</td><br />
<td class="tg-031e">0.13</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Aspall organic cider vinegar</td><br />
<td class="tg-031e">2</td><br />
<td class="tg-031e">Aspall Suffolk</td><br />
<td class="tg-031e">400 ml of a £2.25 500 ml bottle</td><br />
<td class="tg-031e">1.80</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Total</td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv">2.27</td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Product</td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Component</td><br />
<td class="tg-e3zv">Quantity</td><br />
<td class="tg-e3zv">Source</td><br />
<td class="tg-e3zv">Price breakdown (£)</td><br />
<td class="tg-e3zv">Price per g (£)</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Bacterial cellulose yield</td><br />
<td class="tg-031e">60 cm by 40 cm = 0.24 m2</td><br />
<td class="tg-031e">production from single tray</td><br />
<td class="tg-031e">110 g/m2 x 0.24 m2 = 26.4g</td><br />
<td class="tg-e3zv">0.09</td><br />
</tr><br />
</table><br />
<figcaption>Table 1: Cost analysis for production of bacterial cellulose</figcaption><br />
</figure><br />
</section><br />
<section id="references"><br />
<h2>References</h2><br />
<br />
</section><br />
</div><br />
<br />
<br />
<br />
</div><br />
</body><br />
</html><br />
{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/Mass_Production_and_ProcessingTeam:Imperial/Mass Production and Processing2014-10-18T03:07:31Z<p>Xen sm: </p>
<hr />
<div>{{:Team:Imperial/Templates:header}}<br />
<html><br />
<body><br />
<div class="content-wrapper"><br />
<br />
<div class="pure-g"><br />
<br />
<div class="pure-u-1-1 main"><br />
<h1>Mass Production and Processing</h1><br />
<div id="navWrap"><br />
<div id="subNav"><br />
<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<br />
<li><a data-scroll href="#references">References</a><br />
</li><br />
</ul><br />
<br class="clearLeft" /><br />
</div><br />
</div><br />
<br />
<section id="introduction"><br />
<h2>Introduction</h2><br />
<br/> <p> Bacterial cellulose (BC) exhibits a <a href="https://2014.igem.org/Team:Imperial/Project_Background">multitude of different properties</a> depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation steps. </p><br />
<br />
<p> By mass producing cellulose this enables a better understanding of what material properties can be realistically produced during the short duration of iGEM. More importantly, it improves the likelihood of finding suitable processing candidates for the project’s aim of making a customisable ultrafiltration membrane, at the same time as allowing room for creativity and exploration of the remarkable properties of cellulose. </p><br />
<br />
<h3>Minimum requirements</h3><br />
<ol><br />
<li>Treatment of BC requires killing the cells, particularly if the cells are genetically engineered, which is the aim for putting the customisable in ultrafiltration membranes.</li><br />
<li>Based on brainstorming with Central Saint Martins student Zuzana, removing the colour of BC is required as it looks displeasing to the eye otherwise, and seems counter-intuituve to filter clean water with cellulose coloured like turbid water. </li><br />
<li>Removal of the smell of BC has also been raised as a requirement, particularly by producers who work in close contact with the processing facilities.</li><br />
<br />
<h3>Mass Production Methods</h3><br />
<br />
<p>Setting up the mass production of cellulose was done according to the Kombucha media protocol (link), which involved setting up 61 trays with media and G. xylinus and yeast co-culture as shown in figure 3. The trays were left to grow up over 7 days, after which diminishing pellicle growth was detected. Upon harvesting, the pellicles were sorted according to granular pellicles (see figure 2 left) and even pellicles (figure 2 right). All pellicles were kept in distilled water in large plastic buckets or containers. </p><br />
<br />
<figure class="content-image image-right"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/0/04/IC14-post-prod-fig-2-pellicle-comparison.png > <br />
<figcaption>Figure 2. left: A granular pellicle, right: even pellicle</figcaption> <br />
</figure> <br />
<br />
<br />
<img class="content-image image-full" src="https://static.igem.org/mediawiki/2014/b/bf/IC14-mass_production2.png"> <br />
<figure class="content-image image-full"><br />
<table class="tg image-full"><br />
<tr><br />
<th class="tg-e3zv">Component</th><br />
<th class="tg-e3zv">Quantity</th><br />
<th class="tg-e3zv">Source</th><br />
<th class="tg-e3zv">Cost breakdown (£)</th><br />
<th class="tg-e3zv">Cost (£)</th><br />
</tr><br />
<tr><br />
<td class="tg-031e">Water</td><br />
<td class="tg-031e">4l</td><br />
<td class="tg-031e">London South West Water</td><br />
<td class="tg-031e">4 liters of £5.5195 per m3</td><br />
<td class="tg-031e">0.02</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">400 g granulated sugar</td><br />
<td class="tg-031e">400g</td><br />
<td class="tg-031e">Tesco's</td><br />
<td class="tg-031e">79p per 1 kg</td><br />
<td class="tg-031e">0.32</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Clipper green tea tea bags</td><br />
<td class="tg-031e">4</td><br />
<td class="tg-031e">Clipper tea</td><br />
<td class="tg-031e">300 teabags for £9.99</td><br />
<td class="tg-031e">0.13</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Aspall organic cider vinegar</td><br />
<td class="tg-031e">2</td><br />
<td class="tg-031e">Aspall Suffolk</td><br />
<td class="tg-031e">400 ml of a £2.25 500 ml bottle</td><br />
<td class="tg-031e">1.80</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Total</td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv">2.27</td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Product</td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Component</td><br />
<td class="tg-e3zv">Quantity</td><br />
<td class="tg-e3zv">Source</td><br />
<td class="tg-e3zv">Price breakdown (£)</td><br />
<td class="tg-e3zv">Price per g (£)</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Bacterial cellulose yield</td><br />
<td class="tg-031e">60 cm by 40 cm = 0.24 m2</td><br />
<td class="tg-031e">production from single tray</td><br />
<td class="tg-031e">110 g/m2 x 0.24 m2 = 26.4g</td><br />
<td class="tg-e3zv">0.09</td><br />
</tr><br />
</table><br />
<figcaption>Table 1: Cost analysis for production of bacterial cellulose</figcaption><br />
</figure><br />
</section><br />
<section id="references"><br />
<h2>References</h2><br />
<br />
</section><br />
</div><br />
<br />
<br />
<br />
</div><br />
</body><br />
</html><br />
{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/Mass_Production_and_ProcessingTeam:Imperial/Mass Production and Processing2014-10-18T03:04:46Z<p>Xen sm: </p>
<hr />
<div>{{:Team:Imperial/Templates:header}}<br />
<html><br />
<body><br />
<div class="content-wrapper"><br />
<br />
<div class="pure-g"><br />
<br />
<div class="pure-u-1-1 main"><br />
<h1>Mass Production and Processing</h1><br />
<div id="navWrap"><br />
<div id="subNav"><br />
<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<br />
<li><a data-scroll href="#references">References</a><br />
</li><br />
</ul><br />
<br class="clearLeft" /><br />
</div><br />
</div><br />
<br />
<section id="introduction"><br />
<h2>Introduction</h2><br />
<br/> <p> Bacterial cellulose (BC) exhibits a <a href="https://2014.igem.org/Team:Imperial/Project_Background">multitude of different properties</a> depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation steps. </p><br />
<br />
<p> By mass producing cellulose this enables a better understanding of what material properties can be realistically produced during the short duration of iGEM. More importantly, it improves the likelihood of finding suitable processing candidates for the project’s aim of making a customisable ultrafiltration membrane, at the same time as allowing room for creativity and exploration of the remarkable properties of cellulose. </p><br />
<br />
<h3>Minimum requirements</h3><br />
<ol><br />
<li>Treatment of BC requires killing the cells, particularly if the cells are genetically engineered, which is the aim for putting the customisable in ultrafiltration membranes.</li><br />
<li>Based on brainstorming with Central Saint Martins student Zuzana, removing the colour of BC is required as it looks displeasing to the eye otherwise, and seems counter-intuituve to filter clean water with cellulose coloured like turbid water. </li><br />
<li>Removal of the smell of BC has also been raised as a requirement, particularly by producers who work in close contact with the processing facilities.</li><br />
<br />
<br />
<br />
<img class="content-image image-full" src="https://static.igem.org/mediawiki/2014/b/bf/IC14-mass_production2.png"> <br />
<figure class="content-image image-full"><br />
<table class="tg image-full"><br />
<tr><br />
<th class="tg-e3zv">Component</th><br />
<th class="tg-e3zv">Quantity</th><br />
<th class="tg-e3zv">Source</th><br />
<th class="tg-e3zv">Cost breakdown (£)</th><br />
<th class="tg-e3zv">Cost (£)</th><br />
</tr><br />
<tr><br />
<td class="tg-031e">Water</td><br />
<td class="tg-031e">4l</td><br />
<td class="tg-031e">London South West Water</td><br />
<td class="tg-031e">4 liters of £5.5195 per m3</td><br />
<td class="tg-031e">0.02</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">400 g granulated sugar</td><br />
<td class="tg-031e">400g</td><br />
<td class="tg-031e">Tesco's</td><br />
<td class="tg-031e">79p per 1 kg</td><br />
<td class="tg-031e">0.32</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Clipper green tea tea bags</td><br />
<td class="tg-031e">4</td><br />
<td class="tg-031e">Clipper tea</td><br />
<td class="tg-031e">300 teabags for £9.99</td><br />
<td class="tg-031e">0.13</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Aspall organic cider vinegar</td><br />
<td class="tg-031e">2</td><br />
<td class="tg-031e">Aspall Suffolk</td><br />
<td class="tg-031e">400 ml of a £2.25 500 ml bottle</td><br />
<td class="tg-031e">1.80</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Total</td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv">2.27</td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Product</td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Component</td><br />
<td class="tg-e3zv">Quantity</td><br />
<td class="tg-e3zv">Source</td><br />
<td class="tg-e3zv">Price breakdown (£)</td><br />
<td class="tg-e3zv">Price per g (£)</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Bacterial cellulose yield</td><br />
<td class="tg-031e">60 cm by 40 cm = 0.24 m2</td><br />
<td class="tg-031e">production from single tray</td><br />
<td class="tg-031e">110 g/m2 x 0.24 m2 = 26.4g</td><br />
<td class="tg-e3zv">0.09</td><br />
</tr><br />
</table><br />
<figcaption>Table 1: Cost analysis for production of bacterial cellulose</figcaption><br />
</figure><br />
</section><br />
<section id="references"><br />
<h2>References</h2><br />
<br />
</section><br />
</div><br />
<br />
<br />
<br />
</div><br />
</body><br />
</html><br />
{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/Mass_Production_and_ProcessingTeam:Imperial/Mass Production and Processing2014-10-18T03:00:24Z<p>Xen sm: </p>
<hr />
<div>{{:Team:Imperial/Templates:header}}<br />
<html><br />
<body><br />
<div class="content-wrapper"><br />
<br />
<div class="pure-g"><br />
<br />
<div class="pure-u-1-1 main"><br />
<h1>Mass Production and Processing</h1><br />
<div id="navWrap"><br />
<div id="subNav"><br />
<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<br />
<li><a data-scroll href="#references">References</a><br />
</li><br />
</ul><br />
<br class="clearLeft" /><br />
</div><br />
</div><br />
<br />
<section id="introduction"><br />
<h2>Introduction</h2><br />
<br/> <p> Bacterial cellulose (BC) exhibits a <a href="https://2014.igem.org/Team:Imperial/Project_Background">multitude of different properties</a> depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation steps. </p><br />
<br />
<p> By mass producing cellulose this enables a better understanding of what material properties can be realistically produced during the short duration of iGEM. More importantly, it improves the likelihood of finding suitable processing candidates for the project’s aim of making a customisable ultrafiltration membrane, at the same time as allowing room for creativity and exploration of the remarkable properties of cellulose. </p><br />
<br />
<img class="content-image image-full" src="https://static.igem.org/mediawiki/2014/b/bf/IC14-mass_production2.png"> <br />
<figure class="content-image image-full"><br />
<table class="tg image-full"><br />
<tr><br />
<th class="tg-e3zv">Component</th><br />
<th class="tg-e3zv">Quantity</th><br />
<th class="tg-e3zv">Source</th><br />
<th class="tg-e3zv">Cost breakdown (£)</th><br />
<th class="tg-e3zv">Cost (£)</th><br />
</tr><br />
<tr><br />
<td class="tg-031e">Water</td><br />
<td class="tg-031e">4l</td><br />
<td class="tg-031e">London South West Water</td><br />
<td class="tg-031e">4 liters of £5.5195 per m3</td><br />
<td class="tg-031e">0.02</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">400 g granulated sugar</td><br />
<td class="tg-031e">400g</td><br />
<td class="tg-031e">Tesco's</td><br />
<td class="tg-031e">79p per 1 kg</td><br />
<td class="tg-031e">0.32</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Clipper green tea tea bags</td><br />
<td class="tg-031e">4</td><br />
<td class="tg-031e">Clipper tea</td><br />
<td class="tg-031e">300 teabags for £9.99</td><br />
<td class="tg-031e">0.13</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Aspall organic cider vinegar</td><br />
<td class="tg-031e">2</td><br />
<td class="tg-031e">Aspall Suffolk</td><br />
<td class="tg-031e">400 ml of a £2.25 500 ml bottle</td><br />
<td class="tg-031e">1.80</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Total</td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv">2.27</td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Product</td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Component</td><br />
<td class="tg-e3zv">Quantity</td><br />
<td class="tg-e3zv">Source</td><br />
<td class="tg-e3zv">Price breakdown (£)</td><br />
<td class="tg-e3zv">Price per g (£)</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Bacterial cellulose yield</td><br />
<td class="tg-031e">60 cm by 40 cm = 0.24 m2</td><br />
<td class="tg-031e">production from single tray</td><br />
<td class="tg-031e">110 g/m2 x 0.24 m2 = 26.4g</td><br />
<td class="tg-e3zv">0.09</td><br />
</tr><br />
</table><br />
<figcaption>Table 1: Cost analysis for production of bacterial cellulose</figcaption><br />
</figure><br />
</section><br />
<section id="references"><br />
<h2>References</h2><br />
<br />
</section><br />
</div><br />
<br />
<br />
<br />
</div><br />
</body><br />
</html><br />
{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/Mass_Production_and_ProcessingTeam:Imperial/Mass Production and Processing2014-10-18T02:58:17Z<p>Xen sm: </p>
<hr />
<div>{{:Team:Imperial/Templates:header}}<br />
<html><br />
<body><br />
<div class="content-wrapper"><br />
<br />
<div class="pure-g"><br />
<br />
<div class="pure-u-1-1 main"><br />
<h1>Mass Production and Processing</h1><br />
<div id="navWrap"><br />
<div id="subNav"><br />
<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<br />
<li><a data-scroll href="#references">References</a><br />
</li><br />
</ul><br />
<br class="clearLeft" /><br />
</div><br />
</div><br />
<br />
<section id="introduction"><br />
<h2>Introduction</h2><br />
<br/> <p> Bacterial cellulose (BC) exhibits a <a href="https://2014.igem.org/Team:Imperial/Project_Background">multitude of different properties</a> depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation steps. </p><br />
<br />
<img class="content-image image-full" src="https://static.igem.org/mediawiki/2014/b/bf/IC14-mass_production2.png"> <br />
<figure class="content-image image-full"><br />
<table class="tg image-full"><br />
<tr><br />
<th class="tg-e3zv">Component</th><br />
<th class="tg-e3zv">Quantity</th><br />
<th class="tg-e3zv">Source</th><br />
<th class="tg-e3zv">Cost breakdown (£)</th><br />
<th class="tg-e3zv">Cost (£)</th><br />
</tr><br />
<tr><br />
<td class="tg-031e">Water</td><br />
<td class="tg-031e">4l</td><br />
<td class="tg-031e">London South West Water</td><br />
<td class="tg-031e">4 liters of £5.5195 per m3</td><br />
<td class="tg-031e">0.02</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">400 g granulated sugar</td><br />
<td class="tg-031e">400g</td><br />
<td class="tg-031e">Tesco's</td><br />
<td class="tg-031e">79p per 1 kg</td><br />
<td class="tg-031e">0.32</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Clipper green tea tea bags</td><br />
<td class="tg-031e">4</td><br />
<td class="tg-031e">Clipper tea</td><br />
<td class="tg-031e">300 teabags for £9.99</td><br />
<td class="tg-031e">0.13</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Aspall organic cider vinegar</td><br />
<td class="tg-031e">2</td><br />
<td class="tg-031e">Aspall Suffolk</td><br />
<td class="tg-031e">400 ml of a £2.25 500 ml bottle</td><br />
<td class="tg-031e">1.80</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Total</td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv">2.27</td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Product</td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Component</td><br />
<td class="tg-e3zv">Quantity</td><br />
<td class="tg-e3zv">Source</td><br />
<td class="tg-e3zv">Price breakdown (£)</td><br />
<td class="tg-e3zv">Price per g (£)</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Bacterial cellulose yield</td><br />
<td class="tg-031e">60 cm by 40 cm = 0.24 m2</td><br />
<td class="tg-031e">production from single tray</td><br />
<td class="tg-031e">110 g/m2 x 0.24 m2 = 26.4g</td><br />
<td class="tg-e3zv">0.09</td><br />
</tr><br />
</table><br />
<figcaption>Table 1: Cost analysis for production of bacterial cellulose</figcaption><br />
</figure><br />
</section><br />
<section id="references"><br />
<h2>References</h2><br />
<br />
</section><br />
</div><br />
<br />
<br />
<br />
</div><br />
</body><br />
</html><br />
{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/File:IC14-post-prod-fig-2-pellicle-comparison.pngFile:IC14-post-prod-fig-2-pellicle-comparison.png2014-10-18T02:51:07Z<p>Xen sm: </p>
<hr />
<div></div>Xen smhttp://2014.igem.org/File:IC14-Post-prod-fig-1-blue.pngFile:IC14-Post-prod-fig-1-blue.png2014-10-18T02:50:32Z<p>Xen sm: </p>
<hr />
<div></div>Xen smhttp://2014.igem.org/File:IC14-Plateassay2.gifFile:IC14-Plateassay2.gif2014-10-18T02:41:33Z<p>Xen sm: reduced number of pics</p>
<hr />
<div>reduced number of pics</div>Xen smhttp://2014.igem.org/File:IC14-Prelim-plate.gifFile:IC14-Prelim-plate.gif2014-10-18T02:40:58Z<p>Xen sm: prelim assay gif</p>
<hr />
<div>prelim assay gif</div>Xen smhttp://2014.igem.org/File:IC14-Plate-assay.gifFile:IC14-Plate-assay.gif2014-10-18T02:39:52Z<p>Xen sm: all washes</p>
<hr />
<div>all washes</div>Xen smhttp://2014.igem.org/Team:Imperial/FunctionalisationTeam:Imperial/Functionalisation2014-10-18T02:23:03Z<p>Xen sm: </p>
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<h1>Functionalisation</h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#CBDs">CBDs</a><br />
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<li><a data-scroll href="#binding-partners">Functionalisation proteins</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Attaching functional proteins to cellulose can expand the properties of cellulose and allow us to selectivity capture specific contaminants in water. By creating fusion proteins of sfGFP and metal-binding proteins with five different cellulose binding domains, three of which are new to the registry, we were able to characterise the relative affinities of the CBDs and show proof-of-concept removal of contaminants from water.</p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Added three new cellulose binding domains to the registry</li><br />
<li>Performed an assay with sfGFP-fusions to determine the strongest potential binders which could be carried forward for further testing</li><br />
<li>Identified potential metal binding proteins to bind heavy metals from wastewater, including the addition of a new promising polypeptide phytochelatin EC20</li><br />
<li>Tested metal ion capture ability of the CBD-phytochelatin fusion constructs when attached to cellulose</li><br />
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<section id="introduction"><br />
<h2>Introduction</h2><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/a/a0/IC14-func-strategy-both-pursue.png"><br />
<figcaption>Figure 1. Pictorial summary showing the two cellulose functionalization approaches considered in our project. Strategy 1: Cellulose binding domains are expressed as fusions with the protein of interest, separated optionally by a linker sequence. The cellulose binding domains attach to the cellulose by hydrophobic interactions. Strategy 2: Curli fibrils are expressed in parallel with cellulose to form a composite material. The curli fibres coding sequence is modified to contain a ‘SpyTag’ peptide (Zakeri <i>et al</i> 2012) which binds specifically via a covalent isopeptide bond to its protein partner, ‘SpyCatcher’. This SpyCatcher is fused to the proteins of interest. In our project we only had time to pursue strategy 1 but are still excited about the potential of strategy 2 in the future.</figcaption><br />
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<p>Bacterial cellulose is already a useful biomaterial as is due its attractive mechanical properties, with research on applications from loudspeaker diaphragms to protective packaging and wound dressings (Lee 2014). However, our project also aims to extend the properties of cellulose by conjugating functional proteins and peptides to it. This allows us to target specific contaminants in water which can not be filtered by size exclusion alone. (See <a href="https://2014.igem.org/Team:Imperial/Water_Report">water report</a> and <a href="https://2014.igem.org/Team:Imperial/Informing_Design">informing design</a> sections).</p><br />
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<p>We considered two ways of functionalising cellulose (figure 1) but continued to work fully on the cellulose-binding domain (CBD) method. The curli-fibre method would be an exciting avenue to explore in the future; it is feasible since there has already been research into making a “programmable” biomaterial from curli fibres using a peptide tag approach (Nguyen <i>et al</i> 2014), and in combination with cellulose could produce a composite with robust mechanical properties as well.<br />
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<section id="aim"><br />
<h2>Aims</h2><br />
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<p>We aimed to make and characterise new cellulose binding domains, and fuse them to functional proteins. (Incomplete)</p><br />
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<section id="CBDs"><br />
<h2>Cellulose Binding Domains (CBDs)</h2><br />
<p>The cellulose binding domain (CBD) is the link between our functional proteins and the cellulose. Therefore, we wanted to use a CBD which had high affinity and strong binding to cellulose. However, weaker CBDs may still be useful since they could have the potential be controllably eluted under specific conditions, thereby enabling regeneration of the filter when saturated, and controlled disposal of the collected contaminants (refer to water report if relevant?).<br />
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<p>We started our search by looking into the <a href="http://parts.igem.org/">parts registry</a> and found two CBDs already existed in the registry: CBDclos (<a href="http://parts.igem.org/Part:BBa_K863111">BBa_K863111</a>) and CBDcex (<a href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>). We also made three new CBDs to contribute to the registry: dCBD, CBDcipA, and CBDcenA. All the CBDs are in RFC(25) freiberg fusion format to allow for ease of use in protein fusions.</p><br />
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<h3>CBDclos and CBDcex</h3><br />
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<p>These parts were biobricked by <a href="https://2012.igem.org/Team:Bielefeld-Germany/Results/cbc">Bielefeld 2012</a>, please see their part pages (<a href="http://parts.igem.org/Part:BBa_K863111">BBa_K863111</a>a and <a href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>) for detailed information. CBDclos is from the cellulolytic bacterium <em>Clostridium cellulovorans</em> and CBDcex originates from the cellulolytic bacterium <em>Cellulomonas fimi</em>. This is the same organism as for CBDcenA; cex is from the exoglucanase gene and cen is from the endoglucanase gene.</p><br />
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<h3>Double CBD (dCBD) with N-terminal linker</h3><br />
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<p>This part, <a href="http://parts.igem.org/Part:BBa_K1321340">BBa_K1321340</a> is based on the double cellulose-binding domain construct synthesised and characterised by Linder <i>et al</i>1996, who found that this double CBD had higher affinity for cellulose than either of the two CBDs on their own. The main difference is that our part contains an additional linker sequence on the N-terminus of the protein.<br />
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<p>The two CBDs are from the fungus <i>Trichoderma reesei</i> (<i>Hypocrea jecorina</i>) Exocellobiohydrolase (Exoglucanase) I (cbh1), uniprot ID P62694; and Exocellobiohydrolase (Exoglucanase) II, uniprot ID P07987 (cbh2); with a linker peptide between the two CBDs and at the N-terminus of the protein. Both linkers are the same amino acid sequence and are based on the endogenous linker sequences that exists in cbh1 and cbh2 genes. The linker sequence is PGANPPGTTTTSRPATTTGSSPGP which is the same as used by Linder <i>et al</i> 1996. The first three amino acids are from the cbh2 endogenous linker, and the rest is from the cbh1 endogenous linker. CBDcbh1 is placed C-terminal to CBDcbh2 because naturally CBDcbh1 is a C-terminal domain and CBDcbh2 is an N-terminal domain. Both CBDs are from the <a class='iframe' href="http://www.cazy.org/CBM1.html">CBM family 1</a>. The precise location of the CBD within the cbh genes was slightly different according to the uniprot annotations and the sequence used by Linder <i>et al</i> 1996; we chose to use the sequence from the paper since the protein was expressed and characterised successfully.</p><br />
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<h3>CBDcipA with N and C-terminal linker</h3><br />
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<p>This CBD is from the Cellulosomal -scaffolding protein A (cipA) of Clostridium thermocellum including the endogenous linker sequences at the N and C-terminus (<a class='iframe' href="http://www.uniprot.org/uniprot/Q06851">UniProt ID Q06851</a>). It is in RFC25 format to allow for easy use in protein fusions.</p><br />
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<p>The CBD is part of the <a class='iframe' href="http://www.cazy.org/CBM3.html">CBM3 family</a> . This CBD has been used in many application: in fusions with cell adhesion peptides to enhance the properties of cellulose as a cell-growth matrix (Andrade <i>et al </i> 2010a, Andrade <i>et al </i> 2010b), fused to enzymes to remove contaminants from water (Kauffmann <i> et al </i> 2000) and fused to an antimicrobial peptide (Ramos, Domingues & Gama 2010).</p><br />
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<p>At present the cloning for these constructs is still in progress to correct an illegal EcoRI site which was identified in the parts with this CBD. This can be achieved with a silent mutation via site-directed mutagenesis and we aim to send these parts to the registry once thi<br />
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<h3>CBDcenA with C-terminal linker</h3><br />
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<p>This CBD is from the Endoglucanase A (cenA) gene from <i>Cellulomonas fimi</i> and also contains an endogenous C-terminal linker. The cellulose binding domain region occurs at the N-terminus of the cenA gene (UniProt P07984 link to http://www.uniprot.org/uniprot/P07984) and is from the <a class='iframe' href="http://www.cazy.org/CBM2.html">CBM family 2</a><br />
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<p>The linker sequence (PTTSPTPTPTPTTPTPTPTPTPTPTPTVTP) is Pro-Thr box which has an extended conformation and acts as a hinge region (Gilkes <i>et al</i> 1989). The endoglucanase CBDcenA has 50% homology to the exoglucanase CBDcex (<a class="iframe" href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>) and the linker is highly conserved in both, but the order of the catalytic, linker and cellulose-binding regions is reversed (Warren <i>et al</i> 1986). It has been shown that CBDcenA has the highest binding affinity for crystalline cellulose out of the <i>C. fimi</i> CBDs (Kim <i>et al</i> 2013).<br />
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<section id="binding-partners"><br />
<h2>Functionalisation proteins: Binding partners</h2><br />
<h3>Superfolder Green Fluorescent Protein (sfGFP) </h3><br />
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<img class="image-full" src=https://static.igem.org/mediawiki/2014/8/85/IC14-sfGFP-part-table.PNG> <br />
<figcaption>sfGFP-CBD fusion parts</figcaption> <br />
</figure> <p> sfGFP is a more stable mutant of <a href="http://parts.igem.org/Parts:BBa_E0040">mut3GFP</a> and we chose to use this as a robust, easily visible protein to initially fuse to our CBDs. We also designed one of our assays [insert link on 'one of our assays'] for characterising CBD binding using the sfGFP fusion constructs. sfGFP already existed in the registry (<a href="http://parts.igem.org/Part:BBa_I746916">BBa_I746916</a>), detailed information on sfGFP properties is included in this part description) however since we needed to make fusion proteins with our CBDs we modified the part and submitted it in RFC[25] Freiberg fusion format (<a href="http://parts.igem.org/Parts:BBa_K1321337">K1321337</a>). We created the following fusion parts with sfGFP: <br />
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<h3> Metal Binding Proteins </h3><br />
<p>One of the contaminants we can filter out which current size-exclusion technologies can not target, are <a href="https://2014.igem.org/Team:Imperial/Water_Report">heavy metals</a>. We have worked with four different metal binding proteins: the metallothioneins SmtA and fMT, a histidine-rich nickel binding protein (NiBP) from the bacterium <i>Heliobacter pylori</i> and a synthetic phytochelatin (PC) EC20.<br />
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<h3>SmtA and fMT metallothioneins</h3><br />
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<p>These two parts already existed in the BioBrick registry, but since we needed to use them as fusions with the CBDs we improved the parts by making them compatible with the <a class="iframe" href="http://parts.igem.org/Assembly_standard_25">RFC(25) Frieburg Fusion BioBrick standard.</a></p><br />
<p>Metallothioneins (MTs) are genetically encoded cysteine-rich peptides which serve a role in the detoxification of heavy metals in plants, animals and some prokaryotes (Cobbett, 2002). They have a similar structure and function to phytochelatins, but are produced by different mechanisms (Cobbett, 2002). SmtA is from the cyanobacteria <i>Synechococcus sp.</i> and was used by the <a target="_blank" href="https://2011.igem.org/Team:Tokyo-NoKoGen">Tokyo-NoKoGen 2011</a> iGEM team (who submitted the RFC10 part originally, <a class="iframe" href="http://parts.igem.org/Part:BBa_K519010">BBa_K519010</a>) to confer cadmium (Cd2+) tolerance to <em>E. coli</em> cells when expressed. fMT originates from the seaweed <i>Fucus vesiculosus</i> and was submitted by <a target="_blank" href="https://2009.igem.org/Team:Groningen">Groningen 2009 iGEM team </a> and detailed information can be found on the original part page (<a class="iframe" href="http://parts.igem.org/Part:BBa_K190019">BBa_K190019</a>).</p><br />
<p>We successfully <a href="https://2014.igem.org/Team:Imperial/Protocols#general">cloned</a> the following CBD fusion constructs: </p><br />
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<figure class="content-image image-right image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/a/ac/IC14-SmtA-part-table.PNG> <br />
<figcaption>SmtA-CBD fusion parts</figcaption> <br />
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<figure class="content-image image-left image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/6/62/IC14-fMT-part-table.PNG> <br />
<figcaption>fMT-CBD fusion parts</figcaption> <br />
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<h3>Nickel binding protein (NiBP)</h3><br />
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<img class="image-full" src=https://static.igem.org/mediawiki/2014/f/f6/IC14-NiBP-part-table.PNG> <br />
<figcaption>NiBP-CBD fusions parts</figcaption> <br />
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<p><br />
This protein is the histidine-rich nickel-binding protein from <i>Heliobacter pylori</i> (GenBank accession ACX98466.1), and already existed as a BioBrick in the registry (<a class="iframe" href="http://parts.igem.org/Part:K1151001">K1151001</a>). Since we needed to use this part in fusion proteins we improved the part by submitting it as a basic part in RFC(25) (link) Freiberg fusion format (<a class="iframe" href="http://parts.igem.org/Part:K1321009">K1321009</a>).<br />
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<p>We successfully <a href="https://2014.igem.org/Team:Imperial/Protocols#general">cloned</a> the following CBD fusion constructs with NiBP:<br />
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<p>However we discovered late on in the project that the DNA sequence of the NiBP protein in the registry is out of frame, leading to an incorrect protein translation and read-through of the stop codon (figure 2).</p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/8/80/IC14-NiBP-protein-alignment.PNG"><br />
<figcaption>Figure 2. Alignment of BBa_K1151001 NiBP in the registry to the , showing incorrect amino-acid sequence in red compared to <a target="_blank" href="http://www.ncbi.nlm.nih.gov/protein/261838700">native <i>H.pylori</i></a> sequence.<br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/e/e1/IC14-NiBP-DNA-alignment.PNG"><br />
<figcaption>Figure 3. Single nucleotide deletion in BBa_K1151001 compared to native <i>H.pylori</i> sequence (<a target="_blank" href="http://www.ncbi.nlm.nih.gov/nuccore/261837457">GenBank CP000012.1</a> (link ) bp1391851-1392033, reverse complement) resulting in any downstream nucleotides being out of frame.</figcaption><br />
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<p>Further investigation into the error let us to identify a single nucleotide deletion in <a class="iframe" href="http://parts.igem.org/Part:BBa_K1151001">BBa_K1151001</a> compared to the native <i>H.pylori</i> DNA sequence (<a target="_blank" href="http://www.ncbi.nlm.nih.gov/nuccore/261837457">GenBank CP000012.1</a> bp1391851-1392033, reverse complement strand). As seen in Figure 3 the deletion is a ‘C’ at bp 53 of the native sequence. This error was propagated through all our NiBP constructs and means that any subsequent C-terminal fusions are also out of frame and we were unable to express and characterise these constructs. We have noted this on the description pages for each of the parts and hope that in the future they will still be useful, since the error could be corrected relatively easily by a team who wishes to use them by site-directed-mutagenesis PCR to insert the missing nucleotide. </p><br />
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<h3>Synthetic phytochelatin EC20</h3><br />
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<p>This is our new contribution of a metal-binding protein to the parts registry. Synthetic phytochelatins are analogues of phytochelatins (PCs); cysteine-rich peptides of the general structure (γ-GluCys)n-Gly (n~2-5) which bind and sequester heavy metals, with a role in detoxification (Cobbett & Goldsbrough 2002). PCs are found mainly in plants but also in other organisms including diatoms, fungi, algae (including cyanobacteria) and some invertebrates (Cobbett & Goldsbrough 2002, Rea 2012). Natively, the PCs are enzymatically synthesized by phytochelatin synthase (PCS) from a glutathione (GSH) precursor and the amino acids in the resulting peptide are linked by the non-standard gamma peptide bonds (Cobbett & Goldsbrough 2002, Rea 2012). However, genetically encoded analogues (containing normal alpha-peptide bonds) of different number Glu-Cys (EC) repeats have been characterised, with the EC20 having the highest binding (Bae <i>et al</i> 2000).<br />
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<figure class="content-image image-right image-small"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/18/IC14-Biondo-et-al-2012-ion-table.PNG"><br />
<figcaption>Table 1. Metal ion adsorption increase of C. metallidurans cells with PC EC20 displayed on cell surface, compared to negative control. Taken from Biondo <i>et al</i> 2012</figcaption><br />
</figure><br />
<p>The main metal PCs confer tolerance to is Cadmium (Cobbett & Goldsbrough 2002) with a reported stoichiometry of 10 Cd2+ per peptide (Bae <i>et al</i>, . EC20 peptide has still showed good metal ion binding whilst fused to a cellulose binding domain (CBD) for water purification (Xu <i>et al</i> 2002) and when anchored to bacterial cell membrane to confer Cd2+ tolerance (Bae <i>et al</i> 2000, Chaturvedi & Archana 2014). PC EC20 has also been shown to bind many more heavy metals. For example, when displayed on the surface of Cupriavidus metallidurans, the cells capacity to adsorb ions increased by 31 to 219% depending on the ion (table 1, Biondo <i>et al</i> 2012). These binding properties of PC EC20 have been utilised in applications including a regeneratable biosensor of Hg2+, Cd2+, Pb2+, Cu2+ and Zn2+ ions to 100 fM–10 mM (Bontidean <i>et al</i> 2003), and to target CdSe/ZnS quantum dots to streptavidin expressing HeLa cells when biotinylated (Pinaud <i>et al</i> 2004). INSERT TABLE OF THE IONS FROM THE PAPER<br />
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<p>We used this part to <a href="https://2014.igem.org/Team:Imperial/Protocols#general">clone</a> a library of different cellulose-binding domains fused to phytochelatin:<figure class="content-image"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/4/40/IC14-PC-part-table.PNG > <br />
<figcaption>Phytochelatin-CBD fusion parts. </figcaption> <br />
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<h3> Other potential proteins </h3><br />
<p>During our research we also explored other functional proteins to fuse to the CBDs.<br />
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<h3>Laccases</h3><br />
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<p>Laccases are enzymes which can break down aromatic and phenolic compounds such as modified estrogens which can end up unfiltered in the water supply with unknown consequences (Auriol <i>et al</i> 2008). This method of treating estrogen in water was explored by <a target="_blank" href="https://2012.igem.org/Team:Bielefeld-Germany">Bielefeld 2012 iGEM team</a>, and our system also has potential to address this.<br />
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<h3>Nanobodies</h3><br />
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<p>Nanobodies are fragments of camelid antibodies which can be generated against a variety of antigens and can be easily expressed by organisms such as <i>E. coli </i>. Nanobodies could be used to target viruses, which are often too small to be filtered by size exclusion. For example there are already nanobodies which can bind to <i> Vaccinia </i> virus, ricin, <i>Cholera</i> toxin, Stephylococcal enterotoxin B and H5N1 Influenza (Goldman <i>et al </i> 2006, Ibanez <i>et al </i> 2011) to name a few. Additionally, nanobodies are stable and can be selected to resist proteolytic attach (Harmsen <i>et al </i> 2006). Nanobodies can also be functional when as fusions or conjugates to other proteins for example to target enzymes to specific cells (Cortez-Retamozo 2004), so there is much potential for targeting a wide array of contaminants when fused to cellulose binding domains.<br />
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<section id="results"><br />
<h2>Results</h2><br />
<p>The assay for strength of CBD binding utilised our sfGFP-CBD fusion proteins. Strength of binding was assayed between the CBDs by assuming it will be proportional to the level of fluorescence which remains after washing of cellulose coated plates(link to plates protocol)<br />
<div class="accordion"><br />
<h3>Preliminary Assay</h3><br />
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<p>text</p><br />
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<h3>Full Assay</h3><br />
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<p>text</p><br />
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<section id="references"><br />
<h2>References</h2><br />
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<ul><br />
<li>Andrade, F.K., Costa, R., Domingues, L., Soares, R., et al. (2010) Improving bacterial cellulose for blood vessel replacement: Functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide. Acta biomaterialia. [Online] 6 (10), 4034–4041. Available from: doi:10.1016/j.actbio.2010.04.023.</li><br />
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<li>Andrade, F.K., Moreira, S.M.G., Domingues, L. & Gama, F.M.P. (2010) Improving the affinity of fibroblasts for bacterial cellulose using carbohydrate-binding modules fused to RGD. Journal of biomedical materials research. Part A. [Online] 92 (1), 9–17. Available from: doi:10.1002/jbm.a.32284.</li><br />
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<li>Auriol, M., Filali-Meknassi, Y., Adams, C.D., Tyagi, R.D., et al. (2008) Removal of estrogenic activity of natural and synthetic hormones from a municipal wastewater: efficiency of horseradish peroxidase and laccase from Trametes versicolor. Chemosphere. [Online] 70 (3), 445–452. Available from: doi:10.1016/j.chemosphere.2007.06.064.</li><br />
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<li>Bae, W., Chen, W., Mulchandani, A. & Mehra, R.K. (2000) Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnology and bioengineering. 70 (5), 518–524.</li><br />
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<li>Biondo, R., da Silva, F.A., Vicente, E.J., Souza Sarkis, J.E., et al. (2012) Synthetic phytochelatin surface display in Cupriavidus metallidurans CH34 for enhanced metals bioremediation. Environmental science & technology. 46 (15), 8325–8332.</li><br />
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<li>Bontidean, I., Ahlqvist, J., Mulchandani, A., Chen, W., et al. (2003) Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection. Biosensors and Bioelectronics. 18 (5-6), 547–553.</li><br />
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<li>Chaturvedi, R. & Archana, G. (2014) Cytosolic expression of synthetic phytochelatin and bacterial metallothionein genes in Deinococcus radiodurans R1 for enhanced tolerance and bioaccumulation of cadmium. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine. 27 (3), 471–482.</li><br />
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<li>Cobbett, C. & Goldsbrough, P. (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annual review of plant biology. [Online] 53159–182. Available from: doi:10.1146/annurev.arplant.53.100301.135154.</li><br />
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<li>Cortez-Retamozo, V. (2004) Efficient Cancer Therapy with a Nanobody-Based Conjugate. Cancer Research. [Online] 64 (8), 2853–2857. Available from: doi:10.1158/0008-5472.CAN-03-3935.</li><br />
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<li>Gilkes, N.R., Kilburn, D.G., Miller, R.C. & Warren, R.A. (1989) Structural and functional analysis of a bacterial cellulase by proteolysis. The Journal of biological chemistry. 264 (30), 17802–17808.</li><br />
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<li>Harmsen, M.M., van Solt, C.B., van Zijderveld-van Bemmel, A.M., Niewold, T.A., et al. (2006) Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Applied microbiology and biotechnology. [Online] 72 (3), 544–551. Available from: doi:10.1007/s00253-005-0300-7.</li><br />
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<li>Kauffmann, C., Shoseyov, O., Shpigel, E., Bayer, E.A., et al. (2000) Novel Methodology for Enzymatic Removal of Atrazine from Water by CBD-Fusion Protein Immobilized on Cellulose. Environmental Science & Technology. [Online] 34 (7), 1292–1296. Available from: doi:10.1021/es990754h.</li><br />
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<li>Kim, H.-D., Choi, S.-L., Kim, H., Sohn, J.H., et al. (2013) Enzyme-linked assay of cellulose-binding domain functions from Cellulomonas fimi on multi-well microtiter plate. Biotechnology and Bioprocess Engineering. 18 (3), 575–580.</li><br />
<br />
<li>Lee, K.-Y., Buldum, G., Mantalaris, A. and Bismarck, A. (2014), More Than Meets the Eye in Bacterial Cellulose: Biosynthesis, Bioprocessing, and Applications in Advanced Fiber Composites. Macromol. Biosci., 14: 10–32. doi: 10.1002/mabi.201300298</li><br />
<br />
<li>Linder, M.; Salovuori, I.; Ruohonen, L.; Teeri, T.T., 1996. Characterization of a Double Cellulose-binding Domain. SYNERGISTIC HIGH AFFINITY BINDING TO CRYSTALLINE CELLULOSE. Journal of Biological Chemistry, 271(35), pp.21268–21272. Available at: http://www.jbc.org/content/271/35/21268.full</li><br />
<br />
<li>Nguyen, P.Q., Botyanszki, Z., Tay, P.K.R. & Joshi, N.S. (2014) Programmable biofilm-based materials from engineered curli nanofibres. Nature communications. [Online] 54945. Available from: doi:10.1038/ncomms5945.</li><br />
<br />
<li>Pinaud, F., King, D., Moore, H.-P. & Weiss, S. (2004) Bioactivation and cell targeting of semiconductor CdSe/ZnS nanocrystals with phytochelatin-related peptides. Journal of the American Chemical Society. 126 (19), 6115–6123.</li><br />
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<br />
<li>Ramos, R., Domingues, L. & Gama, M. (2010) Escherichia coli expression and purification of LL37 fused to a family III carbohydrate-binding module from Clostridium thermocellum. Protein expression and purification. [Online] 71 (1), 1–7. Available from: doi:10.1016/j.pep.2009.10.016.</li><br />
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<li>Warren, R.A., Beck, C.F., Gilkes, N.R., Kilburn, D.G., et al. (1986) Sequence conservation and region shuffling in an endoglucanase and an exoglucanase from Cellulomonas fimi. Proteins. 1 (4), 335–341.</li><br />
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<li>Xu, Z., Bae, W., Mulchandani, A., Mehra, R.K., et al. (2002) Heavy Metal Removal by Novel CBD-EC20 Sorbents Immobilized on Cellulose. Biomacromolecules. [Online] 3 (3), 462–465.</li><br />
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<li>Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., et al. (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences of the United States of America. [Online] 109 (12), E690–E697. Available from: doi:10.1073/pnas.1115485109.</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/FunctionalisationTeam:Imperial/Functionalisation2014-10-18T02:11:30Z<p>Xen sm: </p>
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<h1>Functionalisation</h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#CBDs">CBDs</a><br />
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<li><a data-scroll href="#binding-partners">Functionalisation proteins</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Attaching functional proteins to cellulose can expand the properties of cellulose and allow us to selectivity capture specific contaminants in water. By creating fusion proteins of sfGFP and metal-binding proteins with five different cellulose binding domains, three of which are new to the registry, we were able to characterise the relative affinities of the CBDs and show proof-of-concept removal of contaminants from water.</p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Added three new cellulose binding domains to the registry</li><br />
<li>Performed an assay with sfGFP-fusions to determine the strongest potential binders which could be carried forward for further testing</li><br />
<li>Identified potential metal binding proteins to bind heavy metals from wastewater, including the addition of a new promising polypeptide phytochelatin EC20</li><br />
<li>Tested metal ion capture ability of the CBD-phytochelatin fusion constructs when attached to cellulose</li><br />
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</section><br />
<section id="introduction"><br />
<h2>Introduction</h2><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/a/a0/IC14-func-strategy-both-pursue.png"><br />
<figcaption>Figure 1. Pictorial summary showing the two cellulose functionalization approaches considered in our project. Strategy 1: Cellulose binding domains are expressed as fusions with the protein of interest, separated optionally by a linker sequence. The cellulose binding domains attach to the cellulose by hydrophobic interactions. Strategy 2: Curli fibrils are expressed in parallel with cellulose to form a composite material. The curli fibres coding sequence is modified to contain a ‘SpyTag’ peptide (Zakeri <i>et al</i> 2012) which binds specifically via a covalent isopeptide bond to its protein partner, ‘SpyCatcher’. This SpyCatcher is fused to the proteins of interest. In our project we only had time to pursue strategy 1 but are still excited about the potential of strategy 2 in the future.</figcaption><br />
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<p>Bacterial cellulose is already a useful biomaterial as is due its attractive mechanical properties, with research on applications from loudspeaker diaphragms to protective packaging and wound dressings (Lee 2014). However, our project also aims to extend the properties of cellulose by conjugating functional proteins and peptides to it. This allows us to target specific contaminants in water which can not be filtered by size exclusion alone. (See <a href="https://2014.igem.org/Team:Imperial/Water_Report">water report</a> and <a href="https://2014.igem.org/Team:Imperial/Informing_Design">informing design</a> sections).</p><br />
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<p>We considered two ways of functionalising cellulose (figure 1) but continued to work fully on the cellulose-binding domain (CBD) method. The curli-fibre method would be an exciting avenue to explore in the future; it is feasible since there has already been research into making a “programmable” biomaterial from curli fibres using a peptide tag approach (Nguyen <i>et al</i> 2014), and in combination with cellulose could produce a composite with robust mechanical properties as well.<br />
</p><br />
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<section id="aim"><br />
<h2>Aims</h2><br />
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<p>We aimed to make and characterise new cellulose binding domains, and fuse them to functional proteins. (Incomplete)</p><br />
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<section id="CBDs"><br />
<h2>Cellulose Binding Domains (CBDs)</h2><br />
<p>The cellulose binding domain (CBD) is the link between our functional proteins and the cellulose. Therefore, we wanted to use a CBD which had high affinity and strong binding to cellulose. However, weaker CBDs may still be useful since they could have the potential be controllably eluted under specific conditions, thereby enabling regeneration of the filter when saturated, and controlled disposal of the collected contaminants (refer to water report if relevant?).<br />
</p><br />
<p>We started our search by looking into the <a href="http://parts.igem.org/">parts registry</a> and found two CBDs already existed in the registry: CBDclos (<a href="http://parts.igem.org/Part:BBa_K863111">BBa_K863111</a>) and CBDcex (<a href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>). We also made three new CBDs to contribute to the registry: dCBD, CBDcipA, and CBDcenA. All the CBDs are in RFC(25) freiberg fusion format to allow for ease of use in protein fusions.</p><br />
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<div class="accordion"><br />
<h3>CBDclos and CBDcex</h3><br />
<div><br />
<p>These parts were biobricked by <a href="https://2012.igem.org/Team:Bielefeld-Germany/Results/cbc">Bielefeld 2012</a>, please see their part pages (<a href="http://parts.igem.org/Part:BBa_K863111">BBa_K863111</a>a and <a href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>) for detailed information. CBDclos is from the cellulolytic bacterium <em>Clostridium cellulovorans</em> and CBDcex originates from the cellulolytic bacterium <em>Cellulomonas fimi</em>. This is the same organism as for CBDcenA; cex is from the exoglucanase gene and cen is from the endoglucanase gene.</p><br />
</div><br />
<h3>Double CBD (dCBD) with N-terminal linker</h3><br />
<div><br />
<p>This part, <a href="http://parts.igem.org/Part:BBa_K1321340">BBa_K1321340</a> is based on the double cellulose-binding domain construct synthesised and characterised by Linder <i>et al</i>1996, who found that this double CBD had higher affinity for cellulose than either of the two CBDs on their own. The main difference is that our part contains an additional linker sequence on the N-terminus of the protein.<br />
</p><br />
<p>The two CBDs are from the fungus <i>Trichoderma reesei</i> (<i>Hypocrea jecorina</i>) Exocellobiohydrolase (Exoglucanase) I (cbh1), uniprot ID P62694; and Exocellobiohydrolase (Exoglucanase) II, uniprot ID P07987 (cbh2); with a linker peptide between the two CBDs and at the N-terminus of the protein. Both linkers are the same amino acid sequence and are based on the endogenous linker sequences that exists in cbh1 and cbh2 genes. The linker sequence is PGANPPGTTTTSRPATTTGSSPGP which is the same as used by Linder <i>et al</i> 1996. The first three amino acids are from the cbh2 endogenous linker, and the rest is from the cbh1 endogenous linker. CBDcbh1 is placed C-terminal to CBDcbh2 because naturally CBDcbh1 is a C-terminal domain and CBDcbh2 is an N-terminal domain. Both CBDs are from the <a class='iframe' href="http://www.cazy.org/CBM1.html">CBM family 1</a>. The precise location of the CBD within the cbh genes was slightly different according to the uniprot annotations and the sequence used by Linder <i>et al</i> 1996; we chose to use the sequence from the paper since the protein was expressed and characterised successfully.</p><br />
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</div><br />
<h3>CBDcipA with N and C-terminal linker</h3><br />
<div><br />
<p>This CBD is from the Cellulosomal -scaffolding protein A (cipA) of Clostridium thermocellum including the endogenous linker sequences at the N and C-terminus (<a class='iframe' href="http://www.uniprot.org/uniprot/Q06851">UniProt ID Q06851</a>). It is in RFC25 format to allow for easy use in protein fusions.</p><br />
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<p>The CBD is part of the <a class='iframe' href="http://www.cazy.org/CBM3.html">CBM3 family</a> . This CBD has been used in many application: in fusions with cell adhesion peptides to enhance the properties of cellulose as a cell-growth matrix (Andrade <i>et al </i> 2010a, Andrade <i>et al </i> 2010b), fused to enzymes to remove contaminants from water (Kauffmann <i> et al </i> 2000) and fused to an antimicrobial peptide (Ramos, Domingues & Gama 2010).</p><br />
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<p>At present the cloning for these constructs is still in progress to correct an illegal EcoRI site which was identified in the parts with this CBD. This can be achieved with a silent mutation via site-directed mutagenesis and we aim to send these parts to the registry once thi<br />
</p><br />
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</div><br />
<h3>CBDcenA with C-terminal linker</h3><br />
<div><br />
<p>This CBD is from the Endoglucanase A (cenA) gene from <i>Cellulomonas fimi</i> and also contains an endogenous C-terminal linker. The cellulose binding domain region occurs at the N-terminus of the cenA gene (UniProt P07984 link to http://www.uniprot.org/uniprot/P07984) and is from the <a class='iframe' href="http://www.cazy.org/CBM2.html">CBM family 2</a><br />
</p><br />
<p>The linker sequence (PTTSPTPTPTPTTPTPTPTPTPTPTPTVTP) is Pro-Thr box which has an extended conformation and acts as a hinge region (Gilkes <i>et al</i> 1989). The endoglucanase CBDcenA has 50% homology to the exoglucanase CBDcex (<a class="iframe" href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>) and the linker is highly conserved in both, but the order of the catalytic, linker and cellulose-binding regions is reversed (Warren <i>et al</i> 1986). It has been shown that CBDcenA has the highest binding affinity for crystalline cellulose out of the <i>C. fimi</i> CBDs (Kim <i>et al</i> 2013).<br />
</p><br />
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</section><br />
<section id="binding-partners"><br />
<h2>Functionalisation proteins: Binding partners</h2><br />
<h3>Superfolder Green Fluorescent Protein (sfGFP) </h3><br />
<figure class="content-image image-right"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/8/85/IC14-sfGFP-part-table.PNG> <br />
<figcaption>sfGFP-CBD fusion parts</figcaption> <br />
</figure> <p> sfGFP is a more stable mutant of <a href="http://parts.igem.org/Parts:BBa_E0040">mut3GFP</a> and we chose to use this as a robust, easily visible protein to initially fuse to our CBDs. We also designed one of our assays [insert link on 'one of our assays'] for characterising CBD binding using the sfGFP fusion constructs. sfGFP already existed in the registry (<a href="http://parts.igem.org/Part:BBa_I746916">BBa_I746916</a>), detailed information on sfGFP properties is included in this part description) however since we needed to make fusion proteins with our CBDs we modified the part and submitted it in RFC[25] Freiberg fusion format (<a href="http://parts.igem.org/Parts:BBa_K1321337">K1321337</a>). We created the following fusion parts with sfGFP: <br />
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</p><br />
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<h3> Metal Binding Proteins </h3><br />
<p>One of the contaminants we can filter out which current size-exclusion technologies can not target, are <a href="https://2014.igem.org/Team:Imperial/Water_Report">heavy metals</a>. We have worked with four different metal binding proteins: the metallothioneins SmtA and fMT, a histidine-rich nickel binding protein (NiBP) from the bacterium <i>Heliobacter pylori</i> and a synthetic phytochelatin (PC) EC20.<br />
</p><br />
<div class="accordion"><br />
<h3>SmtA and fMT metallothioneins</h3><br />
<div><br />
<p>These two parts already existed in the BioBrick registry, but since we needed to use them as fusions with the CBDs we improved the parts by making them compatible with the <a class="iframe" href="http://parts.igem.org/Assembly_standard_25">RFC(25) Frieburg Fusion BioBrick standard.</a></p><br />
<p>Metallothioneins (MTs) are genetically encoded cysteine-rich peptides which serve a role in the detoxification of heavy metals in plants, animals and some prokaryotes (Cobbett, 2002). They have a similar structure and function to phytochelatins, but are produced by different mechanisms (Cobbett, 2002). SmtA is from the cyanobacteria <i>Synechococcus sp.</i> and was used by the <a target="_blank" href="https://2011.igem.org/Team:Tokyo-NoKoGen">Tokyo-NoKoGen 2011</a> iGEM team (who submitted the RFC10 part originally, <a class="iframe" href="http://parts.igem.org/Part:BBa_K519010">BBa_K519010</a>) to confer cadmium (Cd2+) tolerance to <em>E. coli</em> cells when expressed. fMT originates from the seaweed <i>Fucus vesiculosus</i> and was submitted by <a target="_blank" href="https://2009.igem.org/Team:Groningen">Groningen 2009 iGEM team </a> and detailed information can be found on the original part page (<a class="iframe" href="http://parts.igem.org/Part:BBa_K190019">BBa_K190019</a>).</p><br />
<p>We successfully <a href="https://2014.igem.org/Team:Imperial/Protocols#general">cloned</a> the following CBD fusion constructs: </p><br />
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<figure class="content-image image-right image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/a/ac/IC14-SmtA-part-table.PNG> <br />
<figcaption>SmtA-CBD fusion parts</figcaption> <br />
</figure> <br />
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<figure class="content-image image-left image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/6/62/IC14-fMT-part-table.PNG> <br />
<figcaption>fMT-CBD fusion parts</figcaption> <br />
</figure> <br />
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<h3>Nickel binding protein (NiBP)</h3><br />
<div><br />
<figure class="content-image image-right"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/f/f6/IC14-NiBP-part-table.PNG> <br />
<figcaption>NiBP-CBD fusions parts</figcaption> <br />
</figure><br />
<p><br />
This protein is the histidine-rich nickel-binding protein from <i>Heliobacter pylori</i> (GenBank accession ACX98466.1), and already existed as a BioBrick in the registry (<a class="iframe" href="http://parts.igem.org/Part:K1151001">K1151001</a>). Since we needed to use this part in fusion proteins we improved the part by submitting it as a basic part in RFC(25) (link) Freiberg fusion format (<a class="iframe" href="http://parts.igem.org/Part:K1321009">K1321009</a>).<br />
</p><br />
<p>We successfully <a href="https://2014.igem.org/Team:Imperial/Protocols#general">cloned</a> the following CBD fusion constructs with NiBP:<br />
<br />
</p><br />
<p>However we discovered late on in the project that the DNA sequence of the NiBP protein in the registry is out of frame, leading to an incorrect protein translation and read-through of the stop codon (figure 2).</p><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/8/80/IC14-NiBP-protein-alignment.PNG"><br />
<figcaption>Figure 2. Alignment of BBa_K1151001 NiBP in the registry to the , showing incorrect amino-acid sequence in red compared to <a target="_blank" href="http://www.ncbi.nlm.nih.gov/protein/261838700">native <i>H.pylori</i></a> sequence.<br />
</figcaption><br />
</figure><br />
<figure class="content-image image-right"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/e/e1/IC14-NiBP-DNA-alignment.PNG"><br />
<figcaption>Figure 3. Single nucleotide deletion in BBa_K1151001 compared to native <i>H.pylori</i> sequence (<a target="_blank" href="http://www.ncbi.nlm.nih.gov/nuccore/261837457">GenBank CP000012.1</a> (link ) bp1391851-1392033, reverse complement) resulting in any downstream nucleotides being out of frame.</figcaption><br />
</figure><br />
<br />
<p>Further investigation into the error let us to identify a single nucleotide deletion in <a class="iframe" href="http://parts.igem.org/Part:BBa_K1151001">BBa_K1151001</a> compared to the native <i>H.pylori</i> DNA sequence (<a target="_blank" href="http://www.ncbi.nlm.nih.gov/nuccore/261837457">GenBank CP000012.1</a> bp1391851-1392033, reverse complement strand). As seen in Figure 3 the deletion is a ‘C’ at bp 53 of the native sequence. This error was propagated through all our NiBP constructs and means that any subsequent C-terminal fusions are also out of frame and we were unable to express and characterise these constructs. We have noted this on the description pages for each of the parts and hope that in the future they will still be useful, since the error could be corrected relatively easily by a team who wishes to use them by site-directed-mutagenesis PCR to insert the missing nucleotide. </p><br />
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</div><br />
<h3>Synthetic phytochelatin EC20</h3><br />
<div><br />
<p>This is our new contribution of a metal-binding protein to the parts registry. Synthetic phytochelatins are analogues of phytochelatins (PCs); cysteine-rich peptides of the general structure (γ-GluCys)n-Gly (n~2-5) which bind and sequester heavy metals, with a role in detoxification (Cobbett & Goldsbrough 2002). PCs are found mainly in plants but also in other organisms including diatoms, fungi, algae (including cyanobacteria) and some invertebrates (Cobbett & Goldsbrough 2002, Rea 2012). Natively, the PCs are enzymatically synthesized by phytochelatin synthase (PCS) from a glutathione (GSH) precursor and the amino acids in the resulting peptide are linked by the non-standard gamma peptide bonds (Cobbett & Goldsbrough 2002, Rea 2012). However, genetically encoded analogues (containing normal alpha-peptide bonds) of different number Glu-Cys (EC) repeats have been characterised, with the EC20 having the highest binding (Bae <i>et al</i> 2000).<br />
</p><br />
<br />
<br />
<figure class="content-image image-right image-small"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/18/IC14-Biondo-et-al-2012-ion-table.PNG"><br />
<figcaption>Table 1. Metal ion adsorption increase of C. metallidurans cells with PC EC20 displayed on cell surface, compared to negative control. Taken from Biondo <i>et al</i> 2012</figcaption><br />
</figure><br />
<p>The main metal PCs confer tolerance to is Cadmium (Cobbett & Goldsbrough 2002) with a reported stoichiometry of 10 Cd2+ per peptide (Bae <i>et al</i>, . EC20 peptide has still showed good metal ion binding whilst fused to a cellulose binding domain (CBD) for water purification (Xu <i>et al</i> 2002) and when anchored to bacterial cell membrane to confer Cd2+ tolerance (Bae <i>et al</i> 2000, Chaturvedi & Archana 2014). PC EC20 has also been shown to bind many more heavy metals. For example, when displayed on the surface of Cupriavidus metallidurans, the cells capacity to adsorb ions increased by 31 to 219% depending on the ion (table 1, Biondo <i>et al</i> 2012). These binding properties of PC EC20 have been utilised in applications including a regeneratable biosensor of Hg2+, Cd2+, Pb2+, Cu2+ and Zn2+ ions to 100 fM–10 mM (Bontidean <i>et al</i> 2003), and to target CdSe/ZnS quantum dots to streptavidin expressing HeLa cells when biotinylated (Pinaud <i>et al</i> 2004). INSERT TABLE OF THE IONS FROM THE PAPER<br />
</p><br />
<p>We used this part to <a href="https://2014.igem.org/Team:Imperial/Protocols#general">clone</a> a library of different cellulose-binding domains fused to phytochelatin:<figure class="content-image"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/4/40/IC14-PC-part-table.PNG > <br />
<figcaption>Phytochelatin-CBD fusion parts. </figcaption> <br />
</figure> <br />
</p><br />
</div><br />
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</div><br />
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<br />
<br />
<h3> Other potential proteins </h3><br />
<p>During our research we also explored other functional proteins to fuse to the CBDs.<br />
<div class="accordion"><br />
<h3>Laccases</h3><br />
<div><br />
<p>Laccases are enzymes which can break down aromatic and phenolic compounds such as modified estrogens which can end up unfiltered in the water supply with unknown consequences (Auriol <i>et al</i> 2008). This method of treating estrogen in water was explored by <a target="_blank" href="https://2012.igem.org/Team:Bielefeld-Germany">Bielefeld 2012 iGEM team</a>, and our system also has potential to address this.<br />
</p><br />
</div><br />
<br />
<h3>Nanobodies</h3><br />
<div><br />
<p>Nanobodies are fragments of camelid antibodies which can be generated against a variety of antigens and can be easily expressed by organisms such as <i>E. coli </i>. Nanobodies could be used to target viruses, which are often too small to be filtered by size exclusion. For example there are already nanobodies which can bind to <i> Vaccinia </i> virus, ricin, <i>Cholera</i> toxin, Stephylococcal enterotoxin B and H5N1 Influenza (Goldman <i>et al </i> 2006, Ibanez <i>et al </i> 2011) to name a few. Additionally, nanobodies are stable and can be selected to resist proteolytic attach (Harmsen <i>et al </i> 2006). Nanobodies can also be functional when as fusions or conjugates to other proteins for example to target enzymes to specific cells (Cortez-Retamozo 2004), so there is much potential for targeting a wide array of contaminants when fused to cellulose binding domains.<br />
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</p><br />
</div><br />
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</section><br />
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<section id="results"><br />
<h2>Results</h2><br />
<div class="accordion"><br />
<h3>Preliminary Assay</h3><br />
<div><br />
<p>text</p><br />
</div><br />
<h3>Full Assay</h3><br />
<div><br />
<p>text</p><br />
</div><br />
</div><br />
</section><br />
<br />
<section id="references"><br />
<h2>References</h2><br />
<br />
<ul><br />
<li>Andrade, F.K., Costa, R., Domingues, L., Soares, R., et al. (2010) Improving bacterial cellulose for blood vessel replacement: Functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide. Acta biomaterialia. [Online] 6 (10), 4034–4041. Available from: doi:10.1016/j.actbio.2010.04.023.</li><br />
<br />
<li>Andrade, F.K., Moreira, S.M.G., Domingues, L. & Gama, F.M.P. (2010) Improving the affinity of fibroblasts for bacterial cellulose using carbohydrate-binding modules fused to RGD. Journal of biomedical materials research. Part A. [Online] 92 (1), 9–17. Available from: doi:10.1002/jbm.a.32284.</li><br />
<br />
<li>Auriol, M., Filali-Meknassi, Y., Adams, C.D., Tyagi, R.D., et al. (2008) Removal of estrogenic activity of natural and synthetic hormones from a municipal wastewater: efficiency of horseradish peroxidase and laccase from Trametes versicolor. Chemosphere. [Online] 70 (3), 445–452. Available from: doi:10.1016/j.chemosphere.2007.06.064.</li><br />
<br />
<li>Bae, W., Chen, W., Mulchandani, A. & Mehra, R.K. (2000) Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnology and bioengineering. 70 (5), 518–524.</li><br />
<br />
<li>Biondo, R., da Silva, F.A., Vicente, E.J., Souza Sarkis, J.E., et al. (2012) Synthetic phytochelatin surface display in Cupriavidus metallidurans CH34 for enhanced metals bioremediation. Environmental science & technology. 46 (15), 8325–8332.</li><br />
<br />
<li>Bontidean, I., Ahlqvist, J., Mulchandani, A., Chen, W., et al. (2003) Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection. Biosensors and Bioelectronics. 18 (5-6), 547–553.</li><br />
<br />
<li>Chaturvedi, R. & Archana, G. (2014) Cytosolic expression of synthetic phytochelatin and bacterial metallothionein genes in Deinococcus radiodurans R1 for enhanced tolerance and bioaccumulation of cadmium. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine. 27 (3), 471–482.</li><br />
<br />
<br />
<li>Cobbett, C. & Goldsbrough, P. (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annual review of plant biology. [Online] 53159–182. Available from: doi:10.1146/annurev.arplant.53.100301.135154.</li><br />
<br />
<li>Cortez-Retamozo, V. (2004) Efficient Cancer Therapy with a Nanobody-Based Conjugate. Cancer Research. [Online] 64 (8), 2853–2857. Available from: doi:10.1158/0008-5472.CAN-03-3935.</li><br />
<br />
<li>Gilkes, N.R., Kilburn, D.G., Miller, R.C. & Warren, R.A. (1989) Structural and functional analysis of a bacterial cellulase by proteolysis. The Journal of biological chemistry. 264 (30), 17802–17808.</li><br />
<br />
<li>Harmsen, M.M., van Solt, C.B., van Zijderveld-van Bemmel, A.M., Niewold, T.A., et al. (2006) Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Applied microbiology and biotechnology. [Online] 72 (3), 544–551. Available from: doi:10.1007/s00253-005-0300-7.</li><br />
<br />
<li>Kauffmann, C., Shoseyov, O., Shpigel, E., Bayer, E.A., et al. (2000) Novel Methodology for Enzymatic Removal of Atrazine from Water by CBD-Fusion Protein Immobilized on Cellulose. Environmental Science & Technology. [Online] 34 (7), 1292–1296. Available from: doi:10.1021/es990754h.</li><br />
<br />
<li>Kim, H.-D., Choi, S.-L., Kim, H., Sohn, J.H., et al. (2013) Enzyme-linked assay of cellulose-binding domain functions from Cellulomonas fimi on multi-well microtiter plate. Biotechnology and Bioprocess Engineering. 18 (3), 575–580.</li><br />
<br />
<li>Lee, K.-Y., Buldum, G., Mantalaris, A. and Bismarck, A. (2014), More Than Meets the Eye in Bacterial Cellulose: Biosynthesis, Bioprocessing, and Applications in Advanced Fiber Composites. Macromol. Biosci., 14: 10–32. doi: 10.1002/mabi.201300298</li><br />
<br />
<li>Linder, M.; Salovuori, I.; Ruohonen, L.; Teeri, T.T., 1996. Characterization of a Double Cellulose-binding Domain. SYNERGISTIC HIGH AFFINITY BINDING TO CRYSTALLINE CELLULOSE. Journal of Biological Chemistry, 271(35), pp.21268–21272. Available at: http://www.jbc.org/content/271/35/21268.full</li><br />
<br />
<li>Nguyen, P.Q., Botyanszki, Z., Tay, P.K.R. & Joshi, N.S. (2014) Programmable biofilm-based materials from engineered curli nanofibres. Nature communications. [Online] 54945. Available from: doi:10.1038/ncomms5945.</li><br />
<br />
<li>Pinaud, F., King, D., Moore, H.-P. & Weiss, S. (2004) Bioactivation and cell targeting of semiconductor CdSe/ZnS nanocrystals with phytochelatin-related peptides. Journal of the American Chemical Society. 126 (19), 6115–6123.</li><br />
<br />
<br />
<li>Ramos, R., Domingues, L. & Gama, M. (2010) Escherichia coli expression and purification of LL37 fused to a family III carbohydrate-binding module from Clostridium thermocellum. Protein expression and purification. [Online] 71 (1), 1–7. Available from: doi:10.1016/j.pep.2009.10.016.</li><br />
<br />
<li>Warren, R.A., Beck, C.F., Gilkes, N.R., Kilburn, D.G., et al. (1986) Sequence conservation and region shuffling in an endoglucanase and an exoglucanase from Cellulomonas fimi. Proteins. 1 (4), 335–341.</li><br />
<br />
<li>Xu, Z., Bae, W., Mulchandani, A., Mehra, R.K., et al. (2002) Heavy Metal Removal by Novel CBD-EC20 Sorbents Immobilized on Cellulose. Biomacromolecules. [Online] 3 (3), 462–465.</li><br />
<br />
<li>Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., et al. (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences of the United States of America. [Online] 109 (12), E690–E697. Available from: doi:10.1073/pnas.1115485109.</li><br />
</ul><br />
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{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/ProtocolsTeam:Imperial/Protocols2014-10-18T01:32:21Z<p>Xen sm: </p>
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<h1>Protocols</h1><br />
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<ul><br />
<li><a data-scroll href="#gluconacetobacter"><em>G. xylinus</em></a><br />
</li><br />
<li><a data-scroll href="#general">General/<em>E. coli</em></a><br />
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<li><a data-scroll href="#functionalisation">Functionalisation</a><br />
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<p>Here you can find the protocols we used throughout the summer</p><br />
<section id="gluconacetobacter"><br />
<h2><em>G. xylinus</em> Protocols</h2><br />
<div class="accordion"><br />
<h3>Kombucha Protocol</h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Materials<br />
<ul><br />
<li>Organic cider vinegar</li><br />
<li>Granulated sugar</li><br />
<li>One piece of live Kombucha culture</li><br />
<li>Green tea bag</li><br />
<li>Distilled water</li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div class="pure-u-1-2"><br />
<ol><br />
<li>Pour distilled water into 5000 ml or 2000 ml conical flasks, up to 4500 ml or 1800 ml respectively</li><br />
<li>Cover the tops with foil and tape</li><br />
<li>Boil 2L of water to 100 degrees, in the autoclave.</li><br />
<li>Spray with antibacterial spray, and set up trays for growth</li><br />
<li>Pour into container</li><br />
<li>Add green tea and allow to brew for 15 minutes, 1 tea bag per liter of autoclaved water</li><br />
<li>Remove tea bags</li><br />
<li>Add sugar, 100 g per liter</li><br />
<li>Stir until dissolved</li><br />
<li>Cool down to below 30°C</li><br />
<li>Add organic cider vinegar 100 ml per liter of green tea solution</li><br />
<li>Open bag of living Kombucha culture, take pellicle, hold with blue roll</li><br />
<li>Add one piece of Kombucha culture pellicle by cutting a chunk with scissors. It will sink to the bottom of the </li><li>container. Be careful not to add any liquid Kombucha media.</li><br />
<li>Cover growth container with blue roll, be careful not to lean over the trays and keep sterile conditions.</li><br />
<li>Fermentation starts after 48-72h, thin skin and bubbles will be produced and culture will come back out on the surface</li><br />
<li>When product becomes about 2 cm thick, take it out</li><br />
<li>Wash with soaped water</li><br />
<li>Let dry</li><br />
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</ol><br />
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</div><br />
</div><br />
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</div><br />
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<h3><em>G. xylinus</em> HS media and culturing </h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Materials<br />
<ul><br />
<li>For 500ml media:<br />
<li>10g glucose (2%w/v)</li><br />
<li>2.5g yeast extract (0.5% w/v)</li><br />
<li>2.5g peptone (0.5% w/v)</li><br />
<li>1.35g Na2HPO4 (0.27% w/v)</li><br />
<li>0.75g citric acid (0.15% w/v)</li><br />
<li>500ml distilled H20</li><br />
<li>0.5mL cellulase if HS+cellulase media required</li><br />
<li>7.5g of agar (agar-agar) if making HS-agar plates</li><br />
<li>(antibiotics as necessary)</li><br />
<br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div class="pure-u-1-2"><br />
<ol><br />
<li>Add 250ml dH20 to glucose in one bottle and 250ml dH20 to the rest in a second bottle. In incompletely distilled water, glucose will form a solid mass, so stir vigorously immediately after adding water. Autoclave both bottles to sterilize media and pour glucose solution in sterile conditions (next to a Bunsen burner or in a flow hood) into the second bottle. Autoclaving glucose separately from amino acids avoids Maillard reaction, which can result in the formation of toxic byproducts in the media.</li><br />
<li>Streak/inoculate Gluconacetobacter onto plates or into media.</li><br />
<li>Incubate plates at 30°C inverted. Colonies will appear in 48-72 hours.</li><br />
<li>Incubate liquid HS-cultures at 30 °C standing. Standing culturing results in low growth rate, but avoids the formation of cellulose non-producing mutants, which have been reported to appear in shaking conditions.</li><br />
<li>For quick growth, grow with shaking at 180rpm, 30C, with the addition of cellulase. NB! This selects for cellulose non-producing mutants. They can be identified as smooth colonies on plates (cellulose producing colonies have a rough colony morphology - see section <em>Gluconacetobacter</em> for images).</li><br />
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<br />
</ol><br />
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</div><br />
</div><br />
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</div><br />
<h3>Preparing electrocompetent <em>G. xylinus</em> cells</h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Materials<br />
<ul><br />
<li>HS+cellulase media</li><br />
<li>Ice bucket and ice</li><br />
<li>Temperature controlled centrifuge</li><br />
<li>1mM HEPES (ph7.0) 80mL</li><br />
<li>15% glycerol</li><br />
<li>50 mL tubes</li><br />
<li>P1000 pipette and tips</li><br />
<li>Stripettes and automatic pipette for larger volumes</li><br />
<li>Shaker at 30 °C, 180rpm</li><br />
<br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div class="pure-u-1-2"><br />
<ol><br />
<li>If the goal of transformation is to produce cellulose-producing transformed G.xylinus, use regular HS media for culturing. If cellulose production after transformation is not primary and speed is required, HS-cellulase medium can be used. Usage of HS-cellulase medium during growth results in the formation of a higher number of cellulose negative mutants, but results in much higher transformation efficiencies, and requires less time. Even with HS-cellulose medium, cellulose producing colonies can be identified on the plate after transformation, as cellulose-producing colonies differ in morphology from cellulose non-producing colonies (see section <em>Gluconacetobacter</em> for images of colony morphology).</li><br />
<li>Inoculate 5ml of HS or HS+cellulase medium with <em>Gluconacetobacter</em>.<br />
</li><br />
<li>Incubate at 30°C, 180 rpm shaking overnight.</li><br />
<li>Next day, pour 30mL of HS or 15ml of HS+cellulase medium into each of four 50mL tubes. If using regular HS medium, vortex the tubes for 3 minutes to release cells from the cellulose pellicle</li><br />
<li>To each tube, add 1mL of overnight culture and incubate with shaking at 180rpm, 30C.</li><br />
<li>Incubate overnight or until OD600 of around 0.4-0.7 is reached. When using HS-cellulase media, OD600 can reach up to 0.6 -0.7, however when using regular HS medium, OD600 measurement is disturbed by the cellulose pellicle, and can reach up to 0.2. Vortex tubes for 3 minutes before taking the measurements.</li><br />
<li>If using regular HS medium, add 62ul (0.2% v/v) Celluclast cellulase to each tube and incubate at 30C, 180rpm shaking for 2 hours, or until the cellulose pellicle is degraded to completely release the cells. This does not result in higher formation of cel- mutants, as short incubation time and nutrient depletion does not allow for proliferation of cel- mutants.</li><br />
<li>Before continuing, set up the necessary materials:<br />
<ol><br />
<li>Pre-cool centrifuge to 4°C</li><br />
<li>Prepare ice bucket</li><br />
<li>Chill 1mM HEPES buffer and 15% glycerol buffer on ice</li><br />
<br />
</ol><br />
</li><br />
<li>Once the cultures reach desired OD600, take them out of incubation and put them on ice for 10 minutes (the tube should feel cool).</li><br />
<li>From here on, keep cells always cool, at or below 4°C</li><br />
<li>After cooling, spin the tubes in a refrigerated (4°C) centrifuge for 12min at 4100rpm.</li><br />
<li>Pour off supernatant carefully, taking care not to pour off the pellet. <em>G. xylinus</em> does not pellet as easily as E.coli, most likely due to the buffering effects of cellulose. If the pellet is not attached to the wall after centrifugation, smear the pellet onto the wall of the tube and centrifuge again using longer centrifugation times. Re-suspend bacteria in 10mL HEPES: re-suspend first using 1ml HEPES and a P1000 pipette, then add 9ml of HEPES using a stripette; it is much easier to re-suspend the pellet fully using a P1000. Do not use a vortexer.<br />
</li><br />
<li>Pool the samples into a single 50ml tube.</li><br />
<li>Centrifuge again for 14 minutes at 4100 rpm and 4 °C temp. Use a balancer tube with 40ml water.</li><br />
<li>Pour off supernatant again, re-suspend pellet in 10mL ice-cold HEPES on ice as before.</li><br />
<li>Centrifuge again for 14 minutes at 4100 rpm, 4 °C.</li><br />
<li>Pour off supernatant and re-suspend pellet in 4mL ice cold 15% glycerol solution.<br />
</li><br />
<li>Pipette 50ul aliquots into tubes. Label tubes properly.</li><br />
<li>Store samples on ice for immediate use or freeze 50ul aliquots in-80°C. According to some reports, the efficiency of electrocompetent cells reduces after each freezing, so immediate use may result in highest efficiencies.<br />
</li><br />
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<br />
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</ol><br />
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</div><br />
</div><br />
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</div><br />
<h3>gDNA extraction from <em>G.xylinus</em> </h3><br />
<div><br />
<p>This protocol uses the commercially available Qiagen DNA Blood and Tissue kit for gDNA extraction. We have modified it to be amenable for G. xylinus, and deal with the issue of co-purifying contaminants, in order to attain high-quality DNA. If DNA quality is not primary, the original Qiagen protocol may be used. For detailed user-guide and list of required reagents, see Qiagen DNA Blood and Tissue handbook. </p><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Materials<br />
<ul><br />
<li>HS+cellulase media</li><br />
<li>Ice bucket and ice</li><br />
<li>Temperature controlled centrifuge</li><br />
<li>Qiagen DNA Blood and Tissue Kit</li><br />
<li>Zymo DNA Clean and Concentrator 5 kit</li><br />
<li>0.2um filter Millipore filter discs</li><br />
<li>50ml tubes</li><br />
<li>1.5ml tubes</li><br />
<li>Shaker at 30 °C, 180rpm</li><br />
<br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div class="pure-u-1-2"><br />
<ol><br />
<li>Inoculate 4x5ml of HS or HS-cellulase media in 50ml tubes with Gluconacetobacter. I </li><br />
<li>Incubate at 30°C, 180 rpm shaking for 48 hours.</li><br />
<li>Before continuing, set up the necessary materials:<br />
<ol><br />
<li>Pre-cool centrifuge to 4°C</li><br />
<li>Prepare ice bucket</li><br />
<li>Chill HS medium on ice</li><br />
<br />
</ol><br />
</li><br />
<li>Centrifuge 50ml tubes at 4°C, maximum speed for 10 minutes</li><br />
<li>Discard the supernatant and resuspend cells in 5ml of cold HS.</li><br />
<li>Centrifuge and discard the supernatant again as previously</li><br />
<li>Continue with Qiagen DNA Blood and Tissue kit according to the provided handbook until re-suspension with buffer AL.</li><br />
<li>After re-suspension, pool the samples by pipetting the mixture onto a spin column, centrifuging, and pipetting and centrifuging the following mixtures onto the same spin column. Alternatively, different columns can be used, however this results in lower concentrations of DNA in the final preparation.<br />
</li><br />
<li>Continue with DNA Blood and Tissue kit protocol until the elution step.</li><br />
<li>Pre-heat nuclease-free H20 to approximately 50°C by quick microwaving. Pipet 100ul of heated H20 onto the spin column. Wait 15 minutes before centrifugation </li><br />
<li>Repeat the elution step as previously described, elute DNA into a new 1.5ml tube.</li><br />
<li>If high-purity DNA is required, continue with the following steps:</li><br />
<li>Place a 0.2um filter onto distilled water in a Petri dish. Pipet 30ul of extracted DNA onto the filter</li><br />
<li>Wait for 30 minutes to dialyze</li><br />
<li>Pipet DNA into a new tube</li><br />
<li>Use the Zymo Clean and Concentrator kit to further purify the dialyzed DNA, by following the manufacturers instructions until the elution step </li><br />
<li>Elute using 25ul of pre-heated water, wait for 15 minutes before centrifugation. If long-term storage is required, use the elution buffer recommended by the manufacturer. </li><br />
<li>Store the purified DNA at-20°C. </li><br />
<br />
<br />
<br />
</ol><br />
<br />
</div><br />
</div><br />
<br />
<br />
</div><br />
<h3>Transformation of <em>G.xylinus</em> using electroporation<br />
</h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Prepare the following<br />
<ul><br />
<li>Plasmid DNA</li><br />
<li>Electrocompetent <em> G.xylinus cells</em> (in 50µl or 100µl aliquots)</li><br />
<li>Ice bucket and ice</li><br />
<li>1mm path electrocuvettes</li><br />
<li>1.5ml microcentrifuge tubes or PCR tubes</li><br />
<li>An electroporator - set at 2.5kV and 5.9ms</li><br />
<li>HS+cellulase medium</li> <br />
<li>HS agar plates with appropriate antibiotic</li><br />
<li>One aliquot of electrocompetent cells for positive control and one aliquot for negative control.</li><br />
<li>If a plasmid known to work in G. xylinus exists, use this as positive control .</li><br />
<br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div class="pure-u-1-2"><br />
<ol><br />
<li>Set up the electroporator with correct settings: at 2.5kV, 6-8ms, 400ohm resistance, 25microF capacitance.</li><br />
<li>Prepare 800µL HS+cellulase media containing 1.5ml culture tubes and pre-heat to 30 °C. Prepare SOC medium at 37°C. Prepare a space for shaking.</li><br />
<li>Prepare ice bucket, place plasmid DNA and electrocuvettes on ice and thaw electrocompetent cells on ice. <b>NB! Make sure DNA is desalinated before use – ionic solution can otherwise cause arcing.</b><br />
</li><br />
<li>Add <b>2 µL</b> of plasmid DNA to 100 µL of concentrated cells in a cold microcentrifuge or PCR tube and mix well by pipetting. Add plasmid DNA also to one aliquot of electrocompetent E.coli cells (positive control; alternatively also add another known plasmid as pos. control). <b>NB! Do not add plasmid DNA to one aliquot of electrocompetent G. xylinus (negative control)</b><br />
</li><br />
<li>Transfer the cell/DNA mixture and positive and negative controls to a cold 0.2-cm electroporation cuvette. Dry any water condensate outside of the cuvette (using labcoat), place the cuvette into the electroporator, and apply the pulse.<br />
</li><br />
<li>Transfer the pulsed cells into 800 µL of HS+cellulase medium in a culture tube. Transfer E. coli into SOC medium. <b>NB! Prepare everything beforehand and do it quickly.</b><br />
</li><br />
<li>Incubate the culture tubes with shaking (170 rpm) at 30 °C for 4 hours or overnight.</li><br />
<li>When culturing for 4 hours, spin the culture at max available RPM for 10 minutes, resuspend in 200ul and plate on an HS-agar plate with an appropriate antibiotic concentration. If overnight incubation is required, use a 100ul aliquot of the culture for plating, as you will otherwise find a lawn. (The antibiotic concentration of plates is important, be certain to use the antibiotic concentration specific for the G.xylinus strain (see G.xylinus))</li><br />
<li>Grow plates at 30°C inverted, colonies will appear in 24-48 hours </li><br />
<br />
<br />
<br />
</ol><br />
<br />
</div><br />
</div><br />
<br />
<br />
</div><br />
<h3>Quantification of cellulose production</h3><br />
<div><br />
<ol><br />
<li>Add 50ml of HS medium (or other medium of choice) to 250ml conical flask</li><br />
<li>Grow G.xylinus in HS medium for 7 days standing, at 30C. Don’t seal the flasks hermetically in order to allow diffusion of oxygen (seal using foam buns)</li><br />
<li>After 7 days of growth, wash the cellulose twice with distilled water</li><br />
<li>Add 50ml of 0.1M NaOH to cellulose, incubate at 65C for 4 hours</li><br />
<li>Wash the cellulose twice using distilled water</li><br />
<li>Place the formed cellulose pellicle on baking paper and dry the pellicle at 65 degrees for 4 hours-overnight. Before drying, cut out and measure the weight of a piece of baking paper, and dry the pellicle together with the paper. This is because the pellicle will invariably stick to the surface, and removal of it results in loss of cellulose.</li><br />
<li>Place the pellicle+paper into a vacuum desiccator for 2 hours</li><br />
<li>Weigh the pellicle+paper using a high-sensitivity scale. Subtract the weight of the paper to determine the weight of cellulose.</li><br />
<br />
<br />
<br />
</ol><br />
<br />
</div><br />
<h3>gDNA Library preparation for genome sequencing using Illumina Nextera kit </h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-1"><br />
<p>This is a modified protocol for using Illumina Nextera kit for preparation of gDNA library for genome sequencing. We have modified the original Illumina protocol to be amenable for low sample number and have a lower cost. We used this protocol to prepare gDNA libraries of G. xylinus ATCC 53582 strain and the G. xylinus strain we isolated from Kombucha tea at the beginning of this summer. For the full protocol and required materials, see Illumina Nextera Kit user guide.</p><br />
<h4>Preparation</h4><br />
<ol><br />
<li>Prepare 200ul PCR tubes, ice bucket, ice</li><br />
<li>Remove the TD, TDE1, and genomic DNA from -15° to -25°C storage and thaw on ice.</li><br />
<li>Remove RSB from -15° to -25C storage and thaw at room temperature.<br />
</li><br />
<li>After thawing, ensure all reagents are adequately mixed by gently inverting the tubes 3–5 times, followed by a brief spin in a microcentrifuge. NOTE: Ensure the reaction is assembled in the order described for optimal performance. Some sources recommend setting up the reaction on ice, as transposons may exhibit a low activity at room temperature</li><br />
<li>Label the PCR tubes with a smudge resistant pen - important, as labels tend to degrade in a thermocycler.<br />
</li><br />
<li>Add 20 μl of genomic DNA at 2.5 ng/μl (50 ng total) to each PCR tube<br />
</li><br />
<li>Add 25 μl of TD Buffer to the wells containing genomic DNA. Change tips between samples. NOTE:Calculate the total volume of TD for all reactions, and divide among an appropriate number of tubes in an 8-well PCR strip tube.</li><br />
<li>Add 5 μl of TDE1 to the tubes containing genomic DNA</li><br />
<li>Gently pipette up and down 10 times to mix the reaction.<br />
</li><br />
<li>Centrifuge at 280g for 1 minute<br />
</li><br />
<li>Place the PCR tubes into the thermocycler and run the following program:<br />
<ol><br />
<li>Heated lid on</li><br />
<li>55°C for 5 minutes</li><br />
<li>Hold at 10°C</li><br />
</ol><br />
</li><br />
<li>While tagmentation is ongoing, label 1.5ml tubes accordingly for the next step and add 180ul of Zymo DNA binding buffer</li><br />
<li>Also, remove NPM, PPC, and the index primers from -15° to -25°C storage and thaw on a bench at room temperature.</li><br />
<li>Allow approximately 20 minutes to thaw NPM, PPC, and index primers<br />
</li><br />
<li>After tagmentation reaction has finished, proceed immediately to Zymo cleanup - transport samples on ice and do not wait before proceeding to Zymo cleanup, as transposons remain active at lower temperatures, albeit at low level, which may result in overtagmentation and fragments shorter than optimal.</li><br />
<br />
</ol><br />
<h4>Zymo cleanup protocol</h4><br />
<ol><br />
<li>Transfer 50 μl of tagmentation reaction into 1.5ml tubes containing Zymo binding buffer</li><br />
<li>Gently pipette up and down 10x to mix well</li><br />
<li>Transfer the solution to Zymo spin columns</li><br />
<li>Centrifuge at 10000rpm for 1 minute.</li><br />
<li>Discard the flow-through or the collecting tube and place the column into a new collection tube</li><br />
<li>Wash the Zymo spin column twice by:<br />
<ol><br />
<li>Pipette 300ul of Zymo wash buffer to the tubes</li><br />
<li>Centrifuge at 10000rpm for 1 minute, discarding the flow through</li><br />
<li>Repeat the wash step for a total number of 2 washes.</li><br />
<li>Centrifuge at 10000rpm 1 min to ensure no wash buffer remains.</li><br />
<li>Add 25 μl of RSB directly to the column matrix in each well. Confirm visually that RSB has absorbed onto the matrix, and is not on the side of the tube. If it is, flick the tube gently to force the liquid onto the matrix</li><br />
<li>Incubate the tubes for 5 minutes at room temperature. If time is not a limiting factor, increase incubation time to 15 minutes, as this may result in higher elution effieciencies</li><br />
<li>Centrifuge the tubes at 10000rpm for 1 minute.</li><br />
<li>Place the tubes on ice until proceeding to PCR</li><br />
</ol><br />
</li><br />
</ol><br />
<h4>PCR</h4><br />
<ol><br />
<li>After NPM, PPC, and Index primers are completely thawed, gently invert each tube 3–5 times to mix and briefly centrifuge the tubes in a microcentrifuge. Use 1.7 ml Eppendorf tubes as adapters for the microcentrifuge.</li><br />
<li>Label PCR tubes according to your samples</li><br />
<li>Add 5 μl of index 2 primers (white caps) to each tube respectively.</li><br />
<li>Add 5 μl of index 1 primers (orange caps) to each tube respectively.</li><br />
<li>Add 15 μl of NPM (Nextera PCR master mix) to each well of the NAP1 plate containing index primers.</li><br />
<li>Add 5 μl PPC (PCR primer cocktail) to each well containing index primers and NPM.</li><br />
<li>Transfer 20 μl of purified tagmented DNA to the corresponding PCR tube</li><br />
<li>Gently pipette up and down 3–5 times to thoroughly combine the DNA with the PCR mix.</li><br />
<li>Spin down the tubes with quick centrifugation .</li><br />
<li>Ensure that the thermocycler lid is heated during the incubation.Run the PCR using the following program:<br />
<ol><br />
<li>72°C for 3 minutes</li><br />
<li>98°C for 30 seconds</li><br />
<li>5 cycles of:<br />
<ol><br />
<li>98°C for 10 seconds</li><br />
<li>63°C for 30 seconds</li><br />
<li>72°C for 3 minutes</li><br />
<br />
<br />
<br />
</ol><br />
</li><br />
<li>Hold at 10°C</li><br />
<br />
<br />
</ol><br />
</li><br />
<li>Ensure that the 72°C step preceeds the rest of the program.<br />
</li><br />
<li>SAFE STOPPING POINT – can store the DNA in thermocycler overnight, or at 2-8°C for 2 days<br />
</li><br />
<br />
<br />
<br />
<br />
</ol><br />
<h4>Clean-up of PCR using AMPure beads</h4><br />
<ol><br />
<li>Bring the AMPure XP beads to room temperature.</li><br />
<li>Prepare fresh 80% ethanol from absolute ethanol. (Always prepare fresh 80% ethanol for wash steps. Ethanol can absorb water from the air impacting your results.)</li><br />
<li>xCentrifuge the PCR tubes containing the limited cycle PCR product at quickly to spin down the liquid.</li><br />
<li>Label new 1.5ml tubes according to the samples for purification steps</li><br />
<li>Transfer 50 μl of the PCR product from the PCR tubes into new 1.5ml tubes. Change tips between samples.</li><br />
<li>Vortex the AMPure XP beads for 30 seconds to ensure that the beads are evenly dispersed.</li><br />
<li>Add 30 μl of AMPure XP beads to each tube containing the PCR product. For 2x250 runs on the MiSeq, add 25 μl of AMPure XP beads to each tube.</li><br />
<li>Gently pipette mix up and down (gently, so as not to introduce bubbles) until solution is homogeneous. Make sure to pipette, no to vortex the solution, as this results in liquid collecting under the tube cap. This can't be removed easily as centrifugation also results in sedimentation of AMPure beads.</li><br />
<li>Incubate the tubes (containing AMPure beads and PCR product) at room temperature without shaking for 5 minutes.</li><br />
<li>Place the plate on a magnetic stand for 2 minutes or until the supernatant has cleared.</li><br />
<li>With the tubes on the magnetic stand, carefully remove and discard the supernatant. Pieptte carefully, as AMPure beads may follow the surface of the liquid. If any beads are inadvertently aspirated into the tips, dispense the beads back to the plate and let the plate rest on the magnet for 2 minutes and confirm that the supernatant has cleared.</li><br />
<li>With the tubes on the magnetic stand, wash the beads with freshly prepared 80% ethanol as follows:<br />
<ol><br />
<li>Add 200 μl of freshly prepared 80% ethanol to each sample well. You should not resuspend the beads at this time.</li><br />
<li>Incubate the plate on the magnetic stand for 30 seconds or until the supernatant appears clear.</li><br />
<li>Carefully remove and discard the supernatant.</li><br />
</ol><br />
</li><br />
<li>With the tubes on the magnetic stand, perform a second ethanol wash as follows:<br />
<br />
<ol><br />
<li>Add 200 μl of freshly prepared 80% ethanol to each sample.</li><br />
<li>Incubate the plate on the magnetic stand for 30 seconds or until the supernatant appears clear.</li><br />
<li>Carefully remove and discard the supernatant. Use a P20 multichannel pipette with fine pipette tips to remove excess ethanol.</li><br />
</ol><br />
</li><br />
<li>With the tubes still on the magnetic stand, allow the beads to air-dry for 15 minutes.<br />
</li><br />
<li>Remove the tubes from the magnetic stand. Add 32.5 μl of RSB to each well of the NAP2 plate.<br />
</li><br />
<li>Gently pipette mix up and down until the solution is homogenous, changing tips after each column.<br />
</li><br />
<li>Incubate at room temperature for 2 minutes.<br />
</li><br />
<li>Place the tubes on the magnetic stand for 2 minutes or until the supernatant has cleared.</li><br />
<li>Label new 1.5ml tubes accordingly</li><br />
<li>Carefully transfer 30 μl of the supernatant into the new tubes. These will contain your purified library.</li><br />
<li>Store the library in -20C until further processing.</li><br />
<br />
<br />
<br />
<br />
<br />
</ol><br />
<br />
<br />
</div><br />
<br />
</div><br />
<br />
<br />
</div><br />
</div><br />
</section><br />
<br />
<section id="general"><br />
<h2>General/<em>E. coli</em> Protocols</h2><br />
<div class="accordion"><br />
<h3>General Cloning Workflow</h3><br />
<div><br />
<ol> <br />
<li>PCR was often used to generate the insert fragments, for example to add on RFC25 prefix and suffixes to RFC10 parts, or to clone out a desired coding sequence for a new biobrick. In each case primers were designed with overhangs containing the desired prefix and suffix sequences. PCR reactions were set up using Phusion or Q5 (NEB) polymerases, with reactions set up as per manufacturer protocol, with replicates for each construct. PCR programmes varied but typically utilised a touchdown protocol which improves accuracy of primer binding at the beginning of the reaction due to the higher temperatures. ~10% PCR product was ran on agarose gel to confirm it's success, then replicates were pooled and PCR purified (QIAgen kit) </li> <br />
<li>Insert and Vector backbone DNA was digested with the appropriate restriction enzymes, using reaction set-up as recommended in manufacturer protocol</li><br />
<li>Products were Gel-extracted or PCR purified</li><br />
<li>Vector backbone was de-phosphorylated to prevent background re-ligation, setting up reaction as per manufacturers recommendation, then heat-kill the enzyme</li><br />
<li>Optional: PCR purify to remove enzyme and buffer</li><br />
<li>Ligation reactions for the desired combinations of insert and backbone were set up, including negative control reactions which contain no insert DNA. Reactions components and amounts determined as per the manufacturer recommendation, though halving the total reaction was sometimes carried out</li><br />
<li>Transformed with ~2-4 ul ligation reaction into DH10B or NEB-5-alpha cells using the general heat shock protocol as explained previously</li><br />
<li>Picked colonies and innoculated mini-prep cultures. The number picked for each plate depends on the difference between the positive and negative controls. Generally between 2 and 4 were picked. Mini-preps were carried out using the manufacturers protocol (QIAgen QIAspin) </li><br />
<li>Test digest (no more than 10 ul total reaction volume) performed and products analysed using agarose gel electrophoresis to confirm if correct construct was present. </li><br />
<li>Alternativley, instead of picking cultures for mini-prepping, colony PCR was used when large numbers of colonies needed to be screened. Then only the positive clones were mini-prepped. </li><br />
<li>Positive clones were sent for sequencing (Source Bioscience) of the insert using appropriate primers. </li><br />
</ol><br />
<br />
<br />
<br />
<br />
<br />
</div> <br />
<h3>LB Broth Preparation</h3><br />
<div><br />
<ol><br />
<li>Add 25g Luria Broth to 1L demineralised water</li><br />
<li>Autoclave</li><br />
</ol><br />
</div><br />
<h3>LB Agar Preparation</h3><br />
<div><br />
<ol><br />
<li>Add 25g Luria Broth and 15g Agar to 1L demineralised water</li><br />
<li>Autoclave</li><br />
</ol><br />
</div><br />
<h3>Preparation of chemically competent E. coli cells</h3><br />
<div><br />
<p> The basis for the protocol is from http://openwetware.org/wiki/TOP10_chemically_competent_cells with a few differences. This also contains the recipe for the CCMB80 buffer used. The protocol is summarised below:<br />
<ol><br />
<li>Inoculate 2 ml LB broth with an aliquot (~50 ul) of the desired <i>E. coli</i> from the -80degC freezer stock of cells. <br />
</li><br />
<li>Incubate for 2h at 37°C</li><br />
<li>Add the 2 ml seed culture to 250 ml LB broth and grow at 37degC, shaking (~200 rpm) until OD 600 of 0.3 (~5 hours)<br />
</li><br />
<li>Centrifuge at 4degC, (in our case 3000 rpm in Heraeus megafuge, Thermo) for 10 minutes</li><br />
<li>Discard supernatant, then resuspend in 80 ml ice cold CCMB80 buffer (it is easier to resuspend in 1 ml first using a Gilson pipette, then add buffer to the required volume)<br />
</li><br />
<li>Place in ice for 20 minutes<br />
</li><br />
<li>Centrifuge 4degC and discard supernatant</li><br />
<li>Resuspend in 10 ml CCMB80 buffer<br />
</li><br />
<li>Test OD 600 of 200 ul SOC media with 50 ul resuspended cells and based on this calculate the amount of CCMB80 buffer needed to add to the resuspended cells to achieve a final yield of OD 600 1.0-1.5.<br />
</li><br />
<li>Aliquot in volumes as desired (for us ~250 ul) then store at -80 degC</li><br />
</ol><br />
</div><br />
<h3>Preparation of electro-competent E. coli cells</h3><br />
<div><br />
<p> To autoclave </p><br />
<ul><br />
<li>500 mL LB</li><br />
<li>500 mL Water</li><br />
<li>50 mL 10% glycerol </li><br />
<li>Conical Flasks</li><br />
</ul><br />
<p>To grow:5 mL overnight culture containing the required antibiotic, grow under shaking conditions at 37 degC </p> <br />
<ol><br />
<br />
<li>Prepare Eppendorf tubes and keep in the -80 degC freezer until required</li> <br />
<li>Inoculate the autoclaved flasks with 50 mL LB</li> <br />
<li>Add 500 ul of overnight culture into 50 the conical flasks and provide specific antibiotic, if required</li> <br />
<li>Grow for ~1 h and then start taking OD 600 nm readings every half hour. When OD reaches 0.5, proceed to the next step. </li> <br />
<li>Pour culture into falcon tube</li> <br />
<li>Centrifuge for 10 minutes at 4000 rpm and at 4 degC </li> <br />
<li>Discard supernatant and use blue roll remove any left overs. </li> <br />
<li>Add 800 ul of previously chilled, autoclaved water, resuspend cells, then add 9.2 mL to make it up to 10 mL</li> <br />
<li>Centrifuge for 10 minutes at 4000 rpm and at 4 degC </li> <br />
</ol><br />
</div><br />
<h3>General Heat-Shock Transformation</h3><br />
<div><br />
<ol><br />
<li>Add DNA to 50 ul cells on ice (no more than 5 ul, i.e. no more than 10% volume of cells)</li><br />
<li>Incubate on ice 15-30 min</li><br />
<li>Heat shock 42degC 54 s</li><br />
<li>Place samples back on ice for 2 minutes</li><br />
<li>Add 200 ul LB broth, or up to 10x volume of the cells</li><br />
<li>Incubate at 37°C for 60 minutes, shaking</li><br />
<li>Optional: Spin down cells, discard supernatant and resuspend in 100-200 ul LB to concentrate</li><br />
<br />
<li>Plate out cells on LB agar, maximum 200 ul</li><br />
<li>Incubate at 37°C overnight.</li><br />
<br />
<br />
</ol><br />
<br />
<br />
</div><br />
<h3>80% Glycerol Preparation</h3><br />
<div><br />
<ol><br />
<li>Add 80ml 99.7% glycerol to 20ml demineralized water</li><br />
<li>Autoclave</li><br />
</ol><br />
</div><br />
<h3>Glycerol Stock Preparation</h3><br />
<div><br />
<ol><br />
<li>Cultures plated on LB Agar + antibiotic and grown at 37°C overnight.</li><br />
<li>A 5ml LB culture in LB+antibiotic inoculated from a single, freshly growing colony.</li><br />
<li>Cultivate for 16h at 37°C, with constant shaking</li><br />
<li>0.5ml of this culture inoculated into sterile vial</li><br />
<li>Add 0.5ml of 80% glycerol</li><br />
<li>Vortex</li><br />
<li>Spin down</li><br />
<li>Freeze them at -80 degrees</li><br />
<br />
<br />
<br />
</ol><br />
<br />
</div><br />
<h3>QIAprep Spin Miniprep Kit</h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Materials per sample<br />
<ul><br />
<li>250ul P1 buffer (suspension buffer)</li><br />
<li>250ul P2 buffer (Lysis buffer)</li><br />
<li>350ul N3 buffer</li><br />
<li>750ul PE buffer</li><br />
<li>500ul PE buffer</li><br />
<li>Columns</li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div class="pure-u-1-2"><br />
<ol><br />
<li>Spin cells down at 4000rpm for 10 minutes</li><br />
<li>Discard supernatant (LB)</li><br />
<li>Resuspend pellet in P1 buffer</li><br />
<li>Transfer to labeled Eppendorf tube</li><br />
<li>Add P2 buffer. Solution should turn blue</li><br />
<li>Invert tubes 4-6 times, then wait for 2 minutes</li><br />
<li>Stop the reaction by adding N3 buffer and immediately inverting 4-6 times. Solution should turn clear</li><br />
<li>Centrifuge at 13000 rpm for 10 minutesv<br />
<li>Decant/pipette supernatant into mini-prep columns. Discard flow-through</li><br />
<li>Wash with PE buffer (750ul)</li><br />
<li>Centrifuge at 13000 rpm for 1 minutev<br />
<li>Discard flow-through. Add second wash of PE buffer (500ul)v<br />
<li>Centrifuge at 13000 rpm for 1 minute</li><br />
<li>Discard flow-through</li><br />
<li>Centrifuge empty columns at 13000 rpm for 1 minute to eliminate any excess wash buffer</li><br />
<li>Discard flow-through</li><br />
<li>Move columns into a labelled eppendorf</li><br />
<li>Add 30-40ul distilled water and wait for 2-3minutes</li><br />
<li>Elute DNA by centrifuging at 13000 rpm for 1 minute, do not discard flow-through. Discard column.</li><br />
<li>Nanodrop</li><br />
<br />
<br />
</ol><br />
<br />
</div><br />
</div><br />
</div><br />
<br />
<br />
<br />
<h3>1% Agarose Gel</h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Materials<br />
<ul><br />
<li>1g Agarose</li><br />
<li>100mL 1X TAE buffer</li><br />
<li>8uL SYBR Safe</li><br />
<br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div class="pure-u-1-2"><br />
<ol><br />
<li>Mix Agarose and 1x TAE buffer</li><br />
<li>Heat up until Agarose is dissolved</li><br />
<li>Add SYBR Safe</li><br />
<li>Pour into gel tray and let cool</li><br />
<br />
</ol><br />
<br />
</div><br />
</div><br />
<br />
<br />
</div><br />
<br />
<h3>Agarose Gel Electrophoresis</h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Materials<br />
<ul><br />
<li>1% Agarose gel DNA ladder</li><br />
<li>6x loading dye</li><br />
<li>Electrophoresis cuvette</li><br />
<br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div class="pure-u-1-2"><br />
<ol><br />
<li>Set gel tray into cuvette, filled with 1x TAE buffer</li><br />
<li>Inoculate samples, previously dyed with 6x loading dye. Additionally, provided a DNA ladder for further reference of DNA sizes</li><br />
<li>Run gel at 110V for 30-40min</li><br />
<br />
<br />
</ol><br />
<br />
</div><br />
</div><br />
<br />
<br />
</div><br />
<br />
<h3>Overnight Cell Incubation</h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Materials<br />
<ul><br />
<li>5mL Luria Broth</li><br />
<li>5ul specific antibiotic</li><br />
<li>Loops (for colony picking or glycerol stock scraping)</li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div class="pure-u-1-2"><br />
<ol><br />
<li>Add Luria Broth into 50mL tube</li><br />
<li>Inoculate specific antibiotic</li><br />
<li>Scrape/pick glycerol stock surface/colony and transfer into falcon tube</li><br />
<li>Incubate at 37°C overnight</li><br />
<br />
<br />
</ol><br />
<br />
</div><br />
</div><br />
<br />
<br />
</div><br />
<br />
<h3>1x TAE buffer</h3><br />
<div><br />
<ul><br />
<li>1x solution contas 40nM Tris, 20mM acetic acid, 1mM EDTA</li><br />
</ul><br />
</div><br />
<br />
<br />
<br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
</section><br />
<section id="functionalisation"><br />
<h2>Functionalisation Protocols</h2><br />
<div class="accordion"><br />
<h3>Metal binding assay protocol</h3><br />
<div><br />
<br />
<div class="pure-g"><br />
<div class="pure-u-1-1"><br />
<p>Metal binding proteins fused to CBDs were bound onto cellulose in 96-well plates and tested against 3 different metals (Nickel, copper, zinc). First, the fusion protein lysate was incubated overnight in the cellulose wells. Following this, the metal salt solutions are added in excess into the wells. Finally, an EDTA step removes the bound metal ions into solution, and the metal concentration in solution is quantified by mass spectrometer. Multiple washes with PBS and water were done between each binding step, ensuring that metal ions read were released from the metal binding proteins.<br />
</p><br />
</div><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Materials<br />
<ul><br />
<li>Cellulose 96-well plate (see ‘Preparing cellulose 96-well plates’) </li><br />
<li>20mM Zinc chloride/Nickel chloride/Copper chloride solution</li><br />
<li>1x PBS</li><br />
<li>Sterile water</li><br />
<li>Protein(s) of interest in cell lysate(s) (suspended in PBS, see ‘Protein preparation for assays’) or purified protein</li><br />
<li>Mass spectrometer</li><br />
<li>Multichannel pipette</li><br />
<li>25mM EDTA</li><br />
<li>2ml centrifuge tube</li><br />
<br />
<br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Methods<br />
<ol><br />
<li>As we are using mass spectrometry to detect metal bound and eluted, samples must be of 2ml volume. We pool the protein-releasing EDTA solution from 4 wells to make one reading. The remaining volume is made up with water. This should be accounted for when planning repeats and negatives.</li><br />
<li>Apply 200ul cell lysate/purified protein sample in each well on the 96-well plate, accounting for negative controls (wells with no lysate applied).</li><br />
<li>Incubate overnight (9 to 14 hours) at 4°C.</li><br />
<li>Remove the 180ul of cell lysate.</li><br />
<li>Wash thrice with PBS: apply 180ul of 1x PBS into each well. Aspirate 180ul from well and discard. Repeat twice more.</li><br />
<li>Wash twice with water: apply 180ul of water into each well. Aspirate 180ul from well and discard. Repeat once more.</li><br />
<li>Apply 180ul of 20mM metal solution.</li><br />
<li>Incubate for 1 hour at room temperature.</li><br />
<li>Remove the 180ul of metal solution.</li><br />
<li>Wash thrice with water: apply 180ul of water into each well. Aspirate 180ul from well and discard.</li><br />
<li>Apply 180ul of EDTA.</li><br />
<li>Incubate for 30 minutes at room temperature.</li><br />
<li>Transfer the liquids in the wells into a 2ml centrifuge tube, pooling the wells as planned. The sample is ready to read with a mass spectrometer</li><br />
<br />
<br />
<br />
</ol><br />
</li><br />
</ul><br />
<br />
</div><br />
</div><br />
</div><br />
<h3>Preparing Cellulose 96-well Plates<br />
</h3><br />
<div><br />
<br />
<div class="pure-g"><br />
<div class="pure-u-1-1"><br />
<p>40 g of kombucha cellulose was blended with 250 ml sterile water in a conventional kitchen blender. 200 ul of this blend is added into each well of a black 96-well plate. The cellulose is dried in a 37°C incubator overnight until dry and ready to use. <br />
<br />
</p><br />
</div><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Materials<br />
<ul><br />
<li>Black 96-well plate (suitable for fluorescence reading)<br />
</li><br />
<li>40 g Bacterial cellulose, grown from kombucha isolate (choose an even pellicle)</li><br />
<li>250 ml sterile water<br />
</li><br />
<li>Conventional kitchen blender <br />
</li><br />
<li>Multichannel pipette <br />
</li><br />
<br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Methods<br />
<ol><br />
<li>Dab dry your bacterial cellulose. Weigh out 40 g. </li><br />
<li>Add weighed cellulose into blender and add 50 ml of the ddH20. </li><br />
<li>With a combination of pulse and short blends, try to eliminate large chunks. Ensure you scoop chunks that are stuck below the blades. </li><br />
<li>Once the cellulose has begun to homogenise, add the remaining water and blend continuously for 12 mins at the highest setting. </li><br />
<li>Check that the cellulose is fully homogenised. If not, blend for a further 10 minutes. This blended cellulose mix keeps well indefinitely at room temperature in airtight glassware, using parafilm if needed. </li><br />
<li>Using a multichannel pipette, aliquot 200 ul of blended cellulose into each well of a black 96-well plate. It helps to mix between aliquots as settling often occurs. </li><br />
<li>Place in a 37°C incubator until fully dry, usually about 20 hours. Alternatively, place in 60°C oven and monitor for dryness between 3 to 8 hours. </li><br />
<li>Dry plates are ready for immediate use. </li><br />
<br />
<br />
<br />
</ol><br />
</li><br />
</ul><br />
<br />
</div><br />
</div><br />
</div><br />
<h3>Water filtration protocol</h3><br />
<div><br />
<br />
<div class="pure-g"><br />
<div class="pure-u-1-1"><br />
<p>Dried cellulose was exposed to the phytochelatin-CBD protein fusion (<a href="http://parts.igem.org/Part:BBa_K1321110">K1321110</a>) to create a smart cellulose filter with a chelating agent. The filter was set up on a coffee press and secured. Nickel chloride solution (250 µM) was added on the top and let to run through. The filtered liquid was collected and analysed using Mass Spectrometry and using the Nickel Assay Kit (Sigma).<br />
</p><br />
</div><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Materials<br />
<ul><br />
<li>Coffee press </li><br />
<li>ATCC53582 <em>G. xylinus</em> produced cellulose </li><br />
<li>Phytochelatin-CBD in cell lysate</li><br />
<br />
<li>250 µM Nickel chloride solution in MilliQ water</li><br />
<li>Mass spectrometer</li><br />
<li>Nickel Assay Kit</li><br />
<br />
<br />
<br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Methods<br />
<ol><br />
<li><em>G. xylinus</em> (<a href="http://parts.igem.org/Part:BBa_K1321305">K1321305</a>) produced cellulose with yield treatment method was exposed to cell lysate containing phytochelatin-CBD (K1321110) and dried at 37 <sup>O</sup>C.</li><br />
<li>After the solution with the protein on the cellulose has dried, the cellulose was kept at 4 <sup>O</sup>C overnight.</li><br />
<li>The cellulose with the coated protein was gently soaked with milliQ water and secured on a coffee press, as a control, cellulose without coated protein was also secured on a separate coffee press.</li><br />
<li>3mL of 250 µM nickel chloride solution was added into each of the two coffee presses. Increased pressure was set up by the use of the press. The solution was let to filter for a sufficient time to collect at least 100 µl of the filtered solution.</li><br />
<liThe filtered solution was analysed with the Mass Spectrometry and with the Nickel Assay Kit (Sigma), following the provided protocols.</li><br />
</ol><br />
</li><br />
</ul><br />
<br />
</div><br />
</div><br />
</div> <br />
<br />
<h3>CBD binding strength assay, using fluorescence<br />
</h3><br />
<div><br />
<br />
<div class="pure-g"><br />
<div class="pure-u-1-1"><br />
<p>Our selection of CBDs was cloned with sfGFP to make fluorescent fusion proteins that can be easily detected on a plate reader. We incubated the cell lysate on our cellulose plate overnight, and read the comparative reduction in fluorescence after each of the three washes with either water, ethanol, PBS or BSA.</p><br />
</div><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Materials<br />
<ul><br />
<li>- Cellulose 96-well plate (see protocol ‘Preparing cellulose 96-well plates’)<br />
<br />
</li><br />
<li>Cell lysates containing CBD-sfGFP fusions (see protocol ‘Preparation of crude cell lysate for fusion protein assays’) or purified protein sample</li><br />
<li>Fluorescence plate reader<br />
</li><br />
<li>1 x PBS<br />
</li><br />
<li>5% BSA<br />
</li><br />
<li>Sterile water</li><br />
<li>70% ethanol</li><br />
<br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Methods<br />
<ol><br />
<li>Apply 200 ul cell lysate/purified protein sample in each well on the 96-well plate, accounting for negative controls (wells with no lysate applied or reading a blank cellulose plate). </li><br />
<li>Incubate overnight (9 to 14 hours) at 4°C. </li><br />
<li>Read fluorescence of plate with cell lysate still applied. </li><br />
<li>Remove the 180 ul of cell lysate. </li><br />
<li>Read fluorescence of plate with cell lysate removed.</li><br />
<li>First wash with 1 x PBS for all wells: apply 180u l of PBS into each well. Read fluorescence. Aspirate 180 ul from well and discard. Read fluorescence.</li><br />
<li>Optional: compare binding strength of CBDs when exposed to different washes. 4 different washes we’ve tried: water, 1 x PBS, 5% BSA, 70% ethanol. </li><br />
<li>Wash thrice with appropriate reagent: apply 180 ul of wash of choice into each well. Read fluorescence. Aspirate 180 ul from well and discard. Read fluorescence. Repeat twice more. </li><br />
<br />
<br />
<br />
</ol><br />
</li><br />
</ul><br />
<br />
</div><br />
</div><br />
</div><br />
<br />
<h3>Preparation of crude cell lysate for fusion protein assays (for constitutive expression)</h3><br />
<div><br />
<br />
<div class="pure-g"><br />
<div class="pure-u-1-1"><br />
<p>For LacI:<br />
Protein-expressing cells were inoculated into 1litre of LB broth supplemented with antibiotic, and grown in a 37°C shaking incubator overnight until cloudy. The cells were pelleted, then resuspended in 10ml of PBS. The samples were then sonicated, and cell debris pelleted. The resulting clear cell lysate was used for assays and for protein purification. </p><br />
<p>For T7 fusions: <br />
Protein-expressing cells were inoculated into 1litre of LB broth supplemented with antibiotic, and grown in a 37°C shaking incubator to an OD600 between 0.5-1.0 and induced with IPTG. Cultures were returned to the incubator and grown overnight until cloudy. The cells were pelleted, then resuspended in 10ml of PBS. The samples were then sonicated, and cell debris pelleted. The resulting clear cell lysate was used for assays and for protein purification. </p><br />
</div><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Materials<br />
<ul><br />
<li>Verified colony to pick</li><br />
<li>20 ml LB broth, for seed culture</li><br />
<li>2 litre conical flask</li><br />
<li>1 litre autoclaved LB broth, for overnight cell grow up</li><br />
<li>Foam bung</li><br />
<li> Aluminium foil</li><br />
<li>1000x chloramphenicol ( 1ml for each litre of LB)</li><br />
<li>37°C shaking incubator</li><br />
<li>Spectrophotometer set for OD<sub>600</sub>,/li><br />
<li>0.1 M IPTG (for T7 induction)</li><br />
<li>1 litre capacity cooled centrifuge</li><br />
<li>1x PBS</li><br />
<li>50ml Falcon tubes</li><br />
<li>Stripette and pipette controller</li><br />
<li>Beaker, half-filled with ice</li><br />
<li>Sonicator</li><br />
<li>Cooled 50 ml Falcon centrifuge</li><br />
<br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div class="pure-u-1-2"><br />
<ul><br />
<li>Methods<br />
<ol><br />
<li>Pick your verified colony and inoculate into 20 ml LB broth, supplemented with 20 ul of 1000x chloramphenicol.</li> <br />
<li>Grow for 6-10 hours, or until sufficiently cloudy in a 37°C shaking incubator. </li><br />
<li>Prepare 1 litre of autoclaved LB broth in a 2 litre conical flask, cover with foam bung, aluminium foil and label with autoclaved tape prior to autoclaving.</li><br />
<li>When cooled, add 1 ml of 1000x chloramphenicol to broth. Mix well.</li><br />
<li>Pour all of seed culture into prepared broth. Cover again with the same foam bung and aluminium foil. </li><br />
<li>If expression is constitutive, place in the shaking 37°C incubator (180 rpm) to grow overnight, between 12-20 hours. </li><br />
<li>If using T7 promoter, place in a shaking 37°C incubator (180 rpm) and check OD<sub>600</sub> periodically. Induce using 2ul of 0.1 M IPTG per ml, when OD<sub>600</sub> of culture is between 0.5 and 1.0. Return to the shaking 37°C incubator (180 rpm) to grow overnight, between 12-20 hours. </li><br />
<li>Check that growth has occurred and media is sufficiently cloudy. </li><br />
<li>Move culture into 1 litre centrifuge bottle. Balance bottles to the correct 0.1 g. </li><br />
<li>Centrifuge for 20 mins at 4°C, at the speed of 6000 rpm.</li><br />
<li>Check for pellet. If present, discard supernatant. </li><br />
<li>Using a stripette and pipette controller, resuspend in 10 ml 1x PBS, or volume of choice. Transfer to a 50 ml Falcon tube. </li><br />
<li>Using a beaker small enough to fit under the sonicator and half-filled in ice, embed the resuspended cells in a secure upright position within the ice.</li><br />
<li>Remove lid of Falcon tube. Submerge the sonicator tip in the resuspended cells, leaving about 0.5 cm from the bottom. It is important that the tip is not touching the bottom of the tube as this disturbs sonication. </li><br />
<li>Sonicate at 67% for 3:30 minutes in 15 second pulses. Post sonication, keep sample on ice. </li><br />
<li>Using a cooled 50 ml Falcon centrifuge, pellet down cell debris leaving cell lysate. 6000 rpm for 25 minutes at 4°C is sufficient. Check that lysate is clear and that pellet is present.</li><br />
<li>Transfer lysate to a new tube. Cell lysate is ready to use, for assay or further purification purposes. Discard cell debris pellet.</li><br />
<li>Store at 4°C if not using immediately. </li><br />
<br />
<br />
<br />
</ol><br />
</li><br />
</ul><br />
<br />
</div><br />
</div><br />
</div><br />
<br />
<br />
</div><br />
<br />
<br />
</section><br />
<br />
</div><br />
<br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
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<br />
<br />
</div><br />
</html><br />
<br />
{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/File:IC14-avg-nickel-1st-2nd-wash.pngFile:IC14-avg-nickel-1st-2nd-wash.png2014-10-18T01:25:03Z<p>Xen sm: </p>
<hr />
<div></div>Xen smhttp://2014.igem.org/File:IC14-metal-binding-first-wash-EDTA.pngFile:IC14-metal-binding-first-wash-EDTA.png2014-10-18T01:17:33Z<p>Xen sm: </p>
<hr />
<div></div>Xen smhttp://2014.igem.org/File:IC14-metal-binding-avg-conc-second-wash.pngFile:IC14-metal-binding-avg-conc-second-wash.png2014-10-18T01:17:02Z<p>Xen sm: </p>
<hr />
<div></div>Xen smhttp://2014.igem.org/File:IC14-Metal-binding-result-table.PNGFile:IC14-Metal-binding-result-table.PNG2014-10-18T01:15:57Z<p>Xen sm: metal binding assay result table</p>
<hr />
<div>metal binding assay result table</div>Xen smhttp://2014.igem.org/Team:Imperial/PartsTeam:Imperial/Parts2014-10-18T00:56:34Z<p>Xen sm: </p>
<hr />
<div>{{:Team:Imperial/Templates:header}}<br />
<html><br />
<br />
<div class="content-wrapper"><br />
<br />
<div class="pure-g"><br />
<br />
<div class="pure-u-1-1 main"><br />
<h1>Parts</h1><br />
<div id="navWrap"><br />
<div id="subNav"><br />
<ul><br />
<li><a data-scroll href="#featured">Featured Parts</a><br />
</li><br />
<li><a data-scroll href="#table">Table of Parts</a><br />
</li><br />
<br />
</ul><br />
<br class="clearLeft" /><br />
</div><br />
</div><br />
<br />
<section id="featured"><br />
<h2>Featured Parts</h2><br />
<div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321340">BBa_K1321340</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>N-terminal linker + double CBD</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>Double cellulose binding domain (dCBD) using two cellulose binding domains from <i>Trichoderma reesei</i> cellobiohydrolases, with an N-terminal linker and internal linker sequence between the two domains which are derived from the endogenous cellobiohydrolase linker sequence. This part is in RFC(25) Freiberg fusion format to allow for easy use in protein fusions. dCBD binding affinity for cellulose was <a href="https://2014.igem.org/Team:Imperial/Functionalisation"> characterised </a> using sfGFP fusions.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321339">BBa_K1321339</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>CBDcenA + Linker</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>Cellulose binding domain (CBD) of Endoglucanase A (cenA) from <i>Cellulomonas fimi</i> with an endogenous C-terminal linker. The part is in Freiburg format (RFC 25) for ease of use in protein fusions.</p><br />
</div><br />
</div><br />
<br />
<br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321014"> BBa_K1321014</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>CBDcipA with N and C-terminal linker </p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>This CBD is from the Cellulosomal -scaffolding protein A (cipA) of <i>Clostridium thermocellum </i> including the endogenous linker sequences at the N and C-terminus. It is in RFC25 format to allow for easy use in protein fusions. At present the cloning for these constructs is still in progress to correct an illegal EcoRI site which was identified in the parts with this CBD. This can be achieved with a silent mutation via site-directed mutagenesis and we aim to send these parts to the registry once this is complete.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321005">BBa_K1321005</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>Synthetic Phytochelatin (PC) EC20</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>A general heavy metal-binding peptide consisting of 20 Glu-Cys (EC) repeats in Freiburg format (RFC[25]) to allow for easy use in fusion proteins. We created a library of CBD fusions with this part and demonstrated [INSERT FINAL RESULTS SENTENCE HERE]<br />
</p><br />
</div><br />
</div><br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p><i>Vitreoscilla</i> haemoglobin (VHb)</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>This part is a haemoglobin isolated from <i>Vitreoscilla</i> (VHb) which improves the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions. We used this part in <i> G. xylinus </i> to try and improve growth rate and cellulose production.<br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a>, <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>pSEVA331 and pSEVA321 backbones</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>These are two broad host range vector backbones for which we have demonstrated use in <i>E. coli</i> and <i>Gluconacetobacter xylinus </i>.<br />
</p><br />
</div><br />
</div> <br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321334"> BBa_K1321334</a>, <a href="http://parts.igem.org/Part:BBa_K1321335">BBa_K1321335</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>Cellulose synthase operon AcsAB and AcsCD</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>Designed and refactored from <i>G. xylinus</i>, this operon was inserted into <i>E.coli </i> for cellulose synthesis. <br />
</p><br />
</div><br />
</div> <br />
<br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a>,<a href="http://parts.igem.org/Part:BBa_K1321306"> BBa_K1321306</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p><i>Gluconacetobacter xylinus</i> strains ATCC53582 and Kombucha Isolate</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>We created and characterised a library of parts for this organism, with the aim to increase the accessibility the chassis for future work in iGEM. By sharing these strains and their genomes on the registry we hope this will contribute to their ease of use and characterisation.<br />
</p><br />
</div><br />
</div> <br />
</div><br />
</div><br />
<br />
</section><br />
<section id="table"><br />
<h2>Table of Parts</h2><br />
<br />
<br />
<br />
<br />
</section><br />
<br />
</div><br />
<br />
<br />
<br />
</div><br />
<br />
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<br />
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<br />
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<br />
<br />
</div><br />
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</div><br />
</html><br />
<groupparts>iGEM014 Imperial</groupparts><br />
<br />
<br />
{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/FunctionalisationTeam:Imperial/Functionalisation2014-10-18T00:51:38Z<p>Xen sm: added overview and achievements</p>
<hr />
<div>{{:Team:Imperial/Templates:header}}<br />
<html><br />
<body><br />
<div class="content-wrapper"><br />
<br />
<div class="pure-g"><br />
<br />
<div class="pure-u-1-1 main"><br />
<h1>Functionalisation</h1><br />
<div id="navWrap"><br />
<div id="subNav"><br />
<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<li><a data-scroll href="#aim">Aims</a><br />
</li><br />
<li><a data-scroll href="#CBDs">CBDs</a><br />
</li><br />
<li><a data-scroll href="#binding-partners">Functionalisation proteins</a><br />
</li><br />
<br />
<li><a data-scroll href="#results">Results</a><br />
</li><br />
<li><a data-scroll href="#references">References</a><br />
</li><br />
</ul><br />
<br class="clearLeft" /><br />
</div><br />
</div><br />
</div><br />
</div><br />
<section id="overview"><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<h2>Overview</h2><br />
<p>Attaching functional proteins to cellulose can expand the properties of cellulose and allow us to selectivity capture specific contaminants in water. By creating fusion proteins of sfGFP and metal-binding proteins with five different cellulose binding domains, three of which are new to the registry, we were able to characterise the relative affinities of the CBDs and show proof-of-concept removal of contaminants from water.</p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Added three new cellulose binding domains to the registry</li><br />
<li>Performed an assay with sfGFP-fusions to determine the strongest potential binders which could be carried forward for further testing</li><br />
<li>Identified potential metal binding proteins to bind heavy metals from wastewater, including the addition of a new promising polypeptide phytochelatin EC20</li><br />
<li>Tested metal ion capture ability of the CBD-phytochelatin fusion constructs when attached to cellulose</li><br />
<br />
</ul><br />
</div><br />
<br />
<br />
</div><br />
<br />
</section><br />
<section id="introduction"><br />
<h2>Introduction</h2><br />
<figure class="content-image image-right"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/a/a0/IC14-func-strategy-both-pursue.png"><br />
<figcaption>Figure 1. Pictorial summary showing the two cellulose functionalization approaches considered in our project. Strategy 1: Cellulose binding domains are expressed as fusions with the protein of interest, separated optionally by a linker sequence. The cellulose binding domains attach to the cellulose by hydrophobic interactions. Strategy 2: Curli fibrils are expressed in parallel with cellulose to form a composite material. The curli fibres coding sequence is modified to contain a ‘SpyTag’ peptide (Zakeri <i>et al</i> 2012) which binds specifically via a covalent isopeptide bond to its protein partner, ‘SpyCatcher’. This SpyCatcher is fused to the proteins of interest. In our project we only had time to pursue strategy 1 but are still excited about the potential of strategy 2 in the future.</figcaption><br />
</figure><br />
<p>Bacterial cellulose is already a useful biomaterial as is due its attractive mechanical properties, with research on applications from loudspeaker diaphragms to protective packaging and wound dressings (Lee 2014). However, our project also aims to extend the properties of cellulose by conjugating functional proteins and peptides to it. This allows us to target specific contaminants in water which can not be filtered by size exclusion alone. (See <a href="https://2014.igem.org/Team:Imperial/Water_Report">water report</a> and <a href="https://2014.igem.org/Team:Imperial/Informing_Design">informing design</a> sections).</p><br />
<br />
<p>We considered two ways of functionalising cellulose (figure 1) but continued to work fully on the cellulose-binding domain (CBD) method. The curli-fibre method would be an exciting avenue to explore in the future; it is feasible since there has already been research into making a “programmable” biomaterial from curli fibres using a peptide tag approach (Nguyen <i>et al</i> 2014), and in combination with cellulose could produce a composite with robust mechanical properties as well.<br />
</p><br />
<br />
</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>We aimed to make and characterise new cellulose binding domains, and fuse them to functional proteins. (Incomplete)</p><br />
<br />
<br />
</section><br />
<section id="CBDs"><br />
<h2>Cellulose Binding Domains (CBDs)</h2><br />
<p>The cellulose binding domain (CBD) is the link between our functional proteins and the cellulose. Therefore, we wanted to use a CBD which had high affinity and strong binding to cellulose. However, weaker CBDs may still be useful since they could have the potential be controllably eluted under specific conditions, thereby enabling regeneration of the filter when saturated, and controlled disposal of the collected contaminants (refer to water report if relevant?).<br />
</p><br />
<p>We started our search by looking into the <a href="http://parts.igem.org/">parts registry</a> and found two CBDs already existed in the registry: CBDclos (<a href="http://parts.igem.org/Part:BBa_K863111">BBa_K863111</a>) and CBDcex (<a href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>). We also made three new CBDs to contribute to the registry: dCBD, CBDcipA, and CBDcenA. All the CBDs are in RFC(25) freiberg fusion format to allow for ease of use in protein fusions.</p><br />
<br />
<div class="accordion"><br />
<h3>CBDclos and CBDcex</h3><br />
<div><br />
<p>These parts were biobricked by <a href="https://2012.igem.org/Team:Bielefeld-Germany/Results/cbc">Bielefeld 2012</a>, please see their part pages (<a href="http://parts.igem.org/Part:BBa_K863111">BBa_K863111</a>a and <a href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>) for detailed information. CBDclos is from the cellulolytic bacterium <em>Clostridium cellulovorans</em> and CBDcex originates from the cellulolytic bacterium <em>Cellulomonas fimi</em>. This is the same organism as for CBDcenA; cex is from the exoglucanase gene and cen is from the endoglucanase gene.</p><br />
</div><br />
<h3>Double CBD (dCBD) with N-terminal linker</h3><br />
<div><br />
<p>This part, <a href="http://parts.igem.org/Part:BBa_K1321340">BBa_K1321340</a> is based on the double cellulose-binding domain construct synthesised and characterised by Linder <i>et al</i>1996, who found that this double CBD had higher affinity for cellulose than either of the two CBDs on their own. The main difference is that our part contains an additional linker sequence on the N-terminus of the protein.<br />
</p><br />
<p>The two CBDs are from the fungus <i>Trichoderma reesei</i> (<i>Hypocrea jecorina</i>) Exocellobiohydrolase (Exoglucanase) I (cbh1), uniprot ID P62694; and Exocellobiohydrolase (Exoglucanase) II, uniprot ID P07987 (cbh2); with a linker peptide between the two CBDs and at the N-terminus of the protein. Both linkers are the same amino acid sequence and are based on the endogenous linker sequences that exists in cbh1 and cbh2 genes. The linker sequence is PGANPPGTTTTSRPATTTGSSPGP which is the same as used by Linder <i>et al</i> 1996. The first three amino acids are from the cbh2 endogenous linker, and the rest is from the cbh1 endogenous linker. CBDcbh1 is placed C-terminal to CBDcbh2 because naturally CBDcbh1 is a C-terminal domain and CBDcbh2 is an N-terminal domain. Both CBDs are from the <a class='iframe' href="http://www.cazy.org/CBM1.html">CBM family 1</a>. The precise location of the CBD within the cbh genes was slightly different according to the uniprot annotations and the sequence used by Linder <i>et al</i> 1996; we chose to use the sequence from the paper since the protein was expressed and characterised successfully.</p><br />
<br />
</div><br />
<h3>CBDcipA with N and C-terminal linker</h3><br />
<div><br />
<p>This CBD is from the Cellulosomal -scaffolding protein A (cipA) of Clostridium thermocellum including the endogenous linker sequences at the N and C-terminus (<a class='iframe' href="http://www.uniprot.org/uniprot/Q06851">UniProt ID Q06851</a>). It is in RFC25 format to allow for easy use in protein fusions.</p><br />
<br />
<p>The CBD is part of the <a class='iframe' href="http://www.cazy.org/CBM3.html">CBM3 family</a> . This CBD has been used in many application: in fusions with cell adhesion peptides to enhance the properties of cellulose as a cell-growth matrix (Andrade <i>et al </i> 2010a, Andrade <i>et al </i> 2010b), fused to enzymes to remove contaminants from water (Kauffmann <i> et al </i> 2000) and fused to an antimicrobial peptide (Ramos, Domingues & Gama 2010).</p><br />
<br />
<p>At present the cloning for these constructs is still in progress to correct an illegal EcoRI site which was identified in the parts with this CBD. This can be achieved with a silent mutation via site-directed mutagenesis and we aim to send these parts to the registry once thi<br />
</p><br />
<br />
</div><br />
<h3>CBDcenA with C-terminal linker</h3><br />
<div><br />
<p>This CBD is from the Endoglucanase A (cenA) gene from <i>Cellulomonas fimi</i> and also contains an endogenous C-terminal linker. The cellulose binding domain region occurs at the N-terminus of the cenA gene (UniProt P07984 link to http://www.uniprot.org/uniprot/P07984) and is from the <a class='iframe' href="http://www.cazy.org/CBM2.html">CBM family 2</a><br />
</p><br />
<p>The linker sequence (PTTSPTPTPTPTTPTPTPTPTPTPTPTVTP) is Pro-Thr box which has an extended conformation and acts as a hinge region (Gilkes <i>et al</i> 1989). The endoglucanase CBDcenA has 50% homology to the exoglucanase CBDcex (<a class="iframe" href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>) and the linker is highly conserved in both, but the order of the catalytic, linker and cellulose-binding regions is reversed (Warren <i>et al</i> 1986). It has been shown that CBDcenA has the highest binding affinity for crystalline cellulose out of the <i>C. fimi</i> CBDs (Kim <i>et al</i> 2013).<br />
</p><br />
</div><br />
</div><br />
</section><br />
<br />
<br />
</section><br />
<section id="binding-partners"><br />
<h2>Functionalisation proteins: Binding partners</h2><br />
<h3>Superfolder Green Fluorescent Protein (sfGFP) </h3><br />
<figure class="content-image image-right"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/8/85/IC14-sfGFP-part-table.PNG> <br />
<figcaption>sfGFP-CBD fusion parts</figcaption> <br />
</figure> <p> sfGFP is a more stable mutant of <a href="http://parts.igem.org/Parts:BBa_E0040">mut3GFP</a> and we chose to use this as a robust, easily visible protein to initially fuse to our CBDs. We also designed one of our assays [insert link on 'one of our assays'] for characterising CBD binding using the sfGFP fusion constructs. sfGFP already existed in the registry (<a href="http://parts.igem.org/Part:BBa_I746916">BBa_I746916</a>), detailed information on sfGFP properties is included in this part description) however since we needed to make fusion proteins with our CBDs we modified the part and submitted it in RFC[25] Freiberg fusion format (<a href="http://parts.igem.org/Parts:BBa_K1321337">K1321337</a>). We created the following fusion parts with sfGFP: <br />
<br />
<br />
<br />
</p><br />
<br />
<h3> Metal Binding Proteins </h3><br />
<p>One of the contaminants we can filter out which current size-exclusion technologies can not target, are <a href="https://2014.igem.org/Team:Imperial/Water_Report">heavy metals</a>. We have worked with four different metal binding proteins: the metallothioneins SmtA and fMT, a histidine-rich nickel binding protein (NiBP) from the bacterium <i>Heliobacter pylori</i> and a synthetic phytochelatin (PC) EC20.<br />
</p><br />
<div class="accordion"><br />
<h3>SmtA and fMT metallothioneins</h3><br />
<div><br />
<p>These two parts already existed in the BioBrick registry, but since we needed to use them as fusions with the CBDs we improved the parts by making them compatible with the <a class="iframe" href="http://parts.igem.org/Assembly_standard_25">RFC(25) Frieburg Fusion BioBrick standard.</a></p><br />
<p>Metallothioneins (MTs) are genetically encoded cysteine-rich peptides which serve a role in the detoxification of heavy metals in plants, animals and some prokaryotes (Cobbett, 2002). They have a similar structure and function to phytochelatins, but are produced by different mechanisms (Cobbett, 2002). SmtA is from the cyanobacteria <i>Synechococcus sp.</i> and was used by the <a target="_blank" href="https://2011.igem.org/Team:Tokyo-NoKoGen">Tokyo-NoKoGen 2011</a> iGEM team (who submitted the RFC10 part originally, <a class="iframe" href="http://parts.igem.org/Part:BBa_K519010">BBa_K519010</a>) to confer cadmium (Cd2+) tolerance to <em>E. coli</em> cells when expressed. fMT originates from the seaweed <i>Fucus vesiculosus</i> and was submitted by <a target="_blank" href="https://2009.igem.org/Team:Groningen">Groningen 2009 iGEM team </a> and detailed information can be found on the original part page (<a class="iframe" href="http://parts.igem.org/Part:BBa_K190019">BBa_K190019</a>).</p><br />
<p>We successfully <a href="https://2014.igem.org/Team:Imperial/Protocols#general">cloned</a> the following CBD fusion constructs: </p><br />
<br />
<figure class="content-image image-right image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/a/ac/IC14-SmtA-part-table.PNG> <br />
<figcaption>SmtA-CBD fusion parts</figcaption> <br />
</figure> <br />
<br />
<figure class="content-image image-left image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/6/62/IC14-fMT-part-table.PNG> <br />
<figcaption>fMT-CBD fusion parts</figcaption> <br />
</figure> <br />
<br />
<br />
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<br />
</div><br />
<h3>Nickel binding protein (NiBP)</h3><br />
<div><br />
<figure class="content-image image-right"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/f/f6/IC14-NiBP-part-table.PNG> <br />
<figcaption>NiBP-CBD fusions parts</figcaption> <br />
</figure><br />
<p><br />
This protein is the histidine-rich nickel-binding protein from <i>Heliobacter pylori</i> (GenBank accession ACX98466.1), and already existed as a BioBrick in the registry (<a class="iframe" href="http://parts.igem.org/Part:K1151001">K1151001</a>). Since we needed to use this part in fusion proteins we improved the part by submitting it as a basic part in RFC(25) (link) Freiberg fusion format (<a class="iframe" href="http://parts.igem.org/Part:K1321009">K1321009</a>).<br />
</p><br />
<p>We successfully <a href="https://2014.igem.org/Team:Imperial/Protocols#general">cloned</a> the following CBD fusion constructs with NiBP:<br />
<br />
</p><br />
<p>However we discovered late on in the project that the DNA sequence of the NiBP protein in the registry is out of frame, leading to an incorrect protein translation and read-through of the stop codon (figure 2).</p><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/8/80/IC14-NiBP-protein-alignment.PNG"><br />
<figcaption>Figure 2. Alignment of BBa_K1151001 NiBP in the registry to the , showing incorrect amino-acid sequence in red compared to <a target="_blank" href="http://www.ncbi.nlm.nih.gov/protein/261838700">native <i>H.pylori</i></a> sequence.<br />
</figcaption><br />
</figure><br />
<figure class="content-image image-right"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/e/e1/IC14-NiBP-DNA-alignment.PNG"><br />
<figcaption>Figure 3. Single nucleotide deletion in BBa_K1151001 compared to native <i>H.pylori</i> sequence (<a target="_blank" href="http://www.ncbi.nlm.nih.gov/nuccore/261837457">GenBank CP000012.1</a> (link ) bp1391851-1392033, reverse complement) resulting in any downstream nucleotides being out of frame.</figcaption><br />
</figure><br />
<br />
<p>Further investigation into the error let us to identify a single nucleotide deletion in <a class="iframe" href="http://parts.igem.org/Part:BBa_K1151001">BBa_K1151001</a> compared to the native <i>H.pylori</i> DNA sequence (<a target="_blank" href="http://www.ncbi.nlm.nih.gov/nuccore/261837457">GenBank CP000012.1</a> bp1391851-1392033, reverse complement strand). As seen in Figure 3 the deletion is a ‘C’ at bp 53 of the native sequence. This error was propagated through all our NiBP constructs and means that any subsequent C-terminal fusions are also out of frame and we were unable to express and characterise these constructs. We have noted this on the description pages for each of the parts and hope that in the future they will still be useful, since the error could be corrected relatively easily by a team who wishes to use them by site-directed-mutagenesis PCR to insert the missing nucleotide. </p><br />
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<br />
<br />
</div><br />
<h3>Synthetic phytochelatin EC20</h3><br />
<div><br />
<p>This is our new contribution of a metal-binding protein to the parts registry. Synthetic phytochelatins are analogues of phytochelatins (PCs); cysteine-rich peptides of the general structure (γ-GluCys)n-Gly (n~2-5) which bind and sequester heavy metals, with a role in detoxification (Cobbett & Goldsbrough 2002). PCs are found mainly in plants but also in other organisms including diatoms, fungi, algae (including cyanobacteria) and some invertebrates (Cobbett & Goldsbrough 2002, Rea 2012). Natively, the PCs are enzymatically synthesized by phytochelatin synthase (PCS) from a glutathione (GSH) precursor and the amino acids in the resulting peptide are linked by the non-standard gamma peptide bonds (Cobbett & Goldsbrough 2002, Rea 2012). However, genetically encoded analogues (containing normal alpha-peptide bonds) of different number Glu-Cys (EC) repeats have been characterised, with the EC20 having the highest binding (Bae <i>et al</i> 2000).<br />
</p><br />
<br />
<br />
<figure class="content-image image-right image-small"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/18/IC14-Biondo-et-al-2012-ion-table.PNG"><br />
<figcaption>Table 1. Metal ion adsorption increase of C. metallidurans cells with PC EC20 displayed on cell surface, compared to negative control. Taken from Biondo <i>et al</i> 2012</figcaption><br />
</figure><br />
<p>The main metal PCs confer tolerance to is Cadmium (Cobbett & Goldsbrough 2002) with a reported stoichiometry of 10 Cd2+ per peptide (Bae <i>et al</i>, . EC20 peptide has still showed good metal ion binding whilst fused to a cellulose binding domain (CBD) for water purification (Xu <i>et al</i> 2002) and when anchored to bacterial cell membrane to confer Cd2+ tolerance (Bae <i>et al</i> 2000, Chaturvedi & Archana 2014). PC EC20 has also been shown to bind many more heavy metals. For example, when displayed on the surface of Cupriavidus metallidurans, the cells capacity to adsorb ions increased by 31 to 219% depending on the ion (table 1, Biondo <i>et al</i> 2012). These binding properties of PC EC20 have been utilised in applications including a regeneratable biosensor of Hg2+, Cd2+, Pb2+, Cu2+ and Zn2+ ions to 100 fM–10 mM (Bontidean <i>et al</i> 2003), and to target CdSe/ZnS quantum dots to streptavidin expressing HeLa cells when biotinylated (Pinaud <i>et al</i> 2004). INSERT TABLE OF THE IONS FROM THE PAPER<br />
</p><br />
<p>We used this part to <a href="https://2014.igem.org/Team:Imperial/Protocols#general">clone</a> a library of different cellulose-binding domains fused to phytochelatin:<figure class="content-image"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/4/40/IC14-PC-part-table.PNG > <br />
<figcaption>Phytochelatin-CBD fusion parts. </figcaption> <br />
</figure> <br />
</p><br />
</div><br />
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</div><br />
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<br />
<br />
<h3> Other potential proteins </h3><br />
<p>During our research we also explored other functional proteins to fuse to the CBDs.<br />
<div class="accordion"><br />
<h3>Laccases</h3><br />
<div><br />
<p>Laccases are enzymes which can break down aromatic and phenolic compounds such as modified estrogens which can end up unfiltered in the water supply with unknown consequences (Auriol <i>et al</i> 2008). This method of treating estrogen in water was explored by <a target="_blank" href="https://2012.igem.org/Team:Bielefeld-Germany">Bielefeld 2012 iGEM team</a>, and our system also has potential to address this.<br />
</p><br />
</div><br />
<br />
<h3>Nanobodies</h3><br />
<div><br />
<p>Nanobodies are fragments of camelid antibodies which can be generated against a variety of antigens and can be easily expressed by organisms such as <i>E. coli </i>. Nanobodies could be used to target viruses, which are often too small to be filtered by size exclusion. For example there are already nanobodies which can bind to <i> Vaccinia </i> virus, ricin, <i>Cholera</i> toxin, Stephylococcal enterotoxin B and H5N1 Influenza (Goldman <i>et al </i> 2006, Ibanez <i>et al </i> 2011) to name a few. Additionally, nanobodies are stable and can be selected to resist proteolytic attach (Harmsen <i>et al </i> 2006). Nanobodies can also be functional when as fusions or conjugates to other proteins for example to target enzymes to specific cells (Cortez-Retamozo 2004), so there is much potential for targeting a wide array of contaminants when fused to cellulose binding domains.<br />
<br />
</p><br />
</div><br />
</div><br />
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</section><br />
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<br />
<section id="results"><br />
<h2>Results</h2><br />
<p>Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim.</p><br />
</section><br />
<br />
<section id="references"><br />
<h2>References</h2><br />
<br />
<ul><br />
<li>Andrade, F.K., Costa, R., Domingues, L., Soares, R., et al. (2010) Improving bacterial cellulose for blood vessel replacement: Functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide. Acta biomaterialia. [Online] 6 (10), 4034–4041. Available from: doi:10.1016/j.actbio.2010.04.023.</li><br />
<br />
<li>Andrade, F.K., Moreira, S.M.G., Domingues, L. & Gama, F.M.P. (2010) Improving the affinity of fibroblasts for bacterial cellulose using carbohydrate-binding modules fused to RGD. Journal of biomedical materials research. Part A. [Online] 92 (1), 9–17. Available from: doi:10.1002/jbm.a.32284.</li><br />
<br />
<li>Auriol, M., Filali-Meknassi, Y., Adams, C.D., Tyagi, R.D., et al. (2008) Removal of estrogenic activity of natural and synthetic hormones from a municipal wastewater: efficiency of horseradish peroxidase and laccase from Trametes versicolor. Chemosphere. [Online] 70 (3), 445–452. Available from: doi:10.1016/j.chemosphere.2007.06.064.</li><br />
<br />
<li>Bae, W., Chen, W., Mulchandani, A. & Mehra, R.K. (2000) Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnology and bioengineering. 70 (5), 518–524.</li><br />
<br />
<li>Biondo, R., da Silva, F.A., Vicente, E.J., Souza Sarkis, J.E., et al. (2012) Synthetic phytochelatin surface display in Cupriavidus metallidurans CH34 for enhanced metals bioremediation. Environmental science & technology. 46 (15), 8325–8332.</li><br />
<br />
<li>Bontidean, I., Ahlqvist, J., Mulchandani, A., Chen, W., et al. (2003) Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection. Biosensors and Bioelectronics. 18 (5-6), 547–553.</li><br />
<br />
<li>Chaturvedi, R. & Archana, G. (2014) Cytosolic expression of synthetic phytochelatin and bacterial metallothionein genes in Deinococcus radiodurans R1 for enhanced tolerance and bioaccumulation of cadmium. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine. 27 (3), 471–482.</li><br />
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<li>Cobbett, C. & Goldsbrough, P. (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annual review of plant biology. [Online] 53159–182. Available from: doi:10.1146/annurev.arplant.53.100301.135154.</li><br />
<br />
<li>Cortez-Retamozo, V. (2004) Efficient Cancer Therapy with a Nanobody-Based Conjugate. Cancer Research. [Online] 64 (8), 2853–2857. Available from: doi:10.1158/0008-5472.CAN-03-3935.</li><br />
<br />
<li>Gilkes, N.R., Kilburn, D.G., Miller, R.C. & Warren, R.A. (1989) Structural and functional analysis of a bacterial cellulase by proteolysis. The Journal of biological chemistry. 264 (30), 17802–17808.</li><br />
<br />
<li>Harmsen, M.M., van Solt, C.B., van Zijderveld-van Bemmel, A.M., Niewold, T.A., et al. (2006) Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Applied microbiology and biotechnology. [Online] 72 (3), 544–551. Available from: doi:10.1007/s00253-005-0300-7.</li><br />
<br />
<li>Kauffmann, C., Shoseyov, O., Shpigel, E., Bayer, E.A., et al. (2000) Novel Methodology for Enzymatic Removal of Atrazine from Water by CBD-Fusion Protein Immobilized on Cellulose. Environmental Science & Technology. [Online] 34 (7), 1292–1296. Available from: doi:10.1021/es990754h.</li><br />
<br />
<li>Kim, H.-D., Choi, S.-L., Kim, H., Sohn, J.H., et al. (2013) Enzyme-linked assay of cellulose-binding domain functions from Cellulomonas fimi on multi-well microtiter plate. Biotechnology and Bioprocess Engineering. 18 (3), 575–580.</li><br />
<br />
<li>Lee, K.-Y., Buldum, G., Mantalaris, A. and Bismarck, A. (2014), More Than Meets the Eye in Bacterial Cellulose: Biosynthesis, Bioprocessing, and Applications in Advanced Fiber Composites. Macromol. Biosci., 14: 10–32. doi: 10.1002/mabi.201300298</li><br />
<br />
<li>Linder, M.; Salovuori, I.; Ruohonen, L.; Teeri, T.T., 1996. Characterization of a Double Cellulose-binding Domain. SYNERGISTIC HIGH AFFINITY BINDING TO CRYSTALLINE CELLULOSE. Journal of Biological Chemistry, 271(35), pp.21268–21272. Available at: http://www.jbc.org/content/271/35/21268.full</li><br />
<br />
<li>Nguyen, P.Q., Botyanszki, Z., Tay, P.K.R. & Joshi, N.S. (2014) Programmable biofilm-based materials from engineered curli nanofibres. Nature communications. [Online] 54945. Available from: doi:10.1038/ncomms5945.</li><br />
<br />
<li>Pinaud, F., King, D., Moore, H.-P. & Weiss, S. (2004) Bioactivation and cell targeting of semiconductor CdSe/ZnS nanocrystals with phytochelatin-related peptides. Journal of the American Chemical Society. 126 (19), 6115–6123.</li><br />
<br />
<br />
<li>Ramos, R., Domingues, L. & Gama, M. (2010) Escherichia coli expression and purification of LL37 fused to a family III carbohydrate-binding module from Clostridium thermocellum. Protein expression and purification. [Online] 71 (1), 1–7. Available from: doi:10.1016/j.pep.2009.10.016.</li><br />
<br />
<li>Warren, R.A., Beck, C.F., Gilkes, N.R., Kilburn, D.G., et al. (1986) Sequence conservation and region shuffling in an endoglucanase and an exoglucanase from Cellulomonas fimi. Proteins. 1 (4), 335–341.</li><br />
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<li>Xu, Z., Bae, W., Mulchandani, A., Mehra, R.K., et al. (2002) Heavy Metal Removal by Novel CBD-EC20 Sorbents Immobilized on Cellulose. Biomacromolecules. [Online] 3 (3), 462–465.</li><br />
<br />
<li>Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., et al. (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences of the United States of America. [Online] 109 (12), E690–E697. Available from: doi:10.1073/pnas.1115485109.</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/FunctionalisationTeam:Imperial/Functionalisation2014-10-18T00:46:46Z<p>Xen sm: </p>
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<h1>Functionalisation</h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#CBDs">CBDs</a><br />
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<li><a data-scroll href="#binding-partners">Functionalisation proteins</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<h2>Overview</h2><br />
<p>Attaching functional proteins to cellulose can expand the properties of cellulose and allow us to selectivity capture specific contaminants in water. By creating fusion proteins of sfGFP and metal-binding proteins with five different cellulose binding domains, three of which are new to the registry, we were able to characterise the relative affinities of the CBDs and show proof-of-concept removal of contaminants from water.</p><br />
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<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Made cellulose binding domains</li><br />
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<br />
</section><br />
<section id="introduction"><br />
<h2>Introduction</h2><br />
<figure class="content-image image-right"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/a/a0/IC14-func-strategy-both-pursue.png"><br />
<figcaption>Figure 1. Pictorial summary showing the two cellulose functionalization approaches considered in our project. Strategy 1: Cellulose binding domains are expressed as fusions with the protein of interest, separated optionally by a linker sequence. The cellulose binding domains attach to the cellulose by hydrophobic interactions. Strategy 2: Curli fibrils are expressed in parallel with cellulose to form a composite material. The curli fibres coding sequence is modified to contain a ‘SpyTag’ peptide (Zakeri <i>et al</i> 2012) which binds specifically via a covalent isopeptide bond to its protein partner, ‘SpyCatcher’. This SpyCatcher is fused to the proteins of interest. In our project we only had time to pursue strategy 1 but are still excited about the potential of strategy 2 in the future.</figcaption><br />
</figure><br />
<p>Bacterial cellulose is already a useful biomaterial as is due its attractive mechanical properties, with research on applications from loudspeaker diaphragms to protective packaging and wound dressings (Lee 2014). However, our project also aims to extend the properties of cellulose by conjugating functional proteins and peptides to it. This allows us to target specific contaminants in water which can not be filtered by size exclusion alone. (See <a href="https://2014.igem.org/Team:Imperial/Water_Report">water report</a> and <a href="https://2014.igem.org/Team:Imperial/Informing_Design">informing design</a> sections).</p><br />
<br />
<p>We considered two ways of functionalising cellulose (figure 1) but continued to work fully on the cellulose-binding domain (CBD) method. The curli-fibre method would be an exciting avenue to explore in the future; it is feasible since there has already been research into making a “programmable” biomaterial from curli fibres using a peptide tag approach (Nguyen <i>et al</i> 2014), and in combination with cellulose could produce a composite with robust mechanical properties as well.<br />
</p><br />
<br />
</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>We aimed to make and characterise new cellulose binding domains, and fuse them to functional proteins. (Incomplete)</p><br />
<br />
<br />
</section><br />
<section id="CBDs"><br />
<h2>Cellulose Binding Domains (CBDs)</h2><br />
<p>The cellulose binding domain (CBD) is the link between our functional proteins and the cellulose. Therefore, we wanted to use a CBD which had high affinity and strong binding to cellulose. However, weaker CBDs may still be useful since they could have the potential be controllably eluted under specific conditions, thereby enabling regeneration of the filter when saturated, and controlled disposal of the collected contaminants (refer to water report if relevant?).<br />
</p><br />
<p>We started our search by looking into the <a href="http://parts.igem.org/">parts registry</a> and found two CBDs already existed in the registry: CBDclos (<a href="http://parts.igem.org/Part:BBa_K863111">BBa_K863111</a>) and CBDcex (<a href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>). We also made three new CBDs to contribute to the registry: dCBD, CBDcipA, and CBDcenA. All the CBDs are in RFC(25) freiberg fusion format to allow for ease of use in protein fusions.</p><br />
<br />
<div class="accordion"><br />
<h3>CBDclos and CBDcex</h3><br />
<div><br />
<p>These parts were biobricked by <a href="https://2012.igem.org/Team:Bielefeld-Germany/Results/cbc">Bielefeld 2012</a>, please see their part pages (<a href="http://parts.igem.org/Part:BBa_K863111">BBa_K863111</a>a and <a href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>) for detailed information. CBDclos is from the cellulolytic bacterium <em>Clostridium cellulovorans</em> and CBDcex originates from the cellulolytic bacterium <em>Cellulomonas fimi</em>. This is the same organism as for CBDcenA; cex is from the exoglucanase gene and cen is from the endoglucanase gene.</p><br />
</div><br />
<h3>Double CBD (dCBD) with N-terminal linker</h3><br />
<div><br />
<p>This part, <a href="http://parts.igem.org/Part:BBa_K1321340">BBa_K1321340</a> is based on the double cellulose-binding domain construct synthesised and characterised by Linder <i>et al</i>1996, who found that this double CBD had higher affinity for cellulose than either of the two CBDs on their own. The main difference is that our part contains an additional linker sequence on the N-terminus of the protein.<br />
</p><br />
<p>The two CBDs are from the fungus <i>Trichoderma reesei</i> (<i>Hypocrea jecorina</i>) Exocellobiohydrolase (Exoglucanase) I (cbh1), uniprot ID P62694; and Exocellobiohydrolase (Exoglucanase) II, uniprot ID P07987 (cbh2); with a linker peptide between the two CBDs and at the N-terminus of the protein. Both linkers are the same amino acid sequence and are based on the endogenous linker sequences that exists in cbh1 and cbh2 genes. The linker sequence is PGANPPGTTTTSRPATTTGSSPGP which is the same as used by Linder <i>et al</i> 1996. The first three amino acids are from the cbh2 endogenous linker, and the rest is from the cbh1 endogenous linker. CBDcbh1 is placed C-terminal to CBDcbh2 because naturally CBDcbh1 is a C-terminal domain and CBDcbh2 is an N-terminal domain. Both CBDs are from the <a class='iframe' href="http://www.cazy.org/CBM1.html">CBM family 1</a>. The precise location of the CBD within the cbh genes was slightly different according to the uniprot annotations and the sequence used by Linder <i>et al</i> 1996; we chose to use the sequence from the paper since the protein was expressed and characterised successfully.</p><br />
<br />
</div><br />
<h3>CBDcipA with N and C-terminal linker</h3><br />
<div><br />
<p>This CBD is from the Cellulosomal -scaffolding protein A (cipA) of Clostridium thermocellum including the endogenous linker sequences at the N and C-terminus (<a class='iframe' href="http://www.uniprot.org/uniprot/Q06851">UniProt ID Q06851</a>). It is in RFC25 format to allow for easy use in protein fusions.</p><br />
<br />
<p>The CBD is part of the <a class='iframe' href="http://www.cazy.org/CBM3.html">CBM3 family</a> . This CBD has been used in many application: in fusions with cell adhesion peptides to enhance the properties of cellulose as a cell-growth matrix (Andrade <i>et al </i> 2010a, Andrade <i>et al </i> 2010b), fused to enzymes to remove contaminants from water (Kauffmann <i> et al </i> 2000) and fused to an antimicrobial peptide (Ramos, Domingues & Gama 2010).</p><br />
<br />
<p>At present the cloning for these constructs is still in progress to correct an illegal EcoRI site which was identified in the parts with this CBD. This can be achieved with a silent mutation via site-directed mutagenesis and we aim to send these parts to the registry once thi<br />
</p><br />
<br />
</div><br />
<h3>CBDcenA with C-terminal linker</h3><br />
<div><br />
<p>This CBD is from the Endoglucanase A (cenA) gene from <i>Cellulomonas fimi</i> and also contains an endogenous C-terminal linker. The cellulose binding domain region occurs at the N-terminus of the cenA gene (UniProt P07984 link to http://www.uniprot.org/uniprot/P07984) and is from the <a class='iframe' href="http://www.cazy.org/CBM2.html">CBM family 2</a><br />
</p><br />
<p>The linker sequence (PTTSPTPTPTPTTPTPTPTPTPTPTPTVTP) is Pro-Thr box which has an extended conformation and acts as a hinge region (Gilkes <i>et al</i> 1989). The endoglucanase CBDcenA has 50% homology to the exoglucanase CBDcex (<a class="iframe" href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>) and the linker is highly conserved in both, but the order of the catalytic, linker and cellulose-binding regions is reversed (Warren <i>et al</i> 1986). It has been shown that CBDcenA has the highest binding affinity for crystalline cellulose out of the <i>C. fimi</i> CBDs (Kim <i>et al</i> 2013).<br />
</p><br />
</div><br />
</div><br />
</section><br />
<br />
<br />
</section><br />
<section id="binding-partners"><br />
<h2>Functionalisation proteins: Binding partners</h2><br />
<h3>Superfolder Green Fluorescent Protein (sfGFP) </h3><br />
<figure class="content-image image-right"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/8/85/IC14-sfGFP-part-table.PNG> <br />
<figcaption>sfGFP-CBD fusion parts</figcaption> <br />
</figure> <p> sfGFP is a more stable mutant of <a href="http://parts.igem.org/Parts:BBa_E0040">mut3GFP</a> and we chose to use this as a robust, easily visible protein to initially fuse to our CBDs. We also designed one of our assays [insert link on 'one of our assays'] for characterising CBD binding using the sfGFP fusion constructs. sfGFP already existed in the registry (<a href="http://parts.igem.org/Part:BBa_I746916">BBa_I746916</a>), detailed information on sfGFP properties is included in this part description) however since we needed to make fusion proteins with our CBDs we modified the part and submitted it in RFC[25] Freiberg fusion format (<a href="http://parts.igem.org/Parts:BBa_K1321337">K1321337</a>). We created the following fusion parts with sfGFP: <br />
<br />
<br />
<br />
</p><br />
<br />
<h3> Metal Binding Proteins </h3><br />
<p>One of the contaminants we can filter out which current size-exclusion technologies can not target, are <a href="https://2014.igem.org/Team:Imperial/Water_Report">heavy metals</a>. We have worked with four different metal binding proteins: the metallothioneins SmtA and fMT, a histidine-rich nickel binding protein (NiBP) from the bacterium <i>Heliobacter pylori</i> and a synthetic phytochelatin (PC) EC20.<br />
</p><br />
<div class="accordion"><br />
<h3>SmtA and fMT metallothioneins</h3><br />
<div><br />
<p>These two parts already existed in the BioBrick registry, but since we needed to use them as fusions with the CBDs we improved the parts by making them compatible with the <a class="iframe" href="http://parts.igem.org/Assembly_standard_25">RFC(25) Frieburg Fusion BioBrick standard.</a></p><br />
<p>Metallothioneins (MTs) are genetically encoded cysteine-rich peptides which serve a role in the detoxification of heavy metals in plants, animals and some prokaryotes (Cobbett, 2002). They have a similar structure and function to phytochelatins, but are produced by different mechanisms (Cobbett, 2002). SmtA is from the cyanobacteria <i>Synechococcus sp.</i> and was used by the <a target="_blank" href="https://2011.igem.org/Team:Tokyo-NoKoGen">Tokyo-NoKoGen 2011</a> iGEM team (who submitted the RFC10 part originally, <a class="iframe" href="http://parts.igem.org/Part:BBa_K519010">BBa_K519010</a>) to confer cadmium (Cd2+) tolerance to <em>E. coli</em> cells when expressed. fMT originates from the seaweed <i>Fucus vesiculosus</i> and was submitted by <a target="_blank" href="https://2009.igem.org/Team:Groningen">Groningen 2009 iGEM team </a> and detailed information can be found on the original part page (<a class="iframe" href="http://parts.igem.org/Part:BBa_K190019">BBa_K190019</a>).</p><br />
<p>We successfully <a href="https://2014.igem.org/Team:Imperial/Protocols#general">cloned</a> the following CBD fusion constructs: </p><br />
<br />
<figure class="content-image image-right image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/a/ac/IC14-SmtA-part-table.PNG> <br />
<figcaption>SmtA-CBD fusion parts</figcaption> <br />
</figure> <br />
<br />
<figure class="content-image image-left image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/6/62/IC14-fMT-part-table.PNG> <br />
<figcaption>fMT-CBD fusion parts</figcaption> <br />
</figure> <br />
<br />
<br />
<br />
<br />
</div><br />
<h3>Nickel binding protein (NiBP)</h3><br />
<div><br />
<figure class="content-image image-right"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/f/f6/IC14-NiBP-part-table.PNG> <br />
<figcaption>NiBP-CBD fusions parts</figcaption> <br />
</figure><br />
<p><br />
This protein is the histidine-rich nickel-binding protein from <i>Heliobacter pylori</i> (GenBank accession ACX98466.1), and already existed as a BioBrick in the registry (<a class="iframe" href="http://parts.igem.org/Part:K1151001">K1151001</a>). Since we needed to use this part in fusion proteins we improved the part by submitting it as a basic part in RFC(25) (link) Freiberg fusion format (<a class="iframe" href="http://parts.igem.org/Part:K1321009">K1321009</a>).<br />
</p><br />
<p>We successfully <a href="https://2014.igem.org/Team:Imperial/Protocols#general">cloned</a> the following CBD fusion constructs with NiBP:<br />
<br />
</p><br />
<p>However we discovered late on in the project that the DNA sequence of the NiBP protein in the registry is out of frame, leading to an incorrect protein translation and read-through of the stop codon (figure 2).</p><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/8/80/IC14-NiBP-protein-alignment.PNG"><br />
<figcaption>Figure 2. Alignment of BBa_K1151001 NiBP in the registry to the , showing incorrect amino-acid sequence in red compared to <a target="_blank" href="http://www.ncbi.nlm.nih.gov/protein/261838700">native <i>H.pylori</i></a> sequence.<br />
</figcaption><br />
</figure><br />
<figure class="content-image image-right"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/e/e1/IC14-NiBP-DNA-alignment.PNG"><br />
<figcaption>Figure 3. Single nucleotide deletion in BBa_K1151001 compared to native <i>H.pylori</i> sequence (<a target="_blank" href="http://www.ncbi.nlm.nih.gov/nuccore/261837457">GenBank CP000012.1</a> (link ) bp1391851-1392033, reverse complement) resulting in any downstream nucleotides being out of frame.</figcaption><br />
</figure><br />
<br />
<p>Further investigation into the error let us to identify a single nucleotide deletion in <a class="iframe" href="http://parts.igem.org/Part:BBa_K1151001">BBa_K1151001</a> compared to the native <i>H.pylori</i> DNA sequence (<a target="_blank" href="http://www.ncbi.nlm.nih.gov/nuccore/261837457">GenBank CP000012.1</a> bp1391851-1392033, reverse complement strand). As seen in Figure 3 the deletion is a ‘C’ at bp 53 of the native sequence. This error was propagated through all our NiBP constructs and means that any subsequent C-terminal fusions are also out of frame and we were unable to express and characterise these constructs. We have noted this on the description pages for each of the parts and hope that in the future they will still be useful, since the error could be corrected relatively easily by a team who wishes to use them by site-directed-mutagenesis PCR to insert the missing nucleotide. </p><br />
<br />
<br />
<br />
</div><br />
<h3>Synthetic phytochelatin EC20</h3><br />
<div><br />
<p>This is our new contribution of a metal-binding protein to the parts registry. Synthetic phytochelatins are analogues of phytochelatins (PCs); cysteine-rich peptides of the general structure (γ-GluCys)n-Gly (n~2-5) which bind and sequester heavy metals, with a role in detoxification (Cobbett & Goldsbrough 2002). PCs are found mainly in plants but also in other organisms including diatoms, fungi, algae (including cyanobacteria) and some invertebrates (Cobbett & Goldsbrough 2002, Rea 2012). Natively, the PCs are enzymatically synthesized by phytochelatin synthase (PCS) from a glutathione (GSH) precursor and the amino acids in the resulting peptide are linked by the non-standard gamma peptide bonds (Cobbett & Goldsbrough 2002, Rea 2012). However, genetically encoded analogues (containing normal alpha-peptide bonds) of different number Glu-Cys (EC) repeats have been characterised, with the EC20 having the highest binding (Bae <i>et al</i> 2000).<br />
</p><br />
<br />
<br />
<figure class="content-image image-right image-small"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/18/IC14-Biondo-et-al-2012-ion-table.PNG"><br />
<figcaption>Table 1. Metal ion adsorption increase of C. metallidurans cells with PC EC20 displayed on cell surface, compared to negative control. Taken from Biondo <i>et al</i> 2012</figcaption><br />
</figure><br />
<p>The main metal PCs confer tolerance to is Cadmium (Cobbett & Goldsbrough 2002) with a reported stoichiometry of 10 Cd2+ per peptide (Bae <i>et al</i>, . EC20 peptide has still showed good metal ion binding whilst fused to a cellulose binding domain (CBD) for water purification (Xu <i>et al</i> 2002) and when anchored to bacterial cell membrane to confer Cd2+ tolerance (Bae <i>et al</i> 2000, Chaturvedi & Archana 2014). PC EC20 has also been shown to bind many more heavy metals. For example, when displayed on the surface of Cupriavidus metallidurans, the cells capacity to adsorb ions increased by 31 to 219% depending on the ion (table 1, Biondo <i>et al</i> 2012). These binding properties of PC EC20 have been utilised in applications including a regeneratable biosensor of Hg2+, Cd2+, Pb2+, Cu2+ and Zn2+ ions to 100 fM–10 mM (Bontidean <i>et al</i> 2003), and to target CdSe/ZnS quantum dots to streptavidin expressing HeLa cells when biotinylated (Pinaud <i>et al</i> 2004). INSERT TABLE OF THE IONS FROM THE PAPER<br />
</p><br />
<p>We used this part to <a href="https://2014.igem.org/Team:Imperial/Protocols#general">clone</a> a library of different cellulose-binding domains fused to phytochelatin:<figure class="content-image"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/4/40/IC14-PC-part-table.PNG > <br />
<figcaption>Phytochelatin-CBD fusion parts. </figcaption> <br />
</figure> <br />
</p><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<h3> Other potential proteins </h3><br />
<p>During our research we also explored other functional proteins to fuse to the CBDs.<br />
<div class="accordion"><br />
<h3>Laccases</h3><br />
<div><br />
<p>Laccases are enzymes which can break down aromatic and phenolic compounds such as modified estrogens which can end up unfiltered in the water supply with unknown consequences (Auriol <i>et al</i> 2008). This method of treating estrogen in water was explored by <a target="_blank" href="https://2012.igem.org/Team:Bielefeld-Germany">Bielefeld 2012 iGEM team</a>, and our system also has potential to address this.<br />
</p><br />
</div><br />
<br />
<h3>Nanobodies</h3><br />
<div><br />
<p>Nanobodies are fragments of camelid antibodies which can be generated against a variety of antigens and can be easily expressed by organisms such as <i>E. coli </i>. Nanobodies could be used to target viruses, which are often too small to be filtered by size exclusion. For example there are already nanobodies which can bind to <i> Vaccinia </i> virus, ricin, <i>Cholera</i> toxin, Stephylococcal enterotoxin B and H5N1 Influenza (Goldman <i>et al </i> 2006, Ibanez <i>et al </i> 2011) to name a few. Additionally, nanobodies are stable and can be selected to resist proteolytic attach (Harmsen <i>et al </i> 2006). Nanobodies can also be functional when as fusions or conjugates to other proteins for example to target enzymes to specific cells (Cortez-Retamozo 2004), so there is much potential for targeting a wide array of contaminants when fused to cellulose binding domains.<br />
<br />
</p><br />
</div><br />
</div><br />
<br />
</section><br />
<br />
<br />
<section id="results"><br />
<h2>Results</h2><br />
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</section><br />
<br />
<section id="references"><br />
<h2>References</h2><br />
<br />
<ul><br />
<li>Andrade, F.K., Costa, R., Domingues, L., Soares, R., et al. (2010) Improving bacterial cellulose for blood vessel replacement: Functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide. Acta biomaterialia. [Online] 6 (10), 4034–4041. Available from: doi:10.1016/j.actbio.2010.04.023.</li><br />
<br />
<li>Andrade, F.K., Moreira, S.M.G., Domingues, L. & Gama, F.M.P. (2010) Improving the affinity of fibroblasts for bacterial cellulose using carbohydrate-binding modules fused to RGD. Journal of biomedical materials research. Part A. [Online] 92 (1), 9–17. Available from: doi:10.1002/jbm.a.32284.</li><br />
<br />
<li>Auriol, M., Filali-Meknassi, Y., Adams, C.D., Tyagi, R.D., et al. (2008) Removal of estrogenic activity of natural and synthetic hormones from a municipal wastewater: efficiency of horseradish peroxidase and laccase from Trametes versicolor. Chemosphere. [Online] 70 (3), 445–452. Available from: doi:10.1016/j.chemosphere.2007.06.064.</li><br />
<br />
<li>Bae, W., Chen, W., Mulchandani, A. & Mehra, R.K. (2000) Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnology and bioengineering. 70 (5), 518–524.</li><br />
<br />
<li>Biondo, R., da Silva, F.A., Vicente, E.J., Souza Sarkis, J.E., et al. (2012) Synthetic phytochelatin surface display in Cupriavidus metallidurans CH34 for enhanced metals bioremediation. Environmental science & technology. 46 (15), 8325–8332.</li><br />
<br />
<li>Bontidean, I., Ahlqvist, J., Mulchandani, A., Chen, W., et al. (2003) Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection. Biosensors and Bioelectronics. 18 (5-6), 547–553.</li><br />
<br />
<li>Chaturvedi, R. & Archana, G. (2014) Cytosolic expression of synthetic phytochelatin and bacterial metallothionein genes in Deinococcus radiodurans R1 for enhanced tolerance and bioaccumulation of cadmium. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine. 27 (3), 471–482.</li><br />
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<li>Cobbett, C. & Goldsbrough, P. (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annual review of plant biology. [Online] 53159–182. Available from: doi:10.1146/annurev.arplant.53.100301.135154.</li><br />
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<li>Cortez-Retamozo, V. (2004) Efficient Cancer Therapy with a Nanobody-Based Conjugate. Cancer Research. [Online] 64 (8), 2853–2857. Available from: doi:10.1158/0008-5472.CAN-03-3935.</li><br />
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<li>Gilkes, N.R., Kilburn, D.G., Miller, R.C. & Warren, R.A. (1989) Structural and functional analysis of a bacterial cellulase by proteolysis. The Journal of biological chemistry. 264 (30), 17802–17808.</li><br />
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<li>Harmsen, M.M., van Solt, C.B., van Zijderveld-van Bemmel, A.M., Niewold, T.A., et al. (2006) Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Applied microbiology and biotechnology. [Online] 72 (3), 544–551. Available from: doi:10.1007/s00253-005-0300-7.</li><br />
<br />
<li>Kauffmann, C., Shoseyov, O., Shpigel, E., Bayer, E.A., et al. (2000) Novel Methodology for Enzymatic Removal of Atrazine from Water by CBD-Fusion Protein Immobilized on Cellulose. Environmental Science & Technology. [Online] 34 (7), 1292–1296. Available from: doi:10.1021/es990754h.</li><br />
<br />
<li>Kim, H.-D., Choi, S.-L., Kim, H., Sohn, J.H., et al. (2013) Enzyme-linked assay of cellulose-binding domain functions from Cellulomonas fimi on multi-well microtiter plate. Biotechnology and Bioprocess Engineering. 18 (3), 575–580.</li><br />
<br />
<li>Lee, K.-Y., Buldum, G., Mantalaris, A. and Bismarck, A. (2014), More Than Meets the Eye in Bacterial Cellulose: Biosynthesis, Bioprocessing, and Applications in Advanced Fiber Composites. Macromol. Biosci., 14: 10–32. doi: 10.1002/mabi.201300298</li><br />
<br />
<li>Linder, M.; Salovuori, I.; Ruohonen, L.; Teeri, T.T., 1996. Characterization of a Double Cellulose-binding Domain. SYNERGISTIC HIGH AFFINITY BINDING TO CRYSTALLINE CELLULOSE. Journal of Biological Chemistry, 271(35), pp.21268–21272. Available at: http://www.jbc.org/content/271/35/21268.full</li><br />
<br />
<li>Nguyen, P.Q., Botyanszki, Z., Tay, P.K.R. & Joshi, N.S. (2014) Programmable biofilm-based materials from engineered curli nanofibres. Nature communications. [Online] 54945. Available from: doi:10.1038/ncomms5945.</li><br />
<br />
<li>Pinaud, F., King, D., Moore, H.-P. & Weiss, S. (2004) Bioactivation and cell targeting of semiconductor CdSe/ZnS nanocrystals with phytochelatin-related peptides. Journal of the American Chemical Society. 126 (19), 6115–6123.</li><br />
<br />
<br />
<li>Ramos, R., Domingues, L. & Gama, M. (2010) Escherichia coli expression and purification of LL37 fused to a family III carbohydrate-binding module from Clostridium thermocellum. Protein expression and purification. [Online] 71 (1), 1–7. Available from: doi:10.1016/j.pep.2009.10.016.</li><br />
<br />
<li>Warren, R.A., Beck, C.F., Gilkes, N.R., Kilburn, D.G., et al. (1986) Sequence conservation and region shuffling in an endoglucanase and an exoglucanase from Cellulomonas fimi. Proteins. 1 (4), 335–341.</li><br />
<br />
<li>Xu, Z., Bae, W., Mulchandani, A., Mehra, R.K., et al. (2002) Heavy Metal Removal by Novel CBD-EC20 Sorbents Immobilized on Cellulose. Biomacromolecules. [Online] 3 (3), 462–465.</li><br />
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<li>Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., et al. (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences of the United States of America. [Online] 109 (12), E690–E697. Available from: doi:10.1073/pnas.1115485109.</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T00:41:53Z<p>Xen sm: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#engineering">Engineering</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
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<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
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</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost.</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure X for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 3. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
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<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
</div><br />
<h3>Inducible promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p> <br />
</div><br />
<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p>One key drawback of producing cellulose using the obligate aerobe <i>G. xylinus</i> is that it cannot grow below the medium surface in static culture[1], reducing the overall volume of the pellicle that is biologically active. This drawback has been overcome to some extent by transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i>[2], another obligate aerobe that expresses this protein in oxygen-poor environments.</p><br />
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<p><i>Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<p>We cloned the Vitreoscilla hemoglobin gene (<em> Vhb </em>) behind a medium-to-strong Anderson promoter <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass, most likely by increasing oxygen availability to cells. </p><br />
<figure class="content-image"><br />
<img class="image-full image-right" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure Z. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. </figcaption><br />
</figure><br />
</div><br />
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</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
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<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
<br />
<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
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</ul><br />
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</li><br />
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<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
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<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
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<h3>References</h3><br />
<ul><br />
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<li>[1] https://www.researchgate.net/publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T00:40:25Z<p>Xen sm: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#engineering">Engineering</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<section id="introduction"><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
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</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost.</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure X for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 3. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
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<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
</div><br />
<h3>Inducible promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p> <br />
</div><br />
<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p>One key drawback of producing cellulose using the obligate aerobe <i>G. xylinus</i> is that it cannot grow below the medium surface in static culture[1], reducing the overall volume of the pellicle that is biologically active. This drawback has been overcome to some extent by transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i>[2], another obligate aerobe that expresses this protein in oxygen-poor environments.</p><br />
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<p><i>Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<p>We cloned the Vitreoscilla hemoglobin gene (<em> Vhb </em>) behind a medium-to-strong Anderson promoter <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass, most likely by increasing oxygen availability to cells. </p><br />
<figure class="content-image"><br />
<img class="image-full image-right" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure Z. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. </figcaption><br />
</figure><br />
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</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
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<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
<br />
<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
<br />
<br />
</div><br />
<br />
</div><br />
</section><br />
<br />
<br />
<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
<br />
<section id="references"><br />
<h3>References</h3><br />
<ol><br />
<li></li><br />
<li></li><br />
<li></li><br />
<li></li><br />
<li></li><br />
<br />
<br />
<br />
<br />
<br />
</ol><br />
</section><br />
</div><br />
<br />
<br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
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{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/Lab-bookTeam:Imperial/Lab-book2014-10-18T00:30:41Z<p>Xen sm: </p>
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<h1>Lab-books</h1><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Introduction</h2><br />
<p>We kept a record of our lab work across a variety of online documents as well as paper based lab-books. Access our online working documents on this page.</p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>General Mixed LabWork</h2><br />
<p> This <a href="https://static.igem.org/mediawiki/2014/8/8c/Mixed_Labbook.pdf">document</a> contains experimental details for some of the following parts of the project<br />
<ul> <br />
<li>Bulk cellulose growth and processing techniques</li><br />
<li>Coculture experiments </li><br />
<li><i>G. xylinus</i> isolation and culturing</li><br />
<li>Initial cloning work for Acs operon </li><br />
<li>Preliminary work for isolating curli genes </li><br />
</ul></p><br />
<br />
</div><br />
<br />
</div><br />
<br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Primer Inventory</h2><br />
<p>Primers for sequencing, mutagenesis, Gibson Assembly, overlap extension PCR... here is our <a href="https://static.igem.org/mediawiki/2014/d/de/PRIMER-INVENTORY.xls <br />
">inventory</a></p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Functionalisation cloning</h2><br />
<p><a href="https://static.igem.org/mediawiki/2014/9/97/Functionalisation_Labwork_record_GC-CM_collated.pdf">This lab-book </a> is a break-away from the mixed lab-book and contains details of cloning the constructs used in the <a href="https://2014.igem.org/Team:Imperial/Functionalisation">functionalisation</a> part of our project. </p> <br />
<br />
<p> <a href="https://static.igem.org/mediawiki/2014/c/ce/PCR-Digestion-Phos-Ligation-Test_Functionalisation-mainly.xls<br />
">This spreadsheet</a> also details specific digestions and ligation set-ups for these constructs.<br />
<br />
</div><br />
<br />
</div><br />
<br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Interlab study cloning</h2><br />
<p>Precise cloning reaction set-ups are detailed <a href="https://static.igem.org/mediawiki/2014/a/af/Interlab-study-cloning-lab-work-record.xls">here</a></p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2><i>Gluconacetobacter xylinus</i></h2><br />
<p><a href="https://static.igem.org/mediawiki/2014/7/71/Michael%27s_Lab_notebook_-_Copy.pdf">Lab-book</a> exclusively for work on <i>G.xylinus</i>, with parts including:<br />
<ul><br />
<li> Strain growth and isolation </li><br />
<li> Parts library and characterisation</li><br />
<li> Genomic DNA extraction and library preparation </li><br />
</p><br />
<br />
</div><br />
<br />
</div><br />
<br />
<br />
</div><br />
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{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/File:Michael%27s_Lab_notebook_-_Copy.pdfFile:Michael's Lab notebook - Copy.pdf2014-10-18T00:23:10Z<p>Xen sm: Details all parts of project involving G.xylinus</p>
<hr />
<div>Details all parts of project involving G.xylinus</div>Xen smhttp://2014.igem.org/Team:Imperial/Lab-bookTeam:Imperial/Lab-book2014-10-18T00:03:18Z<p>Xen sm: </p>
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<div>{{:Team:Imperial/Templates:header}}<br />
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<body id="labbook"><br />
<div class="content-wrapper"><br />
<h1>Lab-books</h1><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Introduction</h2><br />
<p>We kept a record of our lab work across a variety of online documents as well as paper based lab-books. Access our online working documents on this page.</p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>General Mixed LabWork</h2><br />
<p> This <a href="https://static.igem.org/mediawiki/2014/8/8c/Mixed_Labbook.pdf">document</a> contains experimental details for some of the following parts of the project<br />
<ul> <br />
<li>Bulk cellulose growth and processing techniques</li><br />
<li>Coculture experiments </li><br />
<li><i>G. xylinus</i> isolation and culturing</li><br />
<li>Initial cloning work for Acs operon </li><br />
<li>Preliminary work for isolating curli genes </li><br />
</ul></p><br />
<br />
</div><br />
<br />
</div><br />
<br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Primer Inventory</h2><br />
<p>Primers for sequencing, mutagenesis, Gibson Assembly, overlap extension PCR... here is our <a href="https://static.igem.org/mediawiki/2014/d/de/PRIMER-INVENTORY.xls <br />
">inventory</a></p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Functionalisation cloning</h2><br />
<p><a href="https://static.igem.org/mediawiki/2014/9/97/Functionalisation_Labwork_record_GC-CM_collated.pdf">This lab-book </a> is a break-away from the mixed lab-book and contains details of cloning the constructs used in the <a href="https://2014.igem.org/Team:Imperial/Functionalisation">functionalisation</a> part of our project. </p> <br />
<br />
<p> <a href="https://static.igem.org/mediawiki/2014/c/ce/PCR-Digestion-Phos-Ligation-Test_Functionalisation-mainly.xls<br />
">This spreadsheet</a> also details specific digestions and ligation set-ups for these constructs.<br />
<br />
</div><br />
<br />
</div><br />
<br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Interlab study cloning</h2><br />
<p>Precise cloning reaction set-ups are detailed <a href="https://static.igem.org/mediawiki/2014/a/af/Interlab-study-cloning-lab-work-record.xls">here</a></p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Lab-book 1</h2><br />
<p>LINKS</p><br />
<br />
</div><br />
<br />
</div><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/Lab-bookTeam:Imperial/Lab-book2014-10-18T00:01:07Z<p>Xen sm: </p>
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<div>{{:Team:Imperial/Templates:header}}<br />
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<body id="labbook"><br />
<div class="content-wrapper"><br />
<h1>Lab-books</h1><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Introduction</h2><br />
<p>We kept a record of our lab work across a variety of online documents as well as paper based lab-books. Access our online working documents on this page.</p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>General Mixed LabWork</h2><br />
<p> This <a href="https://static.igem.org/mediawiki/2014/8/8c/Mixed_Labbook.pdf">document</a> contains experimental details for some of the following parts of the project<br />
<ul> <br />
<li>Bulk cellulose growth and processing techniques</li><br />
<li>Coculture experiments </li><br />
<li><i>G. xylinus</i> isolation and culturing</li><br />
<li>Initial cloning work for Acs operon </li><br />
<li>Preliminary work for isolating curli genes </li><br />
</ul></p><br />
<br />
</div><br />
<br />
</div><br />
<br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Primer Inventory</h2><br />
<p>Primers for sequencing, mutagenesis, Gibson Assembly, overlap extension PCR... here is our <a href="https://static.igem.org/mediawiki/2014/d/de/PRIMER-INVENTORY.xls <br />
">inventory</a></p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Functionalisation cloning</h2><br />
<p><a href="https://static.igem.org/mediawiki/2014/9/97/Functionalisation_Labwork_record_GC-CM_collated.pdf">This lab-book </a> is a break-away from the mixed lab-book and contains details of cloning the constructs used in the <a href="https://2014.igem.org/Team:Imperial/Functionalisation">functionalisation</a> part of our project. </p> <br />
<br />
<p> <a href="https://static.igem.org/mediawiki/2014/c/ce/PCR-Digestion-Phos-Ligation-Test_Functionalisation-mainly.xls<br />
">This spreadsheet</a> also details specific digestions and ligation set-ups for these constructs.<br />
<br />
</div><br />
<br />
</div><br />
<br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Lab-book 1</h2><br />
<p>LINKS</p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Lab-book 1</h2><br />
<p>LINKS</p><br />
<br />
</div><br />
<br />
</div><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/Lab-bookTeam:Imperial/Lab-book2014-10-17T23:33:35Z<p>Xen sm: </p>
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<div>{{:Team:Imperial/Templates:header}}<br />
<html><br />
<body id="labbook"><br />
<div class="content-wrapper"><br />
<h1>Lab-books</h1><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Introduction</h2><br />
<p>We kept a record of our lab work across a variety of online documents as well as paper based lab-books. Access our online working documents on this page.</p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>General Mixed LabWork</h2><br />
<p> This <a href="https://static.igem.org/mediawiki/2014/8/8c/Mixed_Labbook.pdf">document</a> contains experimental details for some of the following parts of the project<br />
<ul> <br />
<li>Bulk cellulose growth and processing techniques</li><br />
<li>Coculture experiments </li><br />
<li><i>G. xylinus</i> isolation and culturing</li><br />
<li>Initial cloning work for Acs operon </li><br />
<li>Preliminary work for isolating curli genes </li><br />
</ul></p><br />
<br />
</div><br />
<br />
</div><br />
<br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Primer Inventory</h2><br />
<p>Primers for sequencing, mutagenesis, Gibson Assembly, overlap extension PCR... here is our <a href="https://static.igem.org/mediawiki/2014/d/de/PRIMER-INVENTORY.xls <br />
">inventory</a></p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Functionalisation cloning</h2><br />
<p><a href="https://static.igem.org/mediawiki/2014/a/af/Interlab-study-cloning-lab-work-record.xls">This lab-book </a> is a break-away from the mixed lab-book and contains details of cloning the constructs used in the <a href="https://2014.igem.org/Team:Imperial/Functionalisation">functionalisation</a> part of our project</p><br />
<br />
</div><br />
<br />
</div><br />
<br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Lab-book 1</h2><br />
<p>LINKS</p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Lab-book 1</h2><br />
<p>LINKS</p><br />
<br />
</div><br />
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</div><br />
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<br />
</div><br />
</div><br />
</body><br />
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{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/Lab-bookTeam:Imperial/Lab-book2014-10-17T23:28:30Z<p>Xen sm: </p>
<hr />
<div>{{:Team:Imperial/Templates:header}}<br />
<html><br />
<div class="content-wrapper"><br />
<h1>Lab-books</h1><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Introduction</h2><br />
<p>We kept a record of our lab work across a variety of online documents as well as paper based lab-books. Access our online working documents on this page.</p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>General Mixed LabWork</h2><br />
<p> This <a href="https://static.igem.org/mediawiki/2014/8/8c/Mixed_Labbook.pdf">document</a> contains experimental details for some of the following parts of the project<br />
<ul> <br />
<li>Bulk cellulose growth and processing techniques</li><br />
<li>Coculture experiments </li><br />
<li><i>G. xylinus</i> isolation and culturing</li><br />
<li>Initial cloning work for Acs operon </li><br />
<li>Preliminary work for isolating curli genes </li><br />
</ul></p><br />
<br />
</div><br />
<br />
</div><br />
<br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Primer Inventory</h2><br />
<p>Primers for sequencing, mutagenesis, Gibson Assembly, overlap extension... here is our <a href="https://static.igem.org/mediawiki/2014/d/de/PRIMER-INVENTORY.xls <br />
">inventory</a></p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Lab-book 1</h2><br />
<p>LINKS</p><br />
<br />
</div><br />
<br />
</div><br />
<br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Lab-book 1</h2><br />
<p>LINKS</p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Lab-book 1</h2><br />
<p>LINKS</p><br />
<br />
</div><br />
<br />
</div><br />
<br />
<br />
</div><br />
</div><br />
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{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/Lab-bookTeam:Imperial/Lab-book2014-10-17T22:54:12Z<p>Xen sm: added first link to first labbook</p>
<hr />
<div>{{:Team:Imperial/Templates:header}}<br />
<html><br />
<div class="content-wrapper"><br />
<h1>Lab-books</h1><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Introduction</h2><br />
<p>We kept a record of our lab work across a variety of online documents as well as paper based lab-books. Access our online working documents on this page.</p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>General Mixed LabWork</h2><br />
<p> This <a href="https://static.igem.org/mediawiki/2014/8/8c/Mixed_Labbook.pdf">document</a> contains experimental details for some of the following parts of the project<br />
<ul> <br />
<li>Bulk cellulose growth and processing techniques</li><br />
<li>Coculture experiments </li><br />
<li><i>G. xylinus</i> isolation and culturing</li><br />
<li>Initial cloning work for Acs operon </li><br />
<li>Preliminary work for isolating curli genes </li><br />
</ul></p><br />
<br />
</div><br />
<br />
</div><br />
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<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Lab-book 1</h2><br />
<p>LINKS</p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Lab-book 1</h2><br />
<p>LINKS</p><br />
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</div><br />
<br />
</div><br />
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<div class="pure-u-1-3"><br />
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<h2>Lab-book 1</h2><br />
<p>LINKS</p><br />
<br />
</div><br />
<br />
</div><br />
<div class="pure-u-1-3"><br />
<div class="content content-single"><br />
<h2>Lab-book 1</h2><br />
<p>LINKS</p><br />
<br />
</div><br />
<br />
</div><br />
<br />
<br />
</div><br />
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{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/File:Interlab-study-cloning-lab-work-record.xlsFile:Interlab-study-cloning-lab-work-record.xls2014-10-17T22:28:29Z<p>Xen sm: Cloning reaction and details for the interlab study section of the</p>
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<div>Cloning reaction and details for the interlab study section of the</div>Xen smhttp://2014.igem.org/File:Functionalisation_Labwork_record_GC-CM_collated.pdfFile:Functionalisation Labwork record GC-CM collated.pdf2014-10-17T22:26:51Z<p>Xen sm: Labwork details for the functionalisation part of the project</p>
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<div>Labwork details for the functionalisation part of the project</div>Xen smhttp://2014.igem.org/File:PCR-Digestion-Phos-Ligation-Test_Functionalisation-mainly.xlsFile:PCR-Digestion-Phos-Ligation-Test Functionalisation-mainly.xls2014-10-17T22:24:49Z<p>Xen sm: Cloning reaction details mainly for functionalisation part of project</p>
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<div>Cloning reaction details mainly for functionalisation part of project</div>Xen smhttp://2014.igem.org/File:PRIMER-INVENTORY.xlsFile:PRIMER-INVENTORY.xls2014-10-17T22:16:33Z<p>Xen sm: primers we used in our project</p>
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<div>primers we used in our project</div>Xen smhttp://2014.igem.org/File:Mixed_Labbook.pdfFile:Mixed Labbook.pdf2014-10-17T22:04:52Z<p>Xen sm: Various documented labwork from the team</p>
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<div>Various documented labwork from the team</div>Xen smhttp://2014.igem.org/Team:Imperial/FunctionalisationTeam:Imperial/Functionalisation2014-10-17T21:55:39Z<p>Xen sm: </p>
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<h1>Functionalisation</h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#CBDs">CBDs</a><br />
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<li><a data-scroll href="#binding-partners">Functionalisation proteins</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>By attaching functional proteins to cellulose we can expand it's properties and selectivity capture specific contaminants in water. We used five different cellulose binding domains and fused them to different metal binding proteins, and sfGFP. We performed assays to test the binding of the CBD fusions to our cellulose.</p><br />
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<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Made cellulose binding domains</li><br />
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</section><br />
<section id="introduction"><br />
<h2>Introduction</h2><br />
<figure class="content-image image-right"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/a/a0/IC14-func-strategy-both-pursue.png"><br />
<figcaption>Figure 1. Pictorial summary showing the two cellulose functionalization approaches considered in our project. Strategy 1: Cellulose binding domains are expressed as fusions with the protein of interest, separated optionally by a linker sequence. The cellulose binding domains attach to the cellulose by hydrophobic interactions. Strategy 2: Curli fibrils are expressed in parallel with cellulose to form a composite material. The curli fibres coding sequence is modified to contain a ‘SpyTag’ peptide (Zakeri <i>et al</i> 2012) which binds specifically via a covalent isopeptide bond to its protein partner, ‘SpyCatcher’. This SpyCatcher is fused to the proteins of interest. In our project we only had time to pursue strategy 1 but are still excited about the potential of strategy 2 in the future.</figcaption><br />
</figure><br />
<p>Bacterial cellulose is already a useful biomaterial as is due its attractive mechanical properties, with research on applications from loudspeaker diaphragms to protective packaging and wound dressings (Lee 2014). However, our project also aims to extend the properties of cellulose by conjugating functional proteins and peptides to it. This allows us to target specific contaminants in water which can not be filtered by size exclusion alone. (See <a href="https://2014.igem.org/Team:Imperial/Water_Report">water report</a> and <a href="https://2014.igem.org/Team:Imperial/Informing_Design">informing design</a> sections).</p><br />
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<p>We considered two ways of functionalising cellulose (figure 1) but continued to work fully on the cellulose-binding domain (CBD) method. The curli-fibre method would be an exciting avenue to explore in the future; it is feasible since there has already been research into making a “programmable” biomaterial from curli fibres using a peptide tag approach (Nguyen <i>et al</i> 2014), and in combination with cellulose could produce a composite with robust mechanical properties as well.<br />
</p><br />
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</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
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<p>We aimed to make and characterise new cellulose binding domains, and fuse them to functional proteins. (Incomplete)</p><br />
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</section><br />
<section id="CBDs"><br />
<h2>Cellulose Binding Domains (CBDs)</h2><br />
<p>The cellulose binding domain (CBD) is the link between our functional proteins and the cellulose. Therefore, we wanted to use a CBD which had high affinity and strong binding to cellulose. However, weaker CBDs may still be useful since they could have the potential be controllably eluted under specific conditions, thereby enabling regeneration of the filter when saturated, and controlled disposal of the collected contaminants (refer to water report if relevant?).<br />
</p><br />
<p>We started our search by looking into the <a href="http://parts.igem.org/">parts registry</a> and found two CBDs already existed in the registry: CBDclos (<a href="http://parts.igem.org/Part:BBa_K863111">BBa_K863111</a>) and CBDcex (<a href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>). We also made three new CBDs to contribute to the registry: dCBD, CBDcipA, and CBDcenA. All the CBDs are in RFC(25) freiberg fusion format to allow for ease of use in protein fusions.</p><br />
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<div class="accordion"><br />
<h3>CBDclos and CBDcex</h3><br />
<div><br />
<p>These parts were biobricked by <a href="https://2012.igem.org/Team:Bielefeld-Germany/Results/cbc">Bielefeld 2012</a>, please see their part pages (<a href="http://parts.igem.org/Part:BBa_K863111">BBa_K863111</a>a and <a href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>) for detailed information. CBDclos is from the cellulolytic bacterium <em>Clostridium cellulovorans</em> and CBDcex originates from the cellulolytic bacterium <em>Cellulomonas fimi</em>. This is the same organism as for CBDcenA; cex is from the exoglucanase gene and cen is from the endoglucanase gene.</p><br />
</div><br />
<h3>Double CBD (dCBD) with N-terminal linker</h3><br />
<div><br />
<p>This part, <a href="http://parts.igem.org/Part:BBa_K1321340">BBa_K1321340</a> is based on the double cellulose-binding domain construct synthesised and characterised by Linder <i>et al</i>1996, who found that this double CBD had higher affinity for cellulose than either of the two CBDs on their own. The main difference is that our part contains an additional linker sequence on the N-terminus of the protein.<br />
</p><br />
<p>The two CBDs are from the fungus <i>Trichoderma reesei</i> (<i>Hypocrea jecorina</i>) Exocellobiohydrolase (Exoglucanase) I (cbh1), uniprot ID P62694; and Exocellobiohydrolase (Exoglucanase) II, uniprot ID P07987 (cbh2); with a linker peptide between the two CBDs and at the N-terminus of the protein. Both linkers are the same amino acid sequence and are based on the endogenous linker sequences that exists in cbh1 and cbh2 genes. The linker sequence is PGANPPGTTTTSRPATTTGSSPGP which is the same as used by Linder <i>et al</i> 1996. The first three amino acids are from the cbh2 endogenous linker, and the rest is from the cbh1 endogenous linker. CBDcbh1 is placed C-terminal to CBDcbh2 because naturally CBDcbh1 is a C-terminal domain and CBDcbh2 is an N-terminal domain. Both CBDs are from the <a class='iframe' href="http://www.cazy.org/CBM1.html">CBM family 1</a>. The precise location of the CBD within the cbh genes was slightly different according to the uniprot annotations and the sequence used by Linder <i>et al</i> 1996; we chose to use the sequence from the paper since the protein was expressed and characterised successfully.</p><br />
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</div><br />
<h3>CBDcipA with N and C-terminal linker</h3><br />
<div><br />
<p>This CBD is from the Cellulosomal -scaffolding protein A (cipA) of Clostridium thermocellum including the endogenous linker sequences at the N and C-terminus (<a class='iframe' href="http://www.uniprot.org/uniprot/Q06851">UniProt ID Q06851</a>). It is in RFC25 format to allow for easy use in protein fusions.</p><br />
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<p>The CBD is part of the <a class='iframe' href="http://www.cazy.org/CBM3.html">CBM3 family</a> . This CBD has been used in many application: in fusions with cell adhesion peptides to enhance the properties of cellulose as a cell-growth matrix (Andrade <i>et al </i> 2010a, Andrade <i>et al </i> 2010b), fused to enzymes to remove contaminants from water (Kauffmann <i> et al </i> 2000) and fused to an antimicrobial peptide (Ramos, Domingues & Gama 2010).</p><br />
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<p>At present the cloning for these constructs is still in progress to correct an illegal EcoRI site which was identified in the parts with this CBD. This can be achieved with a silent mutation via site-directed mutagenesis and we aim to send these parts to the registry once thi<br />
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<h3>CBDcenA with C-terminal linker</h3><br />
<div><br />
<p>This CBD is from the Endoglucanase A (cenA) gene from <i>Cellulomonas fimi</i> and also contains an endogenous C-terminal linker. The cellulose binding domain region occurs at the N-terminus of the cenA gene (UniProt P07984 link to http://www.uniprot.org/uniprot/P07984) and is from the <a class='iframe' href="http://www.cazy.org/CBM2.html">CBM family 2</a><br />
</p><br />
<p>The linker sequence (PTTSPTPTPTPTTPTPTPTPTPTPTPTVTP) is Pro-Thr box which has an extended conformation and acts as a hinge region (Gilkes <i>et al</i> 1989). The endoglucanase CBDcenA has 50% homology to the exoglucanase CBDcex (<a class="iframe" href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>) and the linker is highly conserved in both, but the order of the catalytic, linker and cellulose-binding regions is reversed (Warren <i>et al</i> 1986). It has been shown that CBDcenA has the highest binding affinity for crystalline cellulose out of the <i>C. fimi</i> CBDs (Kim <i>et al</i> 2013).<br />
</p><br />
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</section><br />
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<section id="binding-partners"><br />
<h2>Functionalisation proteins: Binding partners</h2><br />
<h3>Superfolder Green Fluorescent Protein (sfGFP) </h3><br />
<figure class="content-image image-right"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/8/85/IC14-sfGFP-part-table.PNG> <br />
<figcaption>sfGFP-CBD fusion parts</figcaption> <br />
</figure> <p> sfGFP is a more stable mutant of <a href="http://parts.igem.org/Parts:BBa_E0040">mut3GFP</a> and we chose to use this as a robust, easily visible protein to initially fuse to our CBDs. We also designed one of our assays [insert link on 'one of our assays'] for characterising CBD binding using the sfGFP fusion constructs. sfGFP already existed in the registry (<a href="http://parts.igem.org/Part:BBa_I746916">BBa_I746916</a>), detailed information on sfGFP properties is included in this part description) however since we needed to make fusion proteins with our CBDs we modified the part and submitted it in RFC[25] Freiberg fusion format (<a href="http://parts.igem.org/Parts:BBa_K1321337">K1321337</a>). We created the following fusion parts with sfGFP: <br />
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<h3> Metal Binding Proteins </h3><br />
<p>One of the contaminants we can filter out which current size-exclusion technologies can not target, are <a href="https://2014.igem.org/Team:Imperial/Water_Report">heavy metals</a>. We have worked with four different metal binding proteins: the metallothioneins SmtA and fMT, a histidine-rich nickel binding protein (NiBP) from the bacterium <i>Heliobacter pylori</i> and a synthetic phytochelatin (PC) EC20.<br />
</p><br />
<div class="accordion"><br />
<h3>SmtA and fMT metallothioneins</h3><br />
<div><br />
<p>These two parts already existed in the BioBrick registry, but since we needed to use them as fusions with the CBDs we improved the parts by making them compatible with the <a class="iframe" href="http://parts.igem.org/Assembly_standard_25">RFC(25) Frieburg Fusion BioBrick standard.</a></p><br />
<p>Metallothioneins (MTs) are genetically encoded cysteine-rich peptides which serve a role in the detoxification of heavy metals in plants, animals and some prokaryotes (Cobbett, 2002). They have a similar structure and function to phytochelatins, but are produced by different mechanisms (Cobbett, 2002). SmtA is from the cyanobacteria <i>Synechococcus sp.</i> and was used by the <a target="_blank" href="https://2011.igem.org/Team:Tokyo-NoKoGen">Tokyo-NoKoGen 2011</a> iGEM team (who submitted the RFC10 part originally, <a class="iframe" href="http://parts.igem.org/Part:BBa_K519010">BBa_K519010</a>) to confer cadmium (Cd2+) tolerance to <em>E. coli</em> cells when expressed. fMT originates from the seaweed <i>Fucus vesiculosus</i> and was submitted by <a target="_blank" href="https://2009.igem.org/Team:Groningen">Groningen 2009 iGEM team </a> and detailed information can be found on the original part page (<a class="iframe" href="http://parts.igem.org/Part:BBa_K190019">BBa_K190019</a>).</p><br />
<p>We successfully <a href="https://2014.igem.org/Team:Imperial/Protocols#general">cloned</a> the following CBD fusion constructs: </p><br />
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<figure class="content-image image-right image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/a/ac/IC14-SmtA-part-table.PNG> <br />
<figcaption>SmtA-CBD fusion parts</figcaption> <br />
</figure> <br />
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<figure class="content-image image-left image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/6/62/IC14-fMT-part-table.PNG> <br />
<figcaption>fMT-CBD fusion parts</figcaption> <br />
</figure> <br />
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<h3>Nickel binding protein (NiBP)</h3><br />
<div><br />
<figure class="content-image image-right"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/f/f6/IC14-NiBP-part-table.PNG> <br />
<figcaption>NiBP-CBD fusions parts</figcaption> <br />
</figure><br />
<p><br />
This protein is the histidine-rich nickel-binding protein from <i>Heliobacter pylori</i> (GenBank accession ACX98466.1), and already existed as a BioBrick in the registry (<a class="iframe" href="http://parts.igem.org/Part:K1151001">K1151001</a>). Since we needed to use this part in fusion proteins we improved the part by submitting it as a basic part in RFC(25) (link) Freiberg fusion format (<a class="iframe" href="http://parts.igem.org/Part:K1321009">K1321009</a>).<br />
</p><br />
<p>We successfully <a href="https://2014.igem.org/Team:Imperial/Protocols#general">cloned</a> the following CBD fusion constructs with NiBP:<br />
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</p><br />
<p>However we discovered late on in the project that the DNA sequence of the NiBP protein in the registry is out of frame, leading to an incorrect protein translation and read-through of the stop codon (figure 2).</p><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/8/80/IC14-NiBP-protein-alignment.PNG"><br />
<figcaption>Figure 2. Alignment of BBa_K1151001 NiBP in the registry to the , showing incorrect amino-acid sequence in red compared to <a target="_blank" href="http://www.ncbi.nlm.nih.gov/protein/261838700">native <i>H.pylori</i></a> sequence.<br />
</figcaption><br />
</figure><br />
<figure class="content-image image-right"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/e/e1/IC14-NiBP-DNA-alignment.PNG"><br />
<figcaption>Figure 3. Single nucleotide deletion in BBa_K1151001 compared to native <i>H.pylori</i> sequence (<a target="_blank" href="http://www.ncbi.nlm.nih.gov/nuccore/261837457">GenBank CP000012.1</a> (link ) bp1391851-1392033, reverse complement) resulting in any downstream nucleotides being out of frame.</figcaption><br />
</figure><br />
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<p>Further investigation into the error let us to identify a single nucleotide deletion in <a class="iframe" href="http://parts.igem.org/Part:BBa_K1151001">BBa_K1151001</a> compared to the native <i>H.pylori</i> DNA sequence (<a target="_blank" href="http://www.ncbi.nlm.nih.gov/nuccore/261837457">GenBank CP000012.1</a> bp1391851-1392033, reverse complement strand). As seen in Figure 3 the deletion is a ‘C’ at bp 53 of the native sequence. This error was propagated through all our NiBP constructs and means that any subsequent C-terminal fusions are also out of frame and we were unable to express and characterise these constructs. We have noted this on the description pages for each of the parts and hope that in the future they will still be useful, since the error could be corrected relatively easily by a team who wishes to use them by site-directed-mutagenesis PCR to insert the missing nucleotide. </p><br />
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<h3>Synthetic phytochelatin EC20</h3><br />
<div><br />
<p>This is our new contribution of a metal-binding protein to the parts registry. Synthetic phytochelatins are analogues of phytochelatins (PCs); cysteine-rich peptides of the general structure (γ-GluCys)n-Gly (n~2-5) which bind and sequester heavy metals, with a role in detoxification (Cobbett & Goldsbrough 2002). PCs are found mainly in plants but also in other organisms including diatoms, fungi, algae (including cyanobacteria) and some invertebrates (Cobbett & Goldsbrough 2002, Rea 2012). Natively, the PCs are enzymatically synthesized by phytochelatin synthase (PCS) from a glutathione (GSH) precursor and the amino acids in the resulting peptide are linked by the non-standard gamma peptide bonds (Cobbett & Goldsbrough 2002, Rea 2012). However, genetically encoded analogues (containing normal alpha-peptide bonds) of different number Glu-Cys (EC) repeats have been characterised, with the EC20 having the highest binding (Bae <i>et al</i> 2000).<br />
</p><br />
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<figure class="content-image image-right image-small"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/18/IC14-Biondo-et-al-2012-ion-table.PNG"><br />
<figcaption>Table 1. Metal ion adsorption increase of C. metallidurans cells with PC EC20 displayed on cell surface, compared to negative control. Taken from Biondo <i>et al</i> 2012</figcaption><br />
</figure><br />
<p>The main metal PCs confer tolerance to is Cadmium (Cobbett & Goldsbrough 2002) with a reported stoichiometry of 10 Cd2+ per peptide (Bae <i>et al</i>, . EC20 peptide has still showed good metal ion binding whilst fused to a cellulose binding domain (CBD) for water purification (Xu <i>et al</i> 2002) and when anchored to bacterial cell membrane to confer Cd2+ tolerance (Bae <i>et al</i> 2000, Chaturvedi & Archana 2014). PC EC20 has also been shown to bind many more heavy metals. For example, when displayed on the surface of Cupriavidus metallidurans, the cells capacity to adsorb ions increased by 31 to 219% depending on the ion (table 1, Biondo <i>et al</i> 2012). These binding properties of PC EC20 have been utilised in applications including a regeneratable biosensor of Hg2+, Cd2+, Pb2+, Cu2+ and Zn2+ ions to 100 fM–10 mM (Bontidean <i>et al</i> 2003), and to target CdSe/ZnS quantum dots to streptavidin expressing HeLa cells when biotinylated (Pinaud <i>et al</i> 2004). INSERT TABLE OF THE IONS FROM THE PAPER<br />
</p><br />
<p>We used this part to <a href="https://2014.igem.org/Team:Imperial/Protocols#general">clone</a> a library of different cellulose-binding domains fused to phytochelatin:<figure class="content-image"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/4/40/IC14-PC-part-table.PNG > <br />
<figcaption>Phytochelatin-CBD fusion parts. </figcaption> <br />
</figure> <br />
</p><br />
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</div><br />
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<h3> Other potential proteins </h3><br />
<p>During our research we also explored other functional proteins to fuse to the CBDs.<br />
<div class="accordion"><br />
<h3>Laccases</h3><br />
<div><br />
<p>Laccases are enzymes which can break down aromatic and phenolic compounds such as modified estrogens which can end up unfiltered in the water supply with unknown consequences (Auriol <i>et al</i> 2008). This method of treating estrogen in water was explored by <a target="_blank" href="https://2012.igem.org/Team:Bielefeld-Germany">Bielefeld 2012 iGEM team</a>, and our system also has potential to address this.<br />
</p><br />
</div><br />
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<h3>Nanobodies</h3><br />
<div><br />
<p>Nanobodies are fragments of camelid antibodies which can be generated against a variety of antigens and can be easily expressed by organisms such as <i>E. coli </i>. Nanobodies could be used to target viruses, which are often too small to be filtered by size exclusion. For example there are already nanobodies which can bind to <i> Vaccinia </i> virus, ricin, <i>Cholera</i> toxin, Stephylococcal enterotoxin B and H5N1 Influenza (Goldman <i>et al </i> 2006, Ibanez <i>et al </i> 2011) to name a few. Additionally, nanobodies are stable and can be selected to resist proteolytic attach (Harmsen <i>et al </i> 2006). Nanobodies can also be functional when as fusions or conjugates to other proteins for example to target enzymes to specific cells (Cortez-Retamozo 2004), so there is much potential for targeting a wide array of contaminants when fused to cellulose binding domains.<br />
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</p><br />
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</section><br />
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<section id="results"><br />
<h2>Results</h2><br />
<p>Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim.</p><br />
</section><br />
<br />
<section id="references"><br />
<h2>References</h2><br />
<br />
<ul><br />
<li>Andrade, F.K., Costa, R., Domingues, L., Soares, R., et al. (2010) Improving bacterial cellulose for blood vessel replacement: Functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide. Acta biomaterialia. [Online] 6 (10), 4034–4041. Available from: doi:10.1016/j.actbio.2010.04.023.</li><br />
<br />
<li>Andrade, F.K., Moreira, S.M.G., Domingues, L. & Gama, F.M.P. (2010) Improving the affinity of fibroblasts for bacterial cellulose using carbohydrate-binding modules fused to RGD. Journal of biomedical materials research. Part A. [Online] 92 (1), 9–17. Available from: doi:10.1002/jbm.a.32284.</li><br />
<br />
<li>Auriol, M., Filali-Meknassi, Y., Adams, C.D., Tyagi, R.D., et al. (2008) Removal of estrogenic activity of natural and synthetic hormones from a municipal wastewater: efficiency of horseradish peroxidase and laccase from Trametes versicolor. Chemosphere. [Online] 70 (3), 445–452. Available from: doi:10.1016/j.chemosphere.2007.06.064.</li><br />
<br />
<li>Bae, W., Chen, W., Mulchandani, A. & Mehra, R.K. (2000) Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnology and bioengineering. 70 (5), 518–524.</li><br />
<br />
<li>Biondo, R., da Silva, F.A., Vicente, E.J., Souza Sarkis, J.E., et al. (2012) Synthetic phytochelatin surface display in Cupriavidus metallidurans CH34 for enhanced metals bioremediation. Environmental science & technology. 46 (15), 8325–8332.</li><br />
<br />
<li>Bontidean, I., Ahlqvist, J., Mulchandani, A., Chen, W., et al. (2003) Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection. Biosensors and Bioelectronics. 18 (5-6), 547–553.</li><br />
<br />
<li>Chaturvedi, R. & Archana, G. (2014) Cytosolic expression of synthetic phytochelatin and bacterial metallothionein genes in Deinococcus radiodurans R1 for enhanced tolerance and bioaccumulation of cadmium. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine. 27 (3), 471–482.</li><br />
<br />
<br />
<li>Cobbett, C. & Goldsbrough, P. (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annual review of plant biology. [Online] 53159–182. Available from: doi:10.1146/annurev.arplant.53.100301.135154.</li><br />
<br />
<li>Cortez-Retamozo, V. (2004) Efficient Cancer Therapy with a Nanobody-Based Conjugate. Cancer Research. [Online] 64 (8), 2853–2857. Available from: doi:10.1158/0008-5472.CAN-03-3935.</li><br />
<br />
<li>Gilkes, N.R., Kilburn, D.G., Miller, R.C. & Warren, R.A. (1989) Structural and functional analysis of a bacterial cellulase by proteolysis. The Journal of biological chemistry. 264 (30), 17802–17808.</li><br />
<br />
<li>Harmsen, M.M., van Solt, C.B., van Zijderveld-van Bemmel, A.M., Niewold, T.A., et al. (2006) Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Applied microbiology and biotechnology. [Online] 72 (3), 544–551. Available from: doi:10.1007/s00253-005-0300-7.</li><br />
<br />
<li>Kauffmann, C., Shoseyov, O., Shpigel, E., Bayer, E.A., et al. (2000) Novel Methodology for Enzymatic Removal of Atrazine from Water by CBD-Fusion Protein Immobilized on Cellulose. Environmental Science & Technology. [Online] 34 (7), 1292–1296. Available from: doi:10.1021/es990754h.</li><br />
<br />
<li>Kim, H.-D., Choi, S.-L., Kim, H., Sohn, J.H., et al. (2013) Enzyme-linked assay of cellulose-binding domain functions from Cellulomonas fimi on multi-well microtiter plate. Biotechnology and Bioprocess Engineering. 18 (3), 575–580.</li><br />
<br />
<li>Lee, K.-Y., Buldum, G., Mantalaris, A. and Bismarck, A. (2014), More Than Meets the Eye in Bacterial Cellulose: Biosynthesis, Bioprocessing, and Applications in Advanced Fiber Composites. Macromol. Biosci., 14: 10–32. doi: 10.1002/mabi.201300298</li><br />
<br />
<li>Linder, M.; Salovuori, I.; Ruohonen, L.; Teeri, T.T., 1996. Characterization of a Double Cellulose-binding Domain. SYNERGISTIC HIGH AFFINITY BINDING TO CRYSTALLINE CELLULOSE. Journal of Biological Chemistry, 271(35), pp.21268–21272. Available at: http://www.jbc.org/content/271/35/21268.full</li><br />
<br />
<li>Nguyen, P.Q., Botyanszki, Z., Tay, P.K.R. & Joshi, N.S. (2014) Programmable biofilm-based materials from engineered curli nanofibres. Nature communications. [Online] 54945. Available from: doi:10.1038/ncomms5945.</li><br />
<br />
<li>Pinaud, F., King, D., Moore, H.-P. & Weiss, S. (2004) Bioactivation and cell targeting of semiconductor CdSe/ZnS nanocrystals with phytochelatin-related peptides. Journal of the American Chemical Society. 126 (19), 6115–6123.</li><br />
<br />
<br />
<li>Ramos, R., Domingues, L. & Gama, M. (2010) Escherichia coli expression and purification of LL37 fused to a family III carbohydrate-binding module from Clostridium thermocellum. Protein expression and purification. [Online] 71 (1), 1–7. Available from: doi:10.1016/j.pep.2009.10.016.</li><br />
<br />
<li>Warren, R.A., Beck, C.F., Gilkes, N.R., Kilburn, D.G., et al. (1986) Sequence conservation and region shuffling in an endoglucanase and an exoglucanase from Cellulomonas fimi. Proteins. 1 (4), 335–341.</li><br />
<br />
<li>Xu, Z., Bae, W., Mulchandani, A., Mehra, R.K., et al. (2002) Heavy Metal Removal by Novel CBD-EC20 Sorbents Immobilized on Cellulose. Biomacromolecules. [Online] 3 (3), 462–465.</li><br />
<br />
<li>Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., et al. (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences of the United States of America. [Online] 109 (12), E690–E697. Available from: doi:10.1073/pnas.1115485109.</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/PartsTeam:Imperial/Parts2014-10-17T18:43:46Z<p>Xen sm: </p>
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<h1>Parts</h1><br />
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<li><a data-scroll href="#featured">Featured Parts</a><br />
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<h2>Featured Parts</h2><br />
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<h3><a href="http://parts.igem.org/Part:BBa_K1321340">BBa_K1321340</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>N-terminal linker + double CBD</p><br />
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<div class="pure-u-2-3"><br />
<p>Double cellulose binding domain (dCBD) using two cellulose binding domains from <i>Trichoderma reesei</i> cellobiohydrolases, with an N-terminal linker and internal linker sequence between the two domains which are derived from the endogenous cellobiohydrolase linker sequence. This part is in RFC(25) Freiberg fusion format to allow for easy use in protein fusions. dCBD binding affinity for cellulose was <a href="https://2014.igem.org/Team:Imperial/Functionalisation"> characterised </a> using sfGFP fusions.<br />
</p><br />
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</div><br />
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</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321339">BBa_K1321339</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>CBDcenA + Linker</p><br />
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<div class="pure-u-2-3"><br />
<p>Cellulose binding domain (CBD) of Endoglucanase A (cenA) from <i>Cellulomonas fimi</i> with an endogenous C-terminal linker. The part is in Freiburg format (RFC 25) for ease of use in protein fusions.</p><br />
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</div><br />
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</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321014"> BBa_K1321014</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>CBDcipA with N and C-terminal linker </p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>This CBD is from the Cellulosomal -scaffolding protein A (cipA) of <i>Clostridium thermocellum </i> including the endogenous linker sequences at the N and C-terminus. It is in RFC25 format to allow for easy use in protein fusions. At present the cloning for these constructs is still in progress to correct an illegal EcoRI site which was identified in the parts with this CBD. This can be achieved with a silent mutation via site-directed mutagenesis and we aim to send these parts to the registry once this is complete.<br />
</p><br />
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</div><br />
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</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321005">BBa_K1321005</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>Synthetic Phytochelatin (PC) EC20</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>A general heavy metal-binding peptide consisting of 20 Glu-Cys (EC) repeats in Freiburg format (RFC[25]) to allow for easy use in fusion proteins. We created a library of CBD fusions with this part and demonstrated [INSERT FINAL RESULTS SENTENCE HERE]<br />
</p><br />
</div><br />
</div><br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p><i>Vitreoscilla</i> haemoglobin (VHb)</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>This part is a haemoglobin isolated from Vitreoscilla (VHb) which increases cell metabolism. We used it as one technique to try and enhance cellulose production in <i> G. xylinus </i>.<br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a>, <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>pSEVA331 and pSEVA321 backbones</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>These are two broad host range vector backbones for which we have demonstrated use in <i>E. coli</i> and <i>Gluconacetobacter xylinus </i>.<br />
</p><br />
</div><br />
</div> <br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321334"> BBa_K1321334</a>, <a href="http://parts.igem.org/Part:BBa_K1321335">BBa_K1321335</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>Cellulose synthase operon AcsAB and AcsCD</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>Designed and refactored from <i>G. xylinus</i>, this operon was inserted into <i>E.coli </i> for cellulose synthesis. <br />
</p><br />
</div><br />
</div> <br />
<br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a>,<a href="http://parts.igem.org/Part:BBa_K1321306"> BBa_K1321306</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p><i>Gluconacetobacter xylinus</i> strains ATCC53582 and Kombucha Isolate</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>We created and characterised a library of parts for this organism, with the aim to increase the accessibility the chassis for future work in iGEM. By sharing these strains and their genomes on the registry we hope this will contribute to their ease of use and characterisation.<br />
</p><br />
</div><br />
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</section><br />
<section id="table"><br />
<h2>Table of Parts</h2><br />
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<groupparts>iGEM014 Imperial</groupparts><br />
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{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/File:IC14-sfGFP-part-table.PNGFile:IC14-sfGFP-part-table.PNG2014-10-17T18:10:48Z<p>Xen sm: uploaded a new version of &quot;File:IC14-sfGFP-part-table.PNG&quot;: udpated</p>
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<div>sfGFP fusion parts made as part of the project</div>Xen smhttp://2014.igem.org/File:IC14-NiBP-part-table.PNGFile:IC14-NiBP-part-table.PNG2014-10-17T17:09:36Z<p>Xen sm: uploaded a new version of &quot;File:IC14-NiBP-part-table.PNG&quot;: updated</p>
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<div>NiBP-CBD fusions made as part of our project</div>Xen smhttp://2014.igem.org/File:IC14-SmtA-part-table.PNGFile:IC14-SmtA-part-table.PNG2014-10-17T16:34:52Z<p>Xen sm: uploaded a new version of &quot;File:IC14-SmtA-part-table.PNG&quot;: updated</p>
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<div>SmtA-CBD fusions we made as part of our project</div>Xen smhttp://2014.igem.org/File:IC14-sfGFP-part-table.PNGFile:IC14-sfGFP-part-table.PNG2014-10-17T16:34:12Z<p>Xen sm: uploaded a new version of &quot;File:IC14-sfGFP-part-table.PNG&quot;: updated</p>
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<div>sfGFP fusion parts made as part of the project</div>Xen smhttp://2014.igem.org/File:IC14-PC-part-table.PNGFile:IC14-PC-part-table.PNG2014-10-17T16:33:00Z<p>Xen sm: uploaded a new version of &quot;File:IC14-PC-part-table.PNG&quot;: updated</p>
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<div>PC-CBD fusions made as part of our project</div>Xen smhttp://2014.igem.org/File:IC14-NiBP-part-table.PNGFile:IC14-NiBP-part-table.PNG2014-10-17T16:32:12Z<p>Xen sm: uploaded a new version of &quot;File:IC14-NiBP-part-table.PNG&quot;: updated</p>
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<div>NiBP-CBD fusions made as part of our project</div>Xen smhttp://2014.igem.org/File:IC14-fMT-part-table.PNGFile:IC14-fMT-part-table.PNG2014-10-17T16:31:19Z<p>Xen sm: uploaded a new version of &quot;File:IC14-fMT-part-table.PNG&quot;: updated</p>
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<div>fMT-CBD fusions made as part of our project</div>Xen smhttp://2014.igem.org/Team:Imperial/FunctionalisationTeam:Imperial/Functionalisation2014-10-17T14:24:35Z<p>Xen sm: </p>
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<h1>Functionalisation</h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<li><a data-scroll href="#aim">Aims</a><br />
</li><br />
<li><a data-scroll href="#CBDs">CBDs</a><br />
</li><br />
<li><a data-scroll href="#binding-partners">Functionalisation proteins</a><br />
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<li><a data-scroll href="#results">Results</a><br />
</li><br />
<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<h2>Overview</h2><br />
<p>(This overview is incomplete). By attaching functional proteins to cellulose we can expand it's properties and can bind specific contaminants in water. We used five different cellulose binding domains and fused them to different metal binding proteins, and sfGFP. We performed assays to test the binding of the CBD fusions to our cellulose.</p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
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<li></li><br />
<li></li><br />
<li></li><br />
<li></li><br />
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</section><br />
<section id="introduction"><br />
<h2>Introduction</h2><br />
<figure class="content-image image-right"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/a/a0/IC14-func-strategy-both-pursue.png"><br />
<figcaption>Figure 1. Pictorial summary showing the two cellulose functionalization approaches considered in our project. Strategy 1: Cellulose binding domains are expressed as fusions with the protein of interest, separated optionally by a linker sequence. The cellulose binding domains attach to the cellulose by hydrophobic interactions. Strategy 2: Curli fibrils are expressed in parallel with cellulose to form a composite material. The curli fibres coding sequence is modified to contain a ‘SpyTag’ peptide (Zakeri <i>et al</i> 2012) which binds specifically via a covalent isopeptide bond to its protein partner, ‘SpyCatcher’. This SpyCatcher is fused to the proteins of interest. In our project we only had time to pursue strategy 1 but are still excited about the potential of strategy 2 in the future.</figcaption><br />
</figure><br />
<p>Bacterial cellulose is already a useful biomaterial as is due its attractive mechanical properties, with research on applications from loudspeaker diaphragms to protective packaging and wound dressings (Lee 2014). However, our project also aims to extend the properties of cellulose by conjugating functional proteins and peptides to it. This allows us to target specific contaminants in water which can not be filtered by size exclusion alone. (See <a href="https://2014.igem.org/Team:Imperial/Water_Report">water report</a> and <a href="https://2014.igem.org/Team:Imperial/Informing_Design">informing design</a> sections).</p><br />
<br />
<p>We considered two ways of functionalising cellulose (figure 1) but continued to work fully on the cellulose-binding domain (CBD) method. The curli-fibre method would be an exciting avenue to explore in the future; it is feasible since there has already been research into making a “programmable” biomaterial from curli fibres using a peptide tag approach (Nguyen <i>et al</i> 2014), and in combination with cellulose could produce a composite with robust mechanical properties as well.<br />
</p><br />
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</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>We aimed to make and characterise new cellulose binding domains, and fuse them to functional proteins. (Incomplete)</p><br />
<br />
<br />
</section><br />
<section id="CBDs"><br />
<h2>Cellulose Binding Domains (CBDs)</h2><br />
<p>The cellulose binding domain (CBD) is the link between our functional proteins and the cellulose. Therefore, we wanted to use a CBD which had high affinity and strong binding to cellulose. However, weaker CBDs may still be useful since they could have the potential be controllably eluted under specific conditions, thereby enabling regeneration of the filter when saturated, and controlled disposal of the collected contaminants (refer to water report if relevant?).<br />
</p><br />
<p>We started our search by looking into the <a href="http://parts.igem.org/">parts registry</a> and found two CBDs already existed in the registry: CBDclos (<a href="http://parts.igem.org/Part:BBa_K863111">BBa_K863111</a>) and CBDcex (<a href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>). We also made three new CBDs to contribute to the registry: dCBD, CBDcipA, and CBDcenA. All the CBDs are in RFC(25) freiberg fusion format to allow for ease of use in protein fusions.</p><br />
<br />
<div class="accordion"><br />
<h3>CBDclos and CBDcex</h3><br />
<div><br />
<p>These parts were biobricked by <a href="https://2012.igem.org/Team:Bielefeld-Germany/Results/cbc">Bielefeld 2012</a>, please see their part pages (<a href="http://parts.igem.org/Part:BBa_K863111">BBa_K863111</a>a and <a href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>) for detailed information. CBDclos is from the cellulolytic bacterium <em>Clostridium cellulovorans</em> and CBDcex originates from the cellulolytic bacterium <em>Cellulomonas fimi</em>. This is the same organism as for CBDcenA; cex is from the exoglucanase gene and cen is from the endoglucanase gene.</p><br />
</div><br />
<h3>Double CBD (dCBD) with N-terminal linker</h3><br />
<div><br />
<p>This part, <a href="http://parts.igem.org/Part:BBa_K1321340">BBa_K1321340</a> is based on the double cellulose-binding domain construct synthesised and characterised by Linder <i>et al</i>1996, who found that this double CBD had higher affinity for cellulose than either of the two CBDs on their own. The main difference is that our part contains an additional linker sequence on the N-terminus of the protein.<br />
</p><br />
<p>The two CBDs are from the fungus <i>Trichoderma reesei</i> (<i>Hypocrea jecorina</i>) Exocellobiohydrolase (Exoglucanase) I (cbh1), uniprot ID P62694; and Exocellobiohydrolase (Exoglucanase) II, uniprot ID P07987 (cbh2); with a linker peptide between the two CBDs and at the N-terminus of the protein. Both linkers are the same amino acid sequence and are based on the endogenous linker sequences that exists in cbh1 and cbh2 genes. The linker sequence is PGANPPGTTTTSRPATTTGSSPGP which is the same as used by Linder <i>et al</i> 1996. The first three amino acids are from the cbh2 endogenous linker, and the rest is from the cbh1 endogenous linker. CBDcbh1 is placed C-terminal to CBDcbh2 because naturally CBDcbh1 is a C-terminal domain and CBDcbh2 is an N-terminal domain. Both CBDs are from the <a class='iframe' href="http://www.cazy.org/CBM1.html">CBM family 1</a>. The precise location of the CBD within the cbh genes was slightly different according to the uniprot annotations and the sequence used by Linder <i>et al</i> 1996; we chose to use the sequence from the paper since the protein was expressed and characterised successfully.</p><br />
<br />
</div><br />
<h3>CBDcipA with N and C-terminal linker</h3><br />
<div><br />
<p>This CBD is from the Cellulosomal -scaffolding protein A (cipA) of Clostridium thermocellum including the endogenous linker sequences at the N and C-terminus (<a class='iframe' href="http://www.uniprot.org/uniprot/Q06851">UniProt ID Q06851</a>). It is in RFC25 format to allow for easy use in protein fusions.</p><br />
<br />
<p>The CBD is part of the <a class='iframe' href="http://www.cazy.org/CBM3.html">CBM3 family</a> . This CBD has been used in many application: in fusions with cell adhesion peptides to enhance the properties of cellulose as a cell-growth matrix (Andrade <i>et al </i> 2010a, Andrade <i>et al </i> 2010b), fused to enzymes to remove contaminants from water (Kauffmann <i> et al </i> 2000) and fused to an antimicrobial peptide (Ramos, Domingues & Gama 2010).</p><br />
<br />
<p>At present the cloning for these constructs is still in progress to correct an illegal EcoRI site which was identified in the parts with this CBD. This can be achieved with a silent mutation via site-directed mutagenesis and we aim to send these parts to the registry once thi<br />
</p><br />
<br />
</div><br />
<h3>CBDcenA with C-terminal linker</h3><br />
<div><br />
<p>This CBD is from the Endoglucanase A (cenA) gene from <i>Cellulomonas fimi</i> and also contains an endogenous C-terminal linker. The cellulose binding domain region occurs at the N-terminus of the cenA gene (UniProt P07984 link to http://www.uniprot.org/uniprot/P07984) and is from the <a class='iframe' href="http://www.cazy.org/CBM2.html">CBM family 2</a><br />
</p><br />
<p>The linker sequence (PTTSPTPTPTPTTPTPTPTPTPTPTPTVTP) is Pro-Thr box which has an extended conformation and acts as a hinge region (Gilkes <i>et al</i> 1989). The endoglucanase CBDcenA has 50% homology to the exoglucanase CBDcex (<a class="iframe" href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>) and the linker is highly conserved in both, but the order of the catalytic, linker and cellulose-binding regions is reversed (Warren <i>et al</i> 1986). It has been shown that CBDcenA has the highest binding affinity for crystalline cellulose out of the <i>C. fimi</i> CBDs (Kim <i>et al</i> 2013).<br />
</p><br />
</div><br />
</div><br />
</section><br />
<br />
<br />
</section><br />
<section id="binding-partners"><br />
<h2>Functionalisation proteins: Binding partners</h2><br />
<h3>Superfolder Green Fluorescent Protein (sfGFP) </h3><br />
<figure class="content-image image-right"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/8/85/IC14-sfGFP-part-table.PNG> <br />
<figcaption>sfGFP-CBD fusion parts</figcaption> <br />
</figure> <p> sfGFP is a more stable mutant of <a href="http://parts.igem.org/Parts:BBa_E0040">mut3GFP</a> and we chose to use this as a robust, easily visible protein to initially fuse to our CBDs. We also designed one of our assays [insert link on 'one of our assays'] for characterising CBD binding using the sfGFP fusion constructs. sfGFP already existed in the registry (<a href="http://parts.igem.org/Part:BBa_I746916">BBa_I746916</a>), detailed information on sfGFP properties is included in this part description) however since we needed to make fusion proteins with our CBDs we modified the part and submitted it in RFC[25] Freiberg fusion format (<a href="http://parts.igem.org/Parts:BBa_K1321337">K1321337</a>). We created the following fusion parts with sfGFP: <br />
<br />
<br />
<br />
</p><br />
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<h3> Metal Binding Proteins </h3><br />
<p>One of the contaminants we can filter out which current size-exclusion technologies can not target, are <a href="https://2014.igem.org/Team:Imperial/Water_Report">heavy metals</a>. We have worked with four different metal binding proteins: the metallothioneins SmtA and fMT, a histidine-rich nickel binding protein (NiBP) from the bacterium <i>Heliobacter pylori</i> and a synthetic phytochelatin (PC) EC20.<br />
</p><br />
<div class="accordion"><br />
<h3>SmtA and fMT metallothioneins</h3><br />
<div><br />
<p>These two parts already existed in the BioBrick registry, but since we needed to use them as fusions with the CBDs we improved the parts by making them compatible with the <a class="iframe" href="http://parts.igem.org/Assembly_standard_25">RFC(25) Frieburg Fusion BioBrick standard.</a></p><br />
<p>Metallothioneins (MTs) are genetically encoded cysteine-rich peptides which serve a role in the detoxification of heavy metals in plants, animals and some prokaryotes (Cobbett, 2002). They have a similar structure and function to phytochelatins, but are produced by different mechanisms (Cobbett, 2002). SmtA is from the cyanobacteria <i>Synechococcus sp.</i> and was used by the <a target="_blank" href="https://2011.igem.org/Team:Tokyo-NoKoGen">Tokyo-NoKoGen 2011</a> iGEM team (who submitted the RFC10 part originally, <a class="iframe" href="http://parts.igem.org/Part:BBa_K519010">BBa_K519010</a>) to confer cadmium (Cd2+) tolerance to <em>E. coli</em> cells when expressed. fMT originates from the seaweed <i>Fucus vesiculosus</i> and was submitted by <a target="_blank" href="https://2009.igem.org/Team:Groningen">Groningen 2009 iGEM team </a> and detailed information can be found on the original part page (<a class="iframe" href="http://parts.igem.org/Part:BBa_K190019">BBa_K190019</a>).</p><br />
<p>We successfully <a href="https://2014.igem.org/Team:Imperial/Protocols#general">cloned</a> the following CBD fusion constructs: </p><br />
<br />
<figure class="content-image image-right image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/a/ac/IC14-SmtA-part-table.PNG> <br />
<figcaption>SmtA-CBD fusion parts</figcaption> <br />
</figure> <br />
<br />
<figure class="content-image image-left image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/6/62/IC14-fMT-part-table.PNG> <br />
<figcaption>fMT-CBD fusion parts</figcaption> <br />
</figure> <br />
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</div><br />
<h3>Nickel binding protein (NiBP)</h3><br />
<div><br />
<figure class="content-image image-right"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/f/f6/IC14-NiBP-part-table.PNG> <br />
<figcaption>NiBP-CBD fusions parts</figcaption> <br />
</figure><br />
<p><br />
This protein is the histidine-rich nickel-binding protein from <i>Heliobacter pylori</i> (GenBank accession ACX98466.1), and already existed as a BioBrick in the registry (<a class="iframe" href="http://parts.igem.org/Part:K1151001">K1151001</a>). Since we needed to use this part in fusion proteins we improved the part by submitting it as a basic part in RFC(25) (link) Freiberg fusion format (<a class="iframe" href="http://parts.igem.org/Part:K1321009">K1321009</a>).<br />
</p><br />
<p>We successfully <a href="https://2014.igem.org/Team:Imperial/Protocols#general">cloned</a> the following CBD fusion constructs with NiBP:<br />
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</p><br />
<p>However we discovered late on in the project that the DNA sequence of the NiBP protein in the registry is out of frame, leading to an incorrect protein translation and read-through of the stop codon (figure 2).</p><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/8/80/IC14-NiBP-protein-alignment.PNG"><br />
<figcaption>Figure 2. Alignment of BBa_K1151001 NiBP in the registry to the , showing incorrect amino-acid sequence in red compared to <a target="_blank" href="http://www.ncbi.nlm.nih.gov/protein/261838700">native <i>H.pylori</i></a> sequence.<br />
</figcaption><br />
</figure><br />
<figure class="content-image image-right"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/e/e1/IC14-NiBP-DNA-alignment.PNG"><br />
<figcaption>Figure 3. Single nucleotide deletion in BBa_K1151001 compared to native <i>H.pylori</i> sequence (<a target="_blank" href="http://www.ncbi.nlm.nih.gov/nuccore/261837457">GenBank CP000012.1</a> (link ) bp1391851-1392033, reverse complement) resulting in any downstream nucleotides being out of frame.</figcaption><br />
</figure><br />
<br />
<p>Further investigation into the error let us to identify a single nucleotide deletion in <a class="iframe" href="http://parts.igem.org/Part:BBa_K1151001">BBa_K1151001</a> compared to the native <i>H.pylori</i> DNA sequence (<a target="_blank" href="http://www.ncbi.nlm.nih.gov/nuccore/261837457">GenBank CP000012.1</a> bp1391851-1392033, reverse complement strand). As seen in Figure 3 the deletion is a ‘C’ at bp 53 of the native sequence. This error was propagated through all our NiBP constructs and means that any subsequent C-terminal fusions are also out of frame and we were unable to express and characterise these constructs. We have noted this on the description pages for each of the parts and hope that in the future they will still be useful, since the error could be corrected relatively easily by a team who wishes to use them by site-directed-mutagenesis PCR to insert the missing nucleotide. </p><br />
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</div><br />
<h3>Synthetic phytochelatin EC20</h3><br />
<div><br />
<p>This is our new contribution of a metal-binding protein to the parts registry. Synthetic phytochelatins are analogues of phytochelatins (PCs); cysteine-rich peptides of the general structure (γ-GluCys)n-Gly (n~2-5) which bind and sequester heavy metals, with a role in detoxification (Cobbett & Goldsbrough 2002). PCs are found mainly in plants but also in other organisms including diatoms, fungi, algae (including cyanobacteria) and some invertebrates (Cobbett & Goldsbrough 2002, Rea 2012). Natively, the PCs are enzymatically synthesized by phytochelatin synthase (PCS) from a glutathione (GSH) precursor and the amino acids in the resulting peptide are linked by the non-standard gamma peptide bonds (Cobbett & Goldsbrough 2002, Rea 2012). However, genetically encoded analogues (containing normal alpha-peptide bonds) of different number Glu-Cys (EC) repeats have been characterised, with the EC20 having the highest binding (Bae <i>et al</i> 2000).<br />
</p><br />
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<figure class="content-image image-right image-small"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/18/IC14-Biondo-et-al-2012-ion-table.PNG"><br />
<figcaption>Table 1. Metal ion adsorption increase of C. metallidurans cells with PC EC20 displayed on cell surface, compared to negative control. Taken from Biondo <i>et al</i> 2012</figcaption><br />
</figure><br />
<p>The main metal PCs confer tolerance to is Cadmium (Cobbett & Goldsbrough 2002) with a reported stoichiometry of 10 Cd2+ per peptide (Bae <i>et al</i>, . EC20 peptide has still showed good metal ion binding whilst fused to a cellulose binding domain (CBD) for water purification (Xu <i>et al</i> 2002) and when anchored to bacterial cell membrane to confer Cd2+ tolerance (Bae <i>et al</i> 2000, Chaturvedi & Archana 2014). PC EC20 has also been shown to bind many more heavy metals. For example, when displayed on the surface of Cupriavidus metallidurans, the cells capacity to adsorb ions increased by 31 to 219% depending on the ion (table 1, Biondo <i>et al</i> 2012). These binding properties of PC EC20 have been utilised in applications including a regeneratable biosensor of Hg2+, Cd2+, Pb2+, Cu2+ and Zn2+ ions to 100 fM–10 mM (Bontidean <i>et al</i> 2003), and to target CdSe/ZnS quantum dots to streptavidin expressing HeLa cells when biotinylated (Pinaud <i>et al</i> 2004). INSERT TABLE OF THE IONS FROM THE PAPER<br />
</p><br />
<p>We used this part to <a href="https://2014.igem.org/Team:Imperial/Protocols#general">clone</a> a library of different cellulose-binding domains fused to phytochelatin:<figure class="content-image"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/4/40/IC14-PC-part-table.PNG > <br />
<figcaption>Phytochelatin-CBD fusion parts. </figcaption> <br />
</figure> <br />
</p><br />
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<br />
<h3> Other potential proteins </h3><br />
<p>During our research we also explored other functional proteins to fuse to the CBDs.<br />
<div class="accordion"><br />
<h3>Laccases</h3><br />
<div><br />
<p>Laccases are enzymes which can break down aromatic and phenolic compounds such as modified estrogens which can end up unfiltered in the water supply with unknown consequences (Auriol <i>et al</i> 2008). This method of treating estrogen in water was explored by <a target="_blank" href="https://2012.igem.org/Team:Bielefeld-Germany">Bielefeld 2012 iGEM team</a>, and our system also has potential to address this.<br />
</p><br />
</div><br />
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<h3>Nanobodies</h3><br />
<div><br />
<p>Nanobodies are fragments of camelid antibodies which can be generated against a variety of antigens and can be easily expressed by organisms such as <i>E. coli </i>. Nanobodies could be used to target viruses, which are often too small to be filtered by size exclusion. For example there are already nanobodies which can bind to <i> Vaccinia </i> virus, ricin, <i>Cholera</i> toxin, Stephylococcal enterotoxin B and H5N1 Influenza (Goldman <i>et al </i> 2006, Ibanez <i>et al </i> 2011) to name a few. Additionally, nanobodies are stable and can be selected to resist proteolytic attach (Harmsen <i>et al </i> 2006). Nanobodies can also be functional when as fusions or conjugates to other proteins for example to target enzymes to specific cells (Cortez-Retamozo 2004), so there is much potential for targeting a wide array of contaminants when fused to cellulose binding domains.<br />
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</p><br />
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<section id="results"><br />
<h2>Results</h2><br />
<p>Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim.</p><br />
</section><br />
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<section id="references"><br />
<h2>References</h2><br />
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<ul><br />
<li>THESE NEED TO BE ALPHABETISED Biondo, R., da Silva, F.A., Vicente, E.J., Souza Sarkis, J.E., et al. (2012) Synthetic phytochelatin surface display in Cupriavidus metallidurans CH34 for enhanced metals bioremediation. Environmental science & technology. 46 (15), 8325–8332.</li><br />
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<li>Bontidean, I., Ahlqvist, J., Mulchandani, A., Chen, W., et al. (2003) Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection. Biosensors and Bioelectronics. 18 (5-6), 547–553.</li><br />
<li>Lee, K.-Y., Buldum, G., Mantalaris, A. and Bismarck, A. (2014), More Than Meets the Eye in Bacterial Cellulose: Biosynthesis, Bioprocessing, and Applications in Advanced Fiber Composites. Macromol. Biosci., 14: 10–32. doi: 10.1002/mabi.201300298</li><br />
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<li>Nguyen, P.Q., Botyanszki, Z., Tay, P.K.R. & Joshi, N.S. (2014) Programmable biofilm-based materials from engineered curli nanofibres. Nature communications. [Online] 54945. Available from: doi:10.1038/ncomms5945.</li><br />
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<li>Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., et al. (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences of the United States of America. [Online] 109 (12), E690–E697. Available from: doi:10.1073/pnas.1115485109.</li><br />
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<li>Linder, M.; Salovuori, I.; Ruohonen, L.; Teeri, T.T., 1996. Characterization of a Double Cellulose-binding Domain. SYNERGISTIC HIGH AFFINITY BINDING TO CRYSTALLINE CELLULOSE. Journal of Biological Chemistry, 271(35), pp.21268–21272. Available at: http://www.jbc.org/content/271/35/21268.full</li><br />
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<li>Andrade, F.K., Moreira, S.M.G., Domingues, L. & Gama, F.M.P. (2010) Improving the affinity of fibroblasts for bacterial cellulose using carbohydrate-binding modules fused to RGD. Journal of biomedical materials research. Part A. [Online] 92 (1), 9–17. Available from: doi:10.1002/jbm.a.32284.</li><br />
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<li>Andrade, F.K., Costa, R., Domingues, L., Soares, R., et al. (2010) Improving bacterial cellulose for blood vessel replacement: Functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide. Acta biomaterialia. [Online] 6 (10), 4034–4041. Available from: doi:10.1016/j.actbio.2010.04.023.</li><br />
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<li>Kauffmann, C., Shoseyov, O., Shpigel, E., Bayer, E.A., et al. (2000) Novel Methodology for Enzymatic Removal of Atrazine from Water by CBD-Fusion Protein Immobilized on Cellulose. Environmental Science & Technology. [Online] 34 (7), 1292–1296. Available from: doi:10.1021/es990754h.</li><br />
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<li>Ramos, R., Domingues, L. & Gama, M. (2010) Escherichia coli expression and purification of LL37 fused to a family III carbohydrate-binding module from Clostridium thermocellum. Protein expression and purification. [Online] 71 (1), 1–7. Available from: doi:10.1016/j.pep.2009.10.016.</li><br />
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<li>Gilkes, N.R., Kilburn, D.G., Miller, R.C. & Warren, R.A. (1989) Structural and functional analysis of a bacterial cellulase by proteolysis. The Journal of biological chemistry. 264 (30), 17802–17808.</li><br />
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<li>Warren, R.A., Beck, C.F., Gilkes, N.R., Kilburn, D.G., et al. (1986) Sequence conservation and region shuffling in an endoglucanase and an exoglucanase from Cellulomonas fimi. Proteins. 1 (4), 335–341.</li><br />
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<li>Kim, H.-D., Choi, S.-L., Kim, H., Sohn, J.H., et al. (2013) Enzyme-linked assay of cellulose-binding domain functions from Cellulomonas fimi on multi-well microtiter plate. Biotechnology and Bioprocess Engineering. 18 (3), 575–580.</li><br />
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<li>Cobbett, C. & Goldsbrough, P. (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annual review of plant biology. [Online] 53159–182. Available from: doi:10.1146/annurev.arplant.53.100301.135154.</li><br />
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<li>Rea, P.A. (2012) Phytochelatin synthase: of a protease a peptide polymerase made. Physiologia plantarum. 145 (1), 154–164.</li><br />
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<li>Bae, W., Chen, W., Mulchandani, A. & Mehra, R.K. (2000) Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnology and bioengineering. 70 (5), 518–524.</li><br />
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<li>Xu, Z., Bae, W., Mulchandani, A., Mehra, R.K., et al. (2002) Heavy Metal Removal by Novel CBD-EC20 Sorbents Immobilized on Cellulose. Biomacromolecules. [Online] 3 (3), 462–465.</li><br />
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<li>Chaturvedi, R. & Archana, G. (2014) Cytosolic expression of synthetic phytochelatin and bacterial metallothionein genes in Deinococcus radiodurans R1 for enhanced tolerance and bioaccumulation of cadmium. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine. 27 (3), 471–482.</li><br />
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<li>Pinaud, F., King, D., Moore, H.-P. & Weiss, S. (2004) Bioactivation and cell targeting of semiconductor CdSe/ZnS nanocrystals with phytochelatin-related peptides. Journal of the American Chemical Society. 126 (19), 6115–6123.</li><br />
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<li>Auriol, M., Filali-Meknassi, Y., Adams, C.D., Tyagi, R.D., et al. (2008) Removal of estrogenic activity of natural and synthetic hormones from a municipal wastewater: efficiency of horseradish peroxidase and laccase from Trametes versicolor. Chemosphere. [Online] 70 (3), 445–452. Available from: doi:10.1016/j.chemosphere.2007.06.064.</li><br />
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<li>Harmsen, M.M., van Solt, C.B., van Zijderveld-van Bemmel, A.M., Niewold, T.A., et al. (2006) Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Applied microbiology and biotechnology. [Online] 72 (3), 544–551. Available from: doi:10.1007/s00253-005-0300-7.</li><br />
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<li>Cortez-Retamozo, V. (2004) Efficient Cancer Therapy with a Nanobody-Based Conjugate. Cancer Research. [Online] 64 (8), 2853–2857. Available from: doi:10.1158/0008-5472.CAN-03-3935.</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Xen smhttp://2014.igem.org/Team:Imperial/FunctionalisationTeam:Imperial/Functionalisation2014-10-17T14:06:15Z<p>Xen sm: correcting typo in cipA name</p>
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<h1>Functionalisation</h1><br />
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<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#CBDs">CBDs</a><br />
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<li><a data-scroll href="#binding-partners">Functionalisation proteins</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<h2>Overview</h2><br />
<p>(This overview is incomplete). By attaching functional proteins to cellulose we can expand it's properties and can bind specific contaminants in water. We used five different cellulose binding domains and fused them to different metal binding proteins, and sfGFP. We performed assays to test the binding of the CBD fusions to our cellulose.</p><br />
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<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
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<section id="introduction"><br />
<h2>Introduction</h2><br />
<figure class="content-image image-right"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/a/a0/IC14-func-strategy-both-pursue.png"><br />
<figcaption>Figure 1. Pictorial summary showing the two cellulose functionalization approaches considered in our project. Strategy 1: Cellulose binding domains are expressed as fusions with the protein of interest, separated optionally by a linker sequence. The cellulose binding domains attach to the cellulose by hydrophobic interactions. Strategy 2: Curli fibrils are expressed in parallel with cellulose to form a composite material. The curli fibres coding sequence is modified to contain a ‘SpyTag’ peptide (Zakeri <i>et al</i> 2012) which binds specifically via a covalent isopeptide bond to its protein partner, ‘SpyCatcher’. This SpyCatcher is fused to the proteins of interest. In our project we only had time to pursue strategy 1 but are still excited about the potential of strategy 2 in the future.</figcaption><br />
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<p>Bacterial cellulose is already a useful biomaterial as is due its attractive mechanical properties, with research on applications from loudspeaker diaphragms to protective packaging and wound dressings (Lee 2014). However, our project also aims to extend the properties of cellulose by conjugating functional proteins and peptides to it. This allows us to target specific contaminants in water which can not be filtered by size exclusion alone. (See <a href="https://2014.igem.org/Team:Imperial/Water_Report">water report</a> and <a href="https://2014.igem.org/Team:Imperial/Informing_Design">informing design</a> sections).</p><br />
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<p>We considered two ways of functionalising cellulose (figure 1) but continued to work fully on the cellulose-binding domain (CBD) method. The curli-fibre method would be an exciting avenue to explore in the future; it is feasible since there has already been research into making a “programmable” biomaterial from curli fibres using a peptide tag approach (Nguyen <i>et al</i> 2014), and in combination with cellulose could produce a composite with robust mechanical properties as well.<br />
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<section id="aim"><br />
<h2>Aims</h2><br />
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<p>Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim. Aliquam lorem ante, dapibus in, viverra quis, feugiat a, tellus. Phasellus viverra nulla ut metus varius laoreet. Quisque rutrum. Aenean imperdiet. Etia.</p><br />
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<section id="CBDs"><br />
<h2>Cellulose Binding Domains (CBDs)</h2><br />
<p>The cellulose binding domain (CBD) is the link between our functional proteins and the cellulose. Therefore, we wanted to use a CBD which had high affinity and strong binding to cellulose. However, weaker CBDs may still be useful since they could have the potential be controllably eluted under specific conditions, thereby enabling regeneration of the filter when saturated, and controlled disposal of the collected contaminants (refer to water report if relevant?).<br />
</p><br />
<p>We started our search by looking into the <a href="http://parts.igem.org/">parts registry</a> and found two CBDs already existed in the registry: CBDclos (<a href="http://parts.igem.org/Part:BBa_K863111">BBa_K863111</a>) and CBDcex (<a href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>). We also made three new CBDs to contribute to the registry: dCBD, CBDcipA, and CBDcenA. All the CBDs are in RFC(25) freiberg fusion format to allow for ease of use in protein fusions.</p><br />
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<div class="accordion"><br />
<h3>CBDclos and CBDcex</h3><br />
<div><br />
<p>These parts were biobricked by <a href="https://2012.igem.org/Team:Bielefeld-Germany/Results/cbc">Bielefeld 2012</a>, please see their part pages (<a href="http://parts.igem.org/Part:BBa_K863111">BBa_K863111</a>a and <a href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>) for detailed information. CBDclos is from the cellulolytic bacterium <em>Clostridium cellulovorans</em> and CBDcex originates from the cellulolytic bacterium <em>Cellulomonas fimi</em>. This is the same organism as for CBDcenA; cex is from the exoglucanase gene and cen is from the endoglucanase gene.</p><br />
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<h3>Double CBD (dCBD) with N-terminal linker</h3><br />
<div><br />
<p>This part, <a href="http://parts.igem.org/Part:BBa_K1321340">BBa_K1321340</a> is based on the double cellulose-binding domain construct synthesised and characterised by Linder <i>et al</i>1996, who found that this double CBD had higher affinity for cellulose than either of the two CBDs on their own. The main difference is that our part contains an additional linker sequence on the N-terminus of the protein.<br />
</p><br />
<p>The two CBDs are from the fungus <i>Trichoderma reesei</i> (<i>Hypocrea jecorina</i>) Exocellobiohydrolase (Exoglucanase) I (cbh1), uniprot ID P62694; and Exocellobiohydrolase (Exoglucanase) II, uniprot ID P07987 (cbh2); with a linker peptide between the two CBDs and at the N-terminus of the protein. Both linkers are the same amino acid sequence and are based on the endogenous linker sequences that exists in cbh1 and cbh2 genes. The linker sequence is PGANPPGTTTTSRPATTTGSSPGP which is the same as used by Linder <i>et al</i> 1996. The first three amino acids are from the cbh2 endogenous linker, and the rest is from the cbh1 endogenous linker. CBDcbh1 is placed C-terminal to CBDcbh2 because naturally CBDcbh1 is a C-terminal domain and CBDcbh2 is an N-terminal domain. Both CBDs are from the <a class='iframe' href="http://www.cazy.org/CBM1.html">CBM family 1</a>. The precise location of the CBD within the cbh genes was slightly different according to the uniprot annotations and the sequence used by Linder <i>et al</i> 1996; we chose to use the sequence from the paper since the protein was expressed and characterised successfully.</p><br />
<br />
</div><br />
<h3>CBDcipA with N and C-terminal linker</h3><br />
<div><br />
<p>This CBD is from the Cellulosomal -scaffolding protein A (cipA) of Clostridium thermocellum including the endogenous linker sequences at the N and C-terminus (<a class='iframe' href="http://www.uniprot.org/uniprot/Q06851">UniProt ID Q06851</a>). It is in RFC25 format to allow for easy use in protein fusions.</p><br />
<br />
<p>The CBD is part of the <a class='iframe' href="http://www.cazy.org/CBM3.html">CBM3 family</a> . This CBD has been used in many application: in fusions with cell adhesion peptides to enhance the properties of cellulose as a cell-growth matrix (Andrade <i>et al </i> 2010a, Andrade <i>et al </i> 2010b), fused to enzymes to remove contaminants from water (Kauffmann <i> et al </i> 2000) and fused to an antimicrobial peptide (Ramos, Domingues & Gama 2010).</p><br />
<br />
<p>At present the cloning for these constructs is still in progress to correct an illegal EcoRI site which was identified in the parts with this CBD. This can be achieved with a silent mutation via site-directed mutagenesis and we aim to send these parts to the registry once thi<br />
</p><br />
<br />
</div><br />
<h3>CBDcenA with C-terminal linker</h3><br />
<div><br />
<p>This CBD is from the Endoglucanase A (cenA) gene from <i>Cellulomonas fimi</i> and also contains an endogenous C-terminal linker. The cellulose binding domain region occurs at the N-terminus of the cenA gene (UniProt P07984 link to http://www.uniprot.org/uniprot/P07984) and is from the <a class='iframe' href="http://www.cazy.org/CBM2.html">CBM family 2</a><br />
</p><br />
<p>The linker sequence (PTTSPTPTPTPTTPTPTPTPTPTPTPTVTP) is Pro-Thr box which has an extended conformation and acts as a hinge region (Gilkes <i>et al</i> 1989). The endoglucanase CBDcenA has 50% homology to the exoglucanase CBDcex (<a class="iframe" href="http://parts.igem.org/Part:BBa_K863101">BBa_K863101</a>) and the linker is highly conserved in both, but the order of the catalytic, linker and cellulose-binding regions is reversed (Warren <i>et al</i> 1986). It has been shown that CBDcenA has the highest binding affinity for crystalline cellulose out of the <i>C. fimi</i> CBDs (Kim <i>et al</i> 2013).<br />
</p><br />
</div><br />
</div><br />
</section><br />
<br />
<br />
</section><br />
<section id="binding-partners"><br />
<h2>Functionalisation proteins: Binding partners</h2><br />
<h3>Superfolder Green Fluorescent Protein (sfGFP) </h3><br />
<figure class="content-image image-right"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/8/85/IC14-sfGFP-part-table.PNG> <br />
<figcaption>sfGFP-CBD fusion parts</figcaption> <br />
</figure> <p> sfGFP is a more stable mutant of <a href="http://parts.igem.org/Parts:BBa_E0040">mut3GFP</a> and we chose to use this as a robust, easily visible protein to initially fuse to our CBDs. We also designed one of our assays [insert link on 'one of our assays'] for characterising CBD binding using the sfGFP fusion constructs. sfGFP already existed in the registry (<a href="http://parts.igem.org/Part:BBa_I746916">BBa_I746916</a>), detailed information on sfGFP properties is included in this part description) however since we needed to make fusion proteins with our CBDs we modified the part and submitted it in RFC[25] Freiberg fusion format (<a href="http://parts.igem.org/Parts:BBa_K1321337">K1321337</a>). We created the following fusion parts with sfGFP: <br />
<br />
<br />
<br />
</p><br />
<br />
<h3> Metal Binding Proteins </h3><br />
<p>One of the contaminants we can filter out which current size-exclusion technologies can not target, are <a href="https://2014.igem.org/Team:Imperial/Water_Report">heavy metals</a>. We have worked with four different metal binding proteins: the metallothioneins SmtA and fMT, a histidine-rich nickel binding protein (NiBP) from the bacterium <i>Heliobacter pylori</i> and a synthetic phytochelatin (PC) EC20.<br />
</p><br />
<div class="accordion"><br />
<h3>SmtA and fMT metallothioneins</h3><br />
<div><br />
<p>These two parts already existed in the BioBrick registry, but since we needed to use them as fusions with the CBDs we improved the parts by making them compatible with the <a class="iframe" href="http://parts.igem.org/Assembly_standard_25">RFC(25) Frieburg Fusion BioBrick standard.</a></p><br />
<p>Metallothioneins (MTs) are genetically encoded cysteine-rich peptides which serve a role in the detoxification of heavy metals in plants, animals and some prokaryotes (Cobbett, 2002). They have a similar structure and function to phytochelatins, but are produced by different mechanisms (Cobbett, 2002). SmtA is from the cyanobacteria <i>Synechococcus sp.</i> and was used by the <a target="_blank" href="https://2011.igem.org/Team:Tokyo-NoKoGen">Tokyo-NoKoGen 2011</a> iGEM team (who submitted the RFC10 part originally, <a class="iframe" href="http://parts.igem.org/Part:BBa_K519010">BBa_K519010</a>) to confer cadmium (Cd2+) tolerance to <em>E. coli</em> cells when expressed. fMT originates from the seaweed <i>Fucus vesiculosus</i> and was submitted by <a target="_blank" href="https://2009.igem.org/Team:Groningen">Groningen 2009 iGEM team </a> and detailed information can be found on the original part page (<a class="iframe" href="http://parts.igem.org/Part:BBa_K190019">BBa_K190019</a>).</p><br />
<p>We successfully <a href="https://2014.igem.org/Team:Imperial/Protocols#general">cloned</a> the following CBD fusion constructs: </p><br />
<br />
<figure class="content-image image-right image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/a/ac/IC14-SmtA-part-table.PNG> <br />
<figcaption>SmtA-CBD fusion parts</figcaption> <br />
</figure> <br />
<br />
<figure class="content-image image-left image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/6/62/IC14-fMT-part-table.PNG> <br />
<figcaption>fMT-CBD fusion parts</figcaption> <br />
</figure> <br />
<br />
<br />
<br />
<br />
</div><br />
<h3>Nickel binding protein (NiBP)</h3><br />
<div><br />
<figure class="content-image image-right"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/f/f6/IC14-NiBP-part-table.PNG> <br />
<figcaption>NiBP-CBD fusions parts</figcaption> <br />
</figure><br />
<p><br />
This protein is the histidine-rich nickel-binding protein from <i>Heliobacter pylori</i> (GenBank accession ACX98466.1), and already existed as a BioBrick in the registry (<a class="iframe" href="http://parts.igem.org/Part:K1151001">K1151001</a>). Since we needed to use this part in fusion proteins we improved the part by submitting it as a basic part in RFC(25) (link) Freiberg fusion format (<a class="iframe" href="http://parts.igem.org/Part:K1321009">K1321009</a>).<br />
</p><br />
<p>We successfully <a href="https://2014.igem.org/Team:Imperial/Protocols#general">cloned</a> the following CBD fusion constructs with NiBP:<br />
<br />
</p><br />
<p>However we discovered late on in the project that the DNA sequence of the NiBP protein in the registry is out of frame, leading to an incorrect protein translation and read-through of the stop codon (figure 2).</p><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/8/80/IC14-NiBP-protein-alignment.PNG"><br />
<figcaption>Figure 2. Alignment of BBa_K1151001 NiBP in the registry to the , showing incorrect amino-acid sequence in red compared to <a target="_blank" href="http://www.ncbi.nlm.nih.gov/protein/261838700">native <i>H.pylori</i></a> sequence.<br />
</figcaption><br />
</figure><br />
<figure class="content-image image-right"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/e/e1/IC14-NiBP-DNA-alignment.PNG"><br />
<figcaption>Figure 3. Single nucleotide deletion in BBa_K1151001 compared to native <i>H.pylori</i> sequence (<a target="_blank" href="http://www.ncbi.nlm.nih.gov/nuccore/261837457">GenBank CP000012.1</a> (link ) bp1391851-1392033, reverse complement) resulting in any downstream nucleotides being out of frame.</figcaption><br />
</figure><br />
<br />
<p>Further investigation into the error let us to identify a single nucleotide deletion in <a class="iframe" href="http://parts.igem.org/Part:BBa_K1151001">BBa_K1151001</a> compared to the native <i>H.pylori</i> DNA sequence (<a target="_blank" href="http://www.ncbi.nlm.nih.gov/nuccore/261837457">GenBank CP000012.1</a> bp1391851-1392033, reverse complement strand). As seen in Figure 3 the deletion is a ‘C’ at bp 53 of the native sequence. This error was propagated through all our NiBP constructs and means that any subsequent C-terminal fusions are also out of frame and we were unable to express and characterise these constructs. We have noted this on the description pages for each of the parts and hope that in the future they will still be useful, since the error could be corrected relatively easily by a team who wishes to use them by site-directed-mutagenesis PCR to insert the missing nucleotide. </p><br />
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<br />
<br />
</div><br />
<h3>Synthetic phytochelatin EC20</h3><br />
<div><br />
<p>This is our new contribution of a metal-binding protein to the parts registry. Synthetic phytochelatins are analogues of phytochelatins (PCs); cysteine-rich peptides of the general structure (γ-GluCys)n-Gly (n~2-5) which bind and sequester heavy metals, with a role in detoxification (Cobbett & Goldsbrough 2002). PCs are found mainly in plants but also in other organisms including diatoms, fungi, algae (including cyanobacteria) and some invertebrates (Cobbett & Goldsbrough 2002, Rea 2012). Natively, the PCs are enzymatically synthesized by phytochelatin synthase (PCS) from a glutathione (GSH) precursor and the amino acids in the resulting peptide are linked by the non-standard gamma peptide bonds (Cobbett & Goldsbrough 2002, Rea 2012). However, genetically encoded analogues (containing normal alpha-peptide bonds) of different number Glu-Cys (EC) repeats have been characterised, with the EC20 having the highest binding (Bae <i>et al</i> 2000).<br />
</p><br />
<br />
<br />
<figure class="content-image image-right image-small"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/18/IC14-Biondo-et-al-2012-ion-table.PNG"><br />
<figcaption>Table 1. Metal ion adsorption increase of C. metallidurans cells with PC EC20 displayed on cell surface, compared to negative control. Taken from Biondo <i>et al</i> 2012</figcaption><br />
</figure><br />
<p>The main metal PCs confer tolerance to is Cadmium (Cobbett & Goldsbrough 2002) with a reported stoichiometry of 10 Cd2+ per peptide (Bae <i>et al</i>, . EC20 peptide has still showed good metal ion binding whilst fused to a cellulose binding domain (CBD) for water purification (Xu <i>et al</i> 2002) and when anchored to bacterial cell membrane to confer Cd2+ tolerance (Bae <i>et al</i> 2000, Chaturvedi & Archana 2014). PC EC20 has also been shown to bind many more heavy metals. For example, when displayed on the surface of Cupriavidus metallidurans, the cells capacity to adsorb ions increased by 31 to 219% depending on the ion (table 1, Biondo <i>et al</i> 2012). These binding properties of PC EC20 have been utilised in applications including a regeneratable biosensor of Hg2+, Cd2+, Pb2+, Cu2+ and Zn2+ ions to 100 fM–10 mM (Bontidean <i>et al</i> 2003), and to target CdSe/ZnS quantum dots to streptavidin expressing HeLa cells when biotinylated (Pinaud <i>et al</i> 2004). INSERT TABLE OF THE IONS FROM THE PAPER<br />
</p><br />
<p>We used this part to <a href="https://2014.igem.org/Team:Imperial/Protocols#general">clone</a> a library of different cellulose-binding domains fused to phytochelatin:<figure class="content-image"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/4/40/IC14-PC-part-table.PNG > <br />
<figcaption>Phytochelatin-CBD fusion parts. </figcaption> <br />
</figure> <br />
</p><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<h3> Other potential proteins </h3><br />
<p>During our research we also explored other functional proteins to fuse to the CBDs.<br />
<div class="accordion"><br />
<h3>Laccases</h3><br />
<div><br />
<p>Laccases are enzymes which can break down aromatic and phenolic compounds such as modified estrogens which can end up unfiltered in the water supply with unknown consequences (Auriol <i>et al</i> 2008). This method of treating estrogen in water was explored by <a target="_blank" href="https://2012.igem.org/Team:Bielefeld-Germany">Bielefeld 2012 iGEM team</a>, and our system also has potential to address this.<br />
</p><br />
</div><br />
<br />
<h3>Nanobodies</h3><br />
<div><br />
<p>Nanobodies are fragments of camelid antibodies which can be generated against a variety of antigens and can be easily expressed by organisms such as <i>E. coli </i>. Nanobodies could be used to target viruses, which are often too small to be filtered by size exclusion. For example there are already nanobodies which can bind to <i> Vaccinia </i> virus, ricin, <i>Cholera</i> toxin, Stephylococcal enterotoxin B and H5N1 Influenza (Goldman <i>et al </i> 2006, Ibanez <i>et al </i> 2011) to name a few. Additionally, nanobodies are stable and can be selected to resist proteolytic attach (Harmsen <i>et al </i> 2006). Nanobodies can also be functional when as fusions or conjugates to other proteins for example to target enzymes to specific cells (Cortez-Retamozo 2004), so there is much potential for targeting a wide array of contaminants when fused to cellulose binding domains.<br />
<br />
</p><br />
</div><br />
</div><br />
<br />
</section><br />
<br />
<br />
<section id="results"><br />
<h2>Results</h2><br />
<p>Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim.</p><br />
</section><br />
<br />
<section id="references"><br />
<h2>References</h2><br />
<br />
<ul><br />
<li>THESE NEED TO BE ALPHABETISED Biondo, R., da Silva, F.A., Vicente, E.J., Souza Sarkis, J.E., et al. (2012) Synthetic phytochelatin surface display in Cupriavidus metallidurans CH34 for enhanced metals bioremediation. Environmental science & technology. 46 (15), 8325–8332.</li><br />
<br />
<li>Bontidean, I., Ahlqvist, J., Mulchandani, A., Chen, W., et al. (2003) Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection. Biosensors and Bioelectronics. 18 (5-6), 547–553.</li><br />
<li>Lee, K.-Y., Buldum, G., Mantalaris, A. and Bismarck, A. (2014), More Than Meets the Eye in Bacterial Cellulose: Biosynthesis, Bioprocessing, and Applications in Advanced Fiber Composites. Macromol. Biosci., 14: 10–32. doi: 10.1002/mabi.201300298</li><br />
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<li>Nguyen, P.Q., Botyanszki, Z., Tay, P.K.R. & Joshi, N.S. (2014) Programmable biofilm-based materials from engineered curli nanofibres. Nature communications. [Online] 54945. Available from: doi:10.1038/ncomms5945.</li><br />
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<li>Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., et al. (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences of the United States of America. [Online] 109 (12), E690–E697. Available from: doi:10.1073/pnas.1115485109.</li><br />
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<li>Linder, M.; Salovuori, I.; Ruohonen, L.; Teeri, T.T., 1996. Characterization of a Double Cellulose-binding Domain. SYNERGISTIC HIGH AFFINITY BINDING TO CRYSTALLINE CELLULOSE. Journal of Biological Chemistry, 271(35), pp.21268–21272. Available at: http://www.jbc.org/content/271/35/21268.full</li><br />
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<li>Andrade, F.K., Moreira, S.M.G., Domingues, L. & Gama, F.M.P. (2010) Improving the affinity of fibroblasts for bacterial cellulose using carbohydrate-binding modules fused to RGD. Journal of biomedical materials research. Part A. [Online] 92 (1), 9–17. Available from: doi:10.1002/jbm.a.32284.</li><br />
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<li>Andrade, F.K., Costa, R., Domingues, L., Soares, R., et al. (2010) Improving bacterial cellulose for blood vessel replacement: Functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide. Acta biomaterialia. [Online] 6 (10), 4034–4041. Available from: doi:10.1016/j.actbio.2010.04.023.</li><br />
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<li>Kauffmann, C., Shoseyov, O., Shpigel, E., Bayer, E.A., et al. (2000) Novel Methodology for Enzymatic Removal of Atrazine from Water by CBD-Fusion Protein Immobilized on Cellulose. Environmental Science & Technology. [Online] 34 (7), 1292–1296. Available from: doi:10.1021/es990754h.</li><br />
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<li>Ramos, R., Domingues, L. & Gama, M. (2010) Escherichia coli expression and purification of LL37 fused to a family III carbohydrate-binding module from Clostridium thermocellum. Protein expression and purification. [Online] 71 (1), 1–7. Available from: doi:10.1016/j.pep.2009.10.016.</li><br />
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<li>Gilkes, N.R., Kilburn, D.G., Miller, R.C. & Warren, R.A. (1989) Structural and functional analysis of a bacterial cellulase by proteolysis. The Journal of biological chemistry. 264 (30), 17802–17808.</li><br />
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<li>Warren, R.A., Beck, C.F., Gilkes, N.R., Kilburn, D.G., et al. (1986) Sequence conservation and region shuffling in an endoglucanase and an exoglucanase from Cellulomonas fimi. Proteins. 1 (4), 335–341.</li><br />
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<li>Kim, H.-D., Choi, S.-L., Kim, H., Sohn, J.H., et al. (2013) Enzyme-linked assay of cellulose-binding domain functions from Cellulomonas fimi on multi-well microtiter plate. Biotechnology and Bioprocess Engineering. 18 (3), 575–580.</li><br />
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<li>Cobbett, C. & Goldsbrough, P. (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annual review of plant biology. [Online] 53159–182. Available from: doi:10.1146/annurev.arplant.53.100301.135154.</li><br />
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<li>Rea, P.A. (2012) Phytochelatin synthase: of a protease a peptide polymerase made. Physiologia plantarum. 145 (1), 154–164.</li><br />
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<li>Bae, W., Chen, W., Mulchandani, A. & Mehra, R.K. (2000) Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnology and bioengineering. 70 (5), 518–524.</li><br />
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<li>Xu, Z., Bae, W., Mulchandani, A., Mehra, R.K., et al. (2002) Heavy Metal Removal by Novel CBD-EC20 Sorbents Immobilized on Cellulose. Biomacromolecules. [Online] 3 (3), 462–465.</li><br />
<br />
<li>Chaturvedi, R. & Archana, G. (2014) Cytosolic expression of synthetic phytochelatin and bacterial metallothionein genes in Deinococcus radiodurans R1 for enhanced tolerance and bioaccumulation of cadmium. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine. 27 (3), 471–482.</li><br />
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<li>Pinaud, F., King, D., Moore, H.-P. & Weiss, S. (2004) Bioactivation and cell targeting of semiconductor CdSe/ZnS nanocrystals with phytochelatin-related peptides. Journal of the American Chemical Society. 126 (19), 6115–6123.</li><br />
<br />
<li>Auriol, M., Filali-Meknassi, Y., Adams, C.D., Tyagi, R.D., et al. (2008) Removal of estrogenic activity of natural and synthetic hormones from a municipal wastewater: efficiency of horseradish peroxidase and laccase from Trametes versicolor. Chemosphere. [Online] 70 (3), 445–452. Available from: doi:10.1016/j.chemosphere.2007.06.064.</li><br />
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<li>Harmsen, M.M., van Solt, C.B., van Zijderveld-van Bemmel, A.M., Niewold, T.A., et al. (2006) Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Applied microbiology and biotechnology. [Online] 72 (3), 544–551. Available from: doi:10.1007/s00253-005-0300-7.</li><br />
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<li>Cortez-Retamozo, V. (2004) Efficient Cancer Therapy with a Nanobody-Based Conjugate. Cancer Research. [Online] 64 (8), 2853–2857. Available from: doi:10.1158/0008-5472.CAN-03-3935.</li><br />
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