Team:Dundee/Biobricks

From 2012.igem.org

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<li class='has-sub'><a href='https://2012.igem.org/Team:Dundee/Wet Lab'><span>Wet Lab</span></a>
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<li class='has-sub'><a href='https://2012.igem.org/Team:Dundee/Strategy'><span>Wet Lab</span></a>
           <ul>
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               <li><a href='https://2012.igem.org/Team:Dundee/Strategy'><span>Strategy</span></a></li>
               <li><a href='https://2012.igem.org/Team:Dundee/Strategy'><span>Strategy</span></a></li>
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        <img src="https://static.igem.org/mediawiki/2012/8/84/Softwareheader.png"><br><br>
 
        
        
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        <div class="boxOuter full" style="padding: 0px; border: 1px solid black; background-color: black;">
 
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            <h3><img src="https://static.igem.org/mediawiki/2012/8/84/Lysistokillheader.png"></h3>
 
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            <div class="boxInner centre clearfix">               
 
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                <div class="pic" style="border-color: transparent; margin: 5px 30px 5px 20px;">
 
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                    <a href="https://static.igem.org/mediawiki/igem.org/5/56/Dundee12lysis.png">
 
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                    <img src="https://static.igem.org/mediawiki/igem.org/5/56/Dundee12lysis.png" width="150px"
 
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                      alt="Lysis To Kill screenshot" />
 
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                    </a>
 
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                </div>
 
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                Lysis To Kill is a game developed for the Android platform. The concept of the game was
 
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                inspired by Splashback which was a Flash game developed for the Cartoon Network
 
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                <a href="https://2012.igem.org/Team:Dundee/References">[1]</a>.
 
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                The original concept involved adding drops of 'goo' to a playing board in order to burst 'blobs',
 
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                creating a cascade of bursts. The aim was to clear the playing board in as few clicks as possible.
 
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                In a similar fashion, Lysis To Kill draws on the same game concept with a twist to mirror the
 
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                iGEM project we have undertaken.
 
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                <br /><br />
 
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                You start the game with 10 clicks. You are in charge of the friendly, synthetically engineered
 
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                E-coli cells (in green), and each time you click a cell it shrinks and eventually bursts.
 
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                Your goal is to destroy all the C.diff cells with &phi;CD27 endolysin, which is fired from your
 
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                E.coli cells  whenever they burst. Each level grants you an extra click, and points are awarded
 
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                for every C.diff cell destroyed. Bonus points are awarded if you finish a level with a clear board.
 
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                The game is free to
 
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                <a href="#dl">download</a>, and the full source code is provided.
 
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            </div>
 
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        </div>
 
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                <p><b> New Biobricks:</b></p>
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<b>1. <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895001" target="_blank">BBa_K895001</a> <i>Salmonella</i> Hcp protein.</b> This biobrick encodes the Hcp protein (STM0279 or <i>tssD</i>). This is a key component of Type VI Secretion Systems and is often found in the cell supernatant so is commonly regarded as a secreted protein.
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            <br /><br />
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        <div class="boxOuter full" style="padding: 0px; border: 1px solid black; background-color: black;">
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<b>2. <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895002" target="_blank">BBa_K895002</a> <i>Salmonella</i> VgrG protein.</b> This biobrick is a gene encoding an important large protein thought to form the tip of the Type VI Secretion/Cell Puncturing device. VgrG (STM0289 or TssI) must have an extracytoplsmic localisation. This would be the initial point of contact between the Type VI Secretion System and any target cell.  
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            <h3><img src="https://static.igem.org/mediawiki/2012/b/b2/Lazyscientist2header.png"></h3>
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            <div class="boxInner centre clearfix">               
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                <div class="pic" style="border-color: transparent; float:right; margin: 5px 20px 5px 30px;">
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                    <a href="https://static.igem.org/mediawiki/igem.org/a/ae/Dundee12lazy.gif">
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                    <img src="https://static.igem.org/mediawiki/igem.org/a/ae/Dundee12lazy.gif" width="150px"
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                      alt="Lazy Scientist II screenshot" />
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                    </a>                    
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                </div>
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                The Dundee iGEM team of 2011 created an Android application named the Lazy Scientist, and this
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                year we chose to follow up the application with a suite of useful tools for biology students.  
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                The main goal was to provide some useful applications and to provide access to the source code
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                to enable developers to add, modify and change the application to suit their needs. The app is
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                free to <a href="#dl">download</a>, and the full source code is provided.
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                 <br /><br />
                 <br /><br />
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                The application currently contains the following tools:
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<b>3. <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895003" target="_blank">BBa_K895003</a> <i>Salmonella</i> TtrRS two-component system.</b> This biobrick encodes a bacterial two-component system. The TtrS protein is a membrane-bound histidine kinase. The kinase senses extracellular tetrathionate levels (a compound produced in the gut when the inflammation process is induced during infection), which activates its autophosphorylation activity. The phosphate is passed on to TtrR, the DNA-binding response regulator, which can then activate transcription of at least one operon its native organism (<i>Salmonella</i>. The divergent <i>ttrB</i> promoter/operator site is included in the biobrick. In the native organism, <i>ttrB</i> is regulated by oxygen levels (an FNR binding site is present in the promoter) and, predominantly, by extracellular tetrathionate levels <i>via</i> TtrRS.
                 <br /><br />
                 <br /><br />
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                <ul style="padding-left: 4%;">
 
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                    <li>Ratio to Percentage/ Percentage to Ratio Convertor</li>
 
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                    <li>OD660 to Cell Count Estimator</li>
 
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                    <li>Simple Dilution Calculator</li>
 
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                    <li>Serial Dilution Checker</li>
 
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                </ul>
 
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                <br />
 
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                <div class="caption" style="width: 80%;">
 
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                    (** Note: The OD660 calculator is based on a single sample set of data that, for obvious reasons,
 
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                    can only provide an estimate of cell count.<br />
 
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                    For more accurate OD calculations, in-situ, calibrated laboratory equipment should be used.)
 
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                </div>
 
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            </div>
 
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        </div>
 
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<b>4. <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895004" target="_blank">BBa_K895004</a> <i>Salmonella</i> Hcp fused to  &phi;CD27 endolysin.</b> This biobrick encodes Hcp (from <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895001" target="_blank">BBa_K895001</a>) fused <i>via</i> an HA epitope tag to a synthetic endolysin based on that from a <i>C. difficile</i> bacteriophage (&phi;CD27).
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                <br /><br />
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        <div class="boxOuter full" style="padding: 0px; border: 1px solid black; background-color: black;">
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<b>5. <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895005" target="_blank">BBa_K895005</a> <i>Salmonella</i> VgrG fused to  &phi;CD27 endolysin.</b> This biobrick encodes VgrG (from <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895002" target="_blank">BBa_K895002</a>) fused <i>via</i> an HA epitope tag to a synthetic endolysin based on that from a <i>C. difficile</i> bacteriophage (&phi;CD27).  
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            <h3><img src="https://static.igem.org/mediawiki/2012/1/1c/Splicerheader.png"></h3>
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            <div class="boxInner centre clearfix">
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                <br />
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                <div class="pic" style="border-color: transparent; margin: 5px 20px 5px 20px;">
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                    <a href="https://static.igem.org/mediawiki/igem.org/3/37/Dundee12splicer.png">
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                    <img src="https://static.igem.org/mediawiki/igem.org/3/37/Dundee12splicer.png" width="400px"  
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                      alt="Splicer Screenshot" />
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                    </a>                    
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                </div>
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                Splicer is a short game of six test levels developed for the Windows desktop. The premise is
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                simple - from a selection of Biobricks, you are tasked with the creation of biological solutions
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                to given problems. By selecting each Biobrick, you insert it into the plasmid in the centre. Once
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                you think you have a viable solution, you click on the 'Transcribe & Translate' button to see if
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                you were right. If you are comfortable with C#, the full source code is included so that you may
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                add more Biobricks and more levels.
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                 <br /><br />
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                The game was developed in Visual Studio using the XNA libraries, and full source code is provided
 
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                in the <a href="#dl">download</a> section, along with a full installable version of the game, which
 
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                can be removed via the Windows control panel as normal.
 
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            </div>
 
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        </div>
 
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        <div class="boxOuter full" style="padding: 0px; border: 1px solid black; background-color: black;">
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<b>6. <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895007" target="_blank">BBa_K895007</a> <i>Salmonella</i> TtrRS two-components system linked to a GFP reporter.</b> This biobrick is a working 'inflammation bisensor' comprising <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895003" target="_blank">BBa_K895003</a> linked to GFP from <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J54103" target="_blank">BBa_J54103</a>. Transcription, and subsequent translation, of GFP is linked to extracellular tetrathionate levels in the <i>E. coli</i> chassis organism.  
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            <h3><img src="https://static.igem.org/mediawiki/2012/c/ce/Cellsimheader.png"></h3>
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            <div class="boxInner centre clearfix">
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                From the onset of the project, the modelling team had to carry out investigative research to
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                understand the biological processes that were involved in the project to ascertain appropriate
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                modelling methodologies to adopt. Due to the unique combination of factors involved in the project,  
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                it became evident that there were considerable quantities of mathematical variables that defined the
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                behaviour of the cellular interaction. From a software development perspective, it was attractive
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                to take the mathematical axioms of behaviour, and to wrap them within a bespoke modelling solution
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                to provide a visual representation of the project.
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                 <br /><br />
                 <br /><br />
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                 <div class="pic" style="border-color: transparent; margin: 5px 20px 5px 20px;">
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                    <a href="https://static.igem.org/mediawiki/igem.org/3/32/Dundee12cellsimanimsmall.gif">
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                 <p><b>Improving and de-bugging old biobricks:</b></p>
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                    <img src="https://static.igem.org/mediawiki/igem.org/3/32/Dundee12cellsimanimsmall.gif" width="400px"
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<b>1. <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895000" target="_blank">BBa_K895000</a> <i>Escherichia coli tat</i> constitutive promoter.</b> This small biobrick was originally designed by the <a href="https://2011.igem.org/Team:Dundee" target="_blank">Dundee iGEM 2011 Team</a>. Though this was cloned as an EcoRI / PstI fragment into pSB1C3, this rookie team mistakenly omitted to include the correct prefix and suffix sequences to meet RFC[10]. In this work we have corrected this error and correctly assembled the <i>tat</i> promoter into pSB1C3.
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                      alt="CellSim Animation" />
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                    </a>                     
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                </div>
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                CellSim is a cross-platform, 2D, cellular automata
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                <a href="https://2012.igem.org/Team:Dundee/References" target="_blank">[4]</a> (CA) modelling
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                tool that was developed
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                to demonstrate, in a graphical manner, how the synthetically modified E-coli and C.diff cells
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                could interact in a simulated environment. The application was designed to simulate the cellular
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                interactions whilst also providing a customisable simulation environment that could enable the tool
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                to be used in other, similar projects. CellSim was developed to enable it to be executed on either
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                Linux or Windows based systems, and is released under the GNU GPL 3 license that permits copying,
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                modification and redistribution of the program. The source code and full class diagram is provided
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                within the download archives for educational purposes as well as encouraging others to build upon
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                the initial release.
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                <div class="clearfix"></div>
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                <br />
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                CellSim initialises the environment by parsing the text data file 'settings.dat' which must
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                be located within the same folder as the program. The download archive contains an example of the
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                settings file which provides all the parameters that are available for user modification.
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                The current development version (1.0) provides an environment in which the following aspects
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                can be simulated and defined by the user:
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                <br />
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                <ul style="padding-left: 4%;">
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                    <li>Cellular Mitosis Rates</li>
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                    <li>Strains</li>
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                    <li>Colony Size, Initial Positioning & Colouration</li>
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                    <li>Medium Flow Effects</li>
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                    <li>Mucus Wall Effects</li>
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                    <li>Time Calibrated Rendering</li>
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                    <li>Nutrient Availability, Supply & Absorption</li>
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                    <li>Low Nutrient Mitosis Suppression</li>
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                </ul>
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                <br />
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                <div class="pic" style="border-color: transparent; float:right; margin: 5px 20px 5px 20px;">
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                    <a href="https://static.igem.org/mediawiki/igem.org/1/10/Dundee12cellsimcd.png">
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                    <img src="https://static.igem.org/mediawiki/igem.org/1/10/Dundee12cellsimcd.png" width="400px"  
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                      alt="CellSim Class Diagram" />
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                    </a>                    
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                </div>
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                The program was developed around an MVC structural design pattern
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                <a href="https://2012.igem.org/Team:Dundee/References" target="_blank">[3]</a>  
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                with a subtle modification.
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                The initial design adopted a classic MVC
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                <a href="https://2012.igem.org/Team:Dundee/References" target="_blank">[3]</a>  
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                architecture, whereby for each generation, each cell
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                was updated within the model classes, and subsequently rendered within the view classes. Due to  
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                the large number of cells that the program is required to simulate, code profiling highlighted
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                the inefficiency if iterating through all cells twice. Furthermore, the status of each cell is
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                partly determined by neighbouring cells, and therefore a method was required to detect the presence
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                of neighbouring cells and their status. To accomplish this through data provided solely within the
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                model, for each cell, all other cells would require iteration and interrogation leading to a time
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                complexity of at least O(n<sup>2</sup>). Due to the mitosis behaviour of cells, the source number of cells
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                also increases in a quadratic manner. The quadratic mitosis behaviour cannot be alleviated, as it
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                represents the real-world scenario, so a solution was sought to alleviate the compound quadratic
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                time complexity that arose from the classic MVC
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                <a href="https://2012.igem.org/Team:Dundee/References" target="_blank">[3]</a>  
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                implementation.
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                 <br /><br />
                 <br /><br />
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                The solution adopted was to carry out cell interrogation, cell update and rendering in a single
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                pass, and establishing the presence of neighbouring cells by direct interrogation of the graphics
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<b>2. <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895006" target="_blank">BBa_K895006</a> Classic GFP-encoding gene cloned to RFC[10] standard in pSB1C3.</b> For our project we decided to use GFP as a reporter protein. We wished to use a GFP-producing biobrick from the 2012 Distribution Kit and chose that from <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J54103" target="_blank">BBa_J54103</a>. We noticed that this was in a ampicillin-resistant vector rather than pSC1C3. We thought it would be helpful to the synthetic biology community to move the open reading frame of the GFP encoded by <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J54103" target="_blank">BBa_J54103</a> onto pSB1C3 following RFC[10] standard rules.
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                memory as opposed to model data interrogation. By calculating the base address of the graphics
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                memory in conjunction with the co-ordinates of the current cell, a direct memory address could be  
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                calculated and accessed to establish the presence of neighbouring cells, alleviating the need to  
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                iterate through all cells. By adopting these measures, the time complexity of the 'cell evolution'
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                section of code was reduced to a constant, greatly improving the speed and number of cells that
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                the program could comfortably emulate.
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                 <br /><br />
                 <br /><br />
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                Cellular movement emulates fluid flow by imposing a circular pressure front that is circular in
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                nature. This provides a simple model that affects each cell's horizontal position by exerting
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<b>3. <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895007" target="_blank">BBa_K895007</a> <i>Salmonella</i> TtrRS two-components system linked to a GFP reporter.</b> This is a working 'inflammation biosensor' comprising <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895003" target="_blank">BBa_K895003</a> linked to GFP from <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J54103" target="_blank">BBa_J54103</a>.
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                a greater force on those nearest to the centre of fluid flow. To enable variable flow rates to be
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                simulated, a normalised force was calculated from the cell's co-ordinates (x, y) relative to the
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                display screens height (Ymax), multiplied by a user adjustable force factor (f) and utilised to
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                modify the horizontal position of the cell. By carrying out this calculation on each cell, a
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                realistic, circular flow pressure is simulated. The new horizontal cell position is calculated
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                from the following formula:
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                 <br /><br />
                 <br /><br />
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                <div class="pic clearfix" style="border-color: transparent; padding-left: 150px;">
 
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                    <a href="https://static.igem.org/mediawiki/igem.org/b/bb/Dundee12flowformula.png">
 
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                    <img src="https://static.igem.org/mediawiki/igem.org/b/bb/Dundee12flowformula.png" width="600px"
 
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                      alt="CellSim Flow Formula" />
 
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                    </a>                     
 
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                </div>             
 
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                <div class="clearfix"></div>
 
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                To provide a realistic and accurate movement of cells within CA environment, each cell contains
 
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                co-ordinate members for the respective 'x' and 'y' position. These class members are defined as
 
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                float data types as opposed to integers to permit gradual movements to be achieved. To render each
 
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                cell, the co-ordinates are simply cast to integer types before being passed to the graphics
 
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                interface. This permits cell movements to be calculable to fractions of a pixel, and provides an
 
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                accurate representation of the behaviour required.
 
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                <br /><br />
 
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                The application adopts the SDL (Simple Directmedia Layer) graphics API that was chosen primarily
 
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                due to its flexibility, and is licensed under GNU LGPL version 2
 
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                <a href="https://2012.igem.org/Team:Dundee/References" target="_blank">[2]. </a>
 
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                Furthermore, the library
 
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                provides binary compilations for a wide variety of operating systems including Windows, Linux
 
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                and MacOS. To encourage development of the program, pre-developed Eclipse and Visual Studio 11
 
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                project files have been created, and are free to download and explore. Alternatively, the packages
 
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                also include the pre-compiled binary versions of the application within the 'Release' folder of
 
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                the download archive (ZIP) files.
 
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                <br />               
 
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            </div>
 
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                <p><b>Favourite Biobricks:</b></p>
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        <div class="boxOuter full" style="padding: 0px; border: 1px solid black; background-color: black;">
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<b>1. <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895007" target="_blank">BBa_K895007</a> <i>Salmonella</i> TtrRS two-components system linked to a GFP reporter.</b> The 'inflammation biosensor' has great potential for health & medicine applications, particularly for devices designed to respond intelligently to the infection process.  
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            <h3><img src="https://static.igem.org/mediawiki/2012/8/86/Downloadsheader.png"></h3>
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                NOTE: To run 'CellSim' under Windows, the Visual C++ Redistributable Package must be installed.  
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                This is free, and can be downloaded from
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                <a href="https://www.microsoft.com/en-us/download/details.aspx?id=30679" target="_blank">
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                https://www.microsoft.com/en-us/download/details.aspx?id=30679
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                </a>
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                Select the file named 'vcredist_x86.exe' (6.2MB), and once downloaded, run the program to install
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                the Visual C++ libraries required prior to running the 'CellSim' program.
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                 <br /><br />
                 <br /><br />
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                <strong>Download Links:</strong><br />
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                Lysis To Kill - Android Package [
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<b>2. <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895001" target="_blank">BBa_K895001</a> <i>Salmonella</i> Hcp protein.</b> The predicted extracellular localisation of this protein makes it potentially useful for future projects. If secretion of the protein can be controlled and characterised then it could be fused to any number of reporters or other enzymes. The <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895001" target="_blank">BBa_K895001</a> polypeptide can be produced stably in <i>E. coli</i>. Fusion proteins are also stable.
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                    <a href="https://dl.dropbox.com/u/52338078/iGEM/Lysis%20To%20Kill.apk">Lysis To Kill.apk</a>
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                ]<br />
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                Lysis To Kill Eclipse Project - Full Source [
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                    <a href="https://dl.dropbox.com/u/52338078/iGEM/Lysis%20To%20Kill.zip">Lysis To Kill.zip</a>
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                ]<br />
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                Lazy Scientist II - Android Package [
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                    <a href="https://dl.dropbox.com/u/52338078/iGEM/Lazy%20Scientist%20II.apk">Lazy Scientist II.apk</a>
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                ]<br />
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                Lazy Scientist II Eclipse Project - Full Source [
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                    <a href="https://dl.dropbox.com/u/52338078/iGEM/Lazy%20Scientist%20II.zip">Lazy Scientist II.zip</a>
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                ]<br />
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                CellSim Linux x86 Eclipse Project - Full Program & Source [
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                    <a href="https://dl.dropbox.com/u/52338078/iGEM/CellSim%20Linux%20x86.zip">CellSim Linux x86.zip</a>
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                ]<br />
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                CellSim Windows x86 Visual Studio 11 Project - Full Program & Source [
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                    <a href="https://dl.dropbox.com/u/52338078/iGEM/CellSim%20Windows%20x86.zip">CellSim Windows x86.zip</a>
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                ]<br />
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                Splicer Source - Visual Studio 10 XNA Project - Full Program & Source [
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                    <a href="https://dl.dropbox.com/u/52338078/iGEM/Splicer%20Source.zip">Splicer Source.zip</a>
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                <br />
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                Splicer Installer - Windows x86 Installer - Full Program [
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                    <a href="https://dl.dropbox.com/u/52338078/iGEM/Splicer%20Installer.zip">Splicer Installer.zip</a>
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                 <br /><br />
                 <br /><br />
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                <div class="caption" style="width: 100%; color: #DD0000;">
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                    All software is provided under the GNU GPL 3 license terms (
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<b>3. <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895002" target="_blank">BBa_K895002</a> <i>Salmonella</i> VgrG protein.</b> The predicted extracellular localisation of this protein makes it potentially useful for future projects. It too could be fused to a broad spectrum of reporter proteins or other enzymes. The <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K895001" target="_blank">BBa_K895002</a> polypeptide can be produced stably in <i>E. coli</i>.
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                    <a href="https://www.gnu.org/licenses/gpl-3.0.txt" target="_blank">full text here</a>), and is
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                    provided 'as-is' without warranty of any kind.<br />By downloading any of the software provided, you
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                    are confirming your agreement to the terms of the license in full.
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                </div>
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                <br />
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                Some or all of the software provided may see further development by the author. Due to the
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                Wiki freeze at the end of the competition, future versions will be released through the
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                author's Blog at <a href="http://nibblesbytes.blogspot.co.uk/">http://nibblesbytes.blogspot.co.uk</a>
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                 <br /><br />
                 <br /><br />
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<div style="margin-left: 40px; text-align: center;"><img style="padding: 10px;" src="https://static.igem.org/mediawiki/2012/b/b7/Homepic2.jpg"><img style="padding: 10px;" src="https://static.igem.org/mediawiki/2012/d/d0/Homepic1.jpg"><img style="padding: 10px;" src="https://static.igem.org/mediawiki/2012/e/ea/Homepic3.jpg"></div><br>
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Latest revision as of 21:07, 26 September 2012

New Biobricks:

1. BBa_K895001 Salmonella Hcp protein. This biobrick encodes the Hcp protein (STM0279 or tssD). This is a key component of Type VI Secretion Systems and is often found in the cell supernatant so is commonly regarded as a secreted protein.

2. BBa_K895002 Salmonella VgrG protein. This biobrick is a gene encoding an important large protein thought to form the tip of the Type VI Secretion/Cell Puncturing device. VgrG (STM0289 or TssI) must have an extracytoplsmic localisation. This would be the initial point of contact between the Type VI Secretion System and any target cell.

3. BBa_K895003 Salmonella TtrRS two-component system. This biobrick encodes a bacterial two-component system. The TtrS protein is a membrane-bound histidine kinase. The kinase senses extracellular tetrathionate levels (a compound produced in the gut when the inflammation process is induced during infection), which activates its autophosphorylation activity. The phosphate is passed on to TtrR, the DNA-binding response regulator, which can then activate transcription of at least one operon its native organism (Salmonella. The divergent ttrB promoter/operator site is included in the biobrick. In the native organism, ttrB is regulated by oxygen levels (an FNR binding site is present in the promoter) and, predominantly, by extracellular tetrathionate levels via TtrRS.

4. BBa_K895004 Salmonella Hcp fused to φCD27 endolysin. This biobrick encodes Hcp (from BBa_K895001) fused via an HA epitope tag to a synthetic endolysin based on that from a C. difficile bacteriophage (φCD27).

5. BBa_K895005 Salmonella VgrG fused to φCD27 endolysin. This biobrick encodes VgrG (from BBa_K895002) fused via an HA epitope tag to a synthetic endolysin based on that from a C. difficile bacteriophage (φCD27).

6. BBa_K895007 Salmonella TtrRS two-components system linked to a GFP reporter. This biobrick is a working 'inflammation bisensor' comprising BBa_K895003 linked to GFP from BBa_J54103. Transcription, and subsequent translation, of GFP is linked to extracellular tetrathionate levels in the E. coli chassis organism.

Improving and de-bugging old biobricks:

1. BBa_K895000 Escherichia coli tat constitutive promoter. This small biobrick was originally designed by the Dundee iGEM 2011 Team. Though this was cloned as an EcoRI / PstI fragment into pSB1C3, this rookie team mistakenly omitted to include the correct prefix and suffix sequences to meet RFC[10]. In this work we have corrected this error and correctly assembled the tat promoter into pSB1C3.

2. BBa_K895006 Classic GFP-encoding gene cloned to RFC[10] standard in pSB1C3. For our project we decided to use GFP as a reporter protein. We wished to use a GFP-producing biobrick from the 2012 Distribution Kit and chose that from BBa_J54103. We noticed that this was in a ampicillin-resistant vector rather than pSC1C3. We thought it would be helpful to the synthetic biology community to move the open reading frame of the GFP encoded by BBa_J54103 onto pSB1C3 following RFC[10] standard rules.

3. BBa_K895007 Salmonella TtrRS two-components system linked to a GFP reporter. This is a working 'inflammation biosensor' comprising BBa_K895003 linked to GFP from BBa_J54103.

Favourite Biobricks:

1. BBa_K895007 Salmonella TtrRS two-components system linked to a GFP reporter. The 'inflammation biosensor' has great potential for health & medicine applications, particularly for devices designed to respond intelligently to the infection process.

2. BBa_K895001 Salmonella Hcp protein. The predicted extracellular localisation of this protein makes it potentially useful for future projects. If secretion of the protein can be controlled and characterised then it could be fused to any number of reporters or other enzymes. The BBa_K895001 polypeptide can be produced stably in E. coli. Fusion proteins are also stable.

3. BBa_K895002 Salmonella VgrG protein. The predicted extracellular localisation of this protein makes it potentially useful for future projects. It too could be fused to a broad spectrum of reporter proteins or other enzymes. The BBa_K895002 polypeptide can be produced stably in E. coli.



Retrieved from "http://2012.igem.org/Team:Dundee/Biobricks"