Difference between revisions of "Team:TAS Taipei/Contribution"

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<html lang="en">
  
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<div class="clear"></div>
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<head>
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    <meta charset="UTF-8">
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    <title>About Us</title>
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    <link href='http://fonts.googleapis.com/css?family=Lato' rel='stylesheet' type='text/css'>
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    <!--
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    <link rel="stylesheet" href="https://2017.igem.org/Template:TAS_Taipei/Bootstrap">
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    <link rel="stylesheet" href="https://2017.igem.org/Template:TAS_Taipei/JqueryJS">
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    <script src="https://2017.igem.org/Template:TAS_Taipei/BootstrapJS"></script>
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    <style type='text/css'>
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        #top_title,
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        #sideMenu {
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            display: none !important;
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        #content {
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<h1>Contribution</h1>
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</head>
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<h3>Bronze Medal Criterion #4</h3>
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<p><b>Standard Tracks:</b> Participate in the Interlab Measurement Study (to be documented on your InterLab page) and/or improve the characterization of an existing BioBrick Part or Device and enter this information on that part's Main Page in the Registry. The part that you are characterizing must NOT be from a 2017 part number range. Teams who are working on improving the characterization of an existing part should document their experimental design here, along with an explanation for why they chose that part to improve. Data can also be shown here, but it MUST also be documented on the part's Main Page in the Registry.
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<br><br>
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<b>Special Tracks:</b> Document at least one new substantial contribution to the iGEM community that showcases a project related to BioBricks. This contribution should be central to your project and equivalent in difficulty to making and submitting a BioBrick part.
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</p>
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<body>
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    <div class="return">
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        <h1>X</h1>
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    </div>
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    <div class="yellow">
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        <div class="box right">
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            <div class="box2 right project" href="https://2017.igem.org/Team:TAS_Taipei/Background">
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                <img src="https://static.igem.org/mediawiki/2017/0/00/T--TAS_Taipei--Project_C.png" id="dna">
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                <h6 class="navCap">Project</h6>
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            </div>
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            <div class="box2 right experiment" href="https://2017.igem.org/Team:TAS_Taipei/Experimental_Summary">
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                <img src="https://static.igem.org/mediawiki/2017/b/b0/T--TAS_Taipei--Exp_C.png" id="dna">
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                <h6 class="navCap">Experiments</h6>
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            </div>
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            <div class="box2 right modeling" href="https://2017.igem.org/Team:TAS_Taipei/Model">
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                <img src="https://static.igem.org/mediawiki/2017/b/be/T--TAS_Taipei--Modeling_C.png" id="dna">
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                <h6 class="navCap">Modeling</h6>
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            </div>
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            <div class="box2 right prototype" href="https://2017.igem.org/Team:TAS_Taipei/Applied_Design">
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                <img src="https://static.igem.org/mediawiki/2017/2/2e/T--TAS_Taipei--Prototype_C.png" id="dna">
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                <h6 class="navCap">Prototype</h6>
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            </div>
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            <div class="box2 right policy" href="https://2017.igem.org/Team:TAS_Taipei/Human_Practices">
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                <img src="https://static.igem.org/mediawiki/2017/4/42/T--TAS_Taipei--HP2_C.png" id="dna">
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                <h6 class="navCap">Human Practices</h6>
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            </div>
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            <div class="box2 right biosafety" href="https://2017.igem.org/Team:TAS_Taipei/Safety">
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                <img src="https://static.igem.org/mediawiki/2017/b/b8/T--TAS_Taipei--Biosafety_C.png" id="dna">
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                <h6 class="navCap">Safety</h6>
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            </div>
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            <div class="box2 right about" href="https://2017.igem.org/Team:TAS_Taipei/Team">
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                <img src="https://static.igem.org/mediawiki/2017/1/1a/T--TAS_Taipei--About_Us_C.png" id="dna">
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                <h6 class="navCap">About Us</h6>
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            </div>
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            <div class="box2 right acknowledgments" href="https://2017.igem.org/Team:TAS_Taipei/Attributions">
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                <img src="https://static.igem.org/mediawiki/2017/5/52/T--TAS_Taipei--Attributions_C.png" id="dna">
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                <h6 class="navCap">Attributions</h6>
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            </div>
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        </div>
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        <div class="blue">
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            <div class="box3 left project" href="https://2017.igem.org/Team:TAS_Taipei/Background">
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                <h1>Project</h1>
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            </div>
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            <div class="box3 left experiment" href="https://2017.igem.org/Team:TAS_Taipei/Experimental_Summary">
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                <h1>Experiment</h1>
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            </div>
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            <div class="box3 left modeling" href="https://2017.igem.org/Team:TAS_Taipei/Model">
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                <h1>Modeling</h1>
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            </div>
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            <div class="box3 left prototype" href="https://2017.igem.org/Team:TAS_Taipei/Applied_Design">
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                <h1>Prototype</h1>
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            </div>
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            <div class="box3 left policy" href="https://2017.igem.org/Team:TAS_Taipei/Human_Practices">
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                <h1>Human Practice</h1>
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            </div>
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            <div class="box3 left biosafety" href="https://2017.igem.org/Team:TAS_Taipei/Safety">
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                <h1>Biosafety</h1>
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            </div>
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            <div class="box3 left about" href="https://2017.igem.org/Team:TAS_Taipei/Team">
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                <h1>About Us</h1>
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            </div>
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            <div class="box3 left acknowledgments" href="https://2017.igem.org/Team:TAS_Taipei/Attributions">
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                <h1>Attributions</h1>
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            </div>
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        </div>
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    </div>
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    <box class="home">
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        <img src="https://static.igem.org/mediawiki/2017/5/56/T--TAS_Taipei--2home.svg" alt="Home" id="home" onclick="location.href='https://2017.igem.org/Team:TAS_Taipei';" style="cursor: pointer;">
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    <div class="cv" id="cv">
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                <ul class="nav">
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                    <li>
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                        <a href="#char" class="pageNavBig">Characterization</a>
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                    </li>
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                    <li>
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                        <a href="#hypo" class="pageNavBig">HYPOTHESIS</a>
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                    </li>
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                    <li>
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                        <a href="#SDS" class="pageNavSm">SDS-PAGE Gel</a>
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                    </li>
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                    <li>
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                        <a href="#CR" class="pageNavSm">Congo Red Assay</a>
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                    </li>
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                <header>
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                        <h1 class="name col-lg-12">Contribution</h1>
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                    </div>
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                    <div class="row">
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                        <h4 class="para col-lg-12">
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                            <b>We improved the characterization of BBa_K342003 and BBa_K805015,</b> which contain the <i>ompR234</i> ORF and <i>csgD</i> ORF, respectively. We chose to characterize these parts because we wanted to use biofilm to capture nanoparticles. We looked through the distribution kit, found these two parts which regulate biofilm production, and tested them. We characterize the protein sizes of OmpR234 (BBa_K342003) and CsgD (BBa_K805015), and show that expression of OmpR234 and CsgD both increase biofilm production.
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                        </h4>
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                <section class="main">
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                    <div class="row" id="char">
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                        <h1 class="col-lg-12 title2">Characterization of BBa_K342003 and BBa_K805015</h1>
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                    </div>
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                    <div class="row">
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                        <h4 class="para col-lg-12">
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                            In order to characterize these existing parts, we cloned them behind a promoter, RBS, and in front of a transcriptional terminator (figures 3-8 and 3-9). PCR checks showed that cloning was successful (figure 3-11). Sequencing results from Tri-I Biotech confirmed that both of our constructs are correct.
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                        </h4>
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                    </div>
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                    <div class="row">
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                        <div class="image_container col-lg-6">
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                            <img src="https://static.igem.org/mediawiki/2017/3/33/T--TAS_Taipei--figure_3-8.jpg" alt="test" id="group">
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                            <h4 class="subtitle">
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                                <b>Figure 3-8 CsgD expression with a strong promoter + strong RBS combination.</b><span class="subCred"> Figure: Justin Y.</span>
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                            </h4>
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                        </div>
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                        <div class="image_container col-lg-6">
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                            <img src="https://static.igem.org/mediawiki/2017/b/b3/T--TAS_Taipei--figure_3-9.jpg" alt="test" id="group">
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                            <h4 class="subtitle"><b> Figure 3-9 OmpR234 expression with a strong promoter + strong RBS combination.</b><span class="subCred"> Figure: Justin Y.</span></h4>
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                        </div>
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                    </div><br>
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                    <div class="row">
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                        <div class="image_container col-lg-8 col-lg-offset-2">
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                            <img src="https://static.igem.org/mediawiki/2017/a/a2/T--TAS_Taipei--figure_3-11.jpg" alt="test" id="group">
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                            <h4 class="subtitle"> <b>Figure 3-11. PCR check for CsgD and OmpR full constructs. </b> The expected size of CsgD full construct is 1100 bp (orange box) and OmpR full construct is 1200 bp (blue box).<span class="subCred"> Cloning: Catherine Y., Dylan L., Justin Y.</span></h4>
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                        </div>
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                    </div><br>
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                    <div class="row">
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                        <h4 class="para col-lg-12">
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                            To make the final composite, a strong RBS (BBa_B0034) was inserted in front of BBa_S05398 to make BBa_S05399. FInally, BBa_S05397 was inserted before BBa_S05399 to complete the full construct BBa_K2229300 (figure 3-13). Sequencing results from Tri-I Biotech confirmed that our final construct is correct.
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                        </h4>
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                    </div>
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                    <div class="row" id="SDS">
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                        <h1 class="col-lg-12 section-title">SDS-PAGE Gel</h1>
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                    </div>
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                    <div class="row">
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                        <div class="image_container col-lg-12">
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                            <img src="https://static.igem.org/mediawiki/2017/1/14/T--TAS_Taipei--figure_3-15.jpg" alt="test" id="group">
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                            <h4 class="subtitle"><b>Figure 3-15 SDS-PAGE shows <i>E. coli</i> overexpressing CsgD or OmpR234.</b> Predicted sizes of the curli proteins are listed on the right, and <i>E. coli</i> expressing GFP was used as a positive control.<span class="subCred"> Protein Gel & Figure: Justin Y.</span></h4>
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                        </div>
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                    <div class="row">
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                        <h4 class="para col-lg-12">
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                            SDS-PAGE was performed using transformed and lysed <i>E. coli</i> cultures. <b>Bacteria overexpressing CsgD and OmpR234 showed darker bands at 25 kDa and 27 kDa, respectively,</b> compared to controls that only contain the respective ORFs.
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                        </h4>
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                    </div>
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                    <div class="row" id="CR">
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                        <h1 class="col-lg-12 section-title">Congo Red Assay</h1>
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                    </div>
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                    <div class="row">
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                        <h4 class="para col-lg-12">
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                            After confirming protein expression, we wanted to test if our constructs actually lead to faster and more robust biofilm production. We used <b>Congo Red (CR)</b>, a dye commonly used to measure biofilm production (Reinke & Gestwicki 2011). CR solution mixed with bacterial liquid cultures were transferred to 12-well plates with glass coverslips and incubated at 37˚C for one day. The samples were then washed with PBS and dried. Any stained biofilm on the glass coverslips was solubilized in ethanol, and absorbance was measured at 500 nm (figures 3-19). If biofilms were present, the solution would appear red, which could be quantified by an absorbance value.
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                        </h4>
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                    </div>
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                    <div class="row">
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                        <h4 class="para col-lg-12">
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                            To test if the overexpression of CsgD or OmpR234 actually lead to faster and more robust biofilm production, we used Congo Red (CR), a dye commonly used to measure biofilm production (Reinke & Gestwicki 2011). CR solution mixed with bacterial liquid cultures were grown in 12-well plates with glass coverslips, and incubated at 37˚C for one day. The samples were then washed with PBS and dried. Any stained biofilm on the glass coverslips was solubilized in ethanol, and absorbance was measured at 500 nm (figures 3-16 and 3-17). If biofilms were present, the solution would appear red, which could be quantified by an absorbance value.
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                        </h4>
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                    </div>
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                    <div class="row">
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                        <h4 class="para col-lg-12">
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                            We find that <b>overexpressing either CsgD or OmpR234 increases biofilm production,</b> as we hypothesized (figure 3-16 and 3-17). In our experiments, overexpression of CsgD leads to about twice the amount of biofilm compared to BBa_K850015 (figure 3-16), whereas overexpression of OmpR234 leads to about 8 times more biofilm compared to BBa_K342003 (figure 3-17). When bacteria expressing OmpR234 was grown in petri dishes, biofilms appeared thicker compared to controls (figure 3-18).
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                        </h4>
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                        <div class="image_container col-lg-6">
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                            <img src="https://static.igem.org/mediawiki/2017/7/71/T--TAS_Taipei--3-16_new-min.jpg" alt="test" id="group">
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                            <h4 class="subtitle"><b> Figure 3-16: Overexpression of CsgD (BBa_K2229100) doubles biofilm production </b> A) Congo red assay stains biofilm (red). B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm.<span class="subCred"> Experiment & Figure: Yvonne W.</span></h4>
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                        </div>
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                        <div class="image_container col-lg-6">
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                            <img src="https://static.igem.org/mediawiki/2017/a/a2/T--TAS_Taipei--figure_3-17.jpg" alt="test" id="group">
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                            <h4 class="subtitle"><b> Figure 3-17 Overexpression of OmpR234 (BBa_K2229200) leads to ~8 times more biofilm production than control (BBa. </b> A) Congo red assay stains biofilm (red). B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm.<span class="subCred"> Experiment & Figure: Yvonne W.</span></h4>
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                        </div>
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                    </div>
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                    <div class="row">
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                        <div class="image_container col-lg-8 col-lg-offset-2">
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                            <img src="https://static.igem.org/mediawiki/2017/7/70/T--TAS_Taipei--figure_3-18.jpg" alt="test" id="group">
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                            <h4 class="subtitle"><b> Figure 3-18 Overexpression of OmpR234 (BBa_K2229200) produces more biofilm compared to BBa_K342003, which contains just the ORF.</b><span class="subCred"> Experiment &amp; Figure: Yvonne W.</span></h4>
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                        </div>
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                    </div>
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Revision as of 08:04, 22 October 2017

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Contribution

We improved the characterization of BBa_K342003 and BBa_K805015, which contain the ompR234 ORF and csgD ORF, respectively. We chose to characterize these parts because we wanted to use biofilm to capture nanoparticles. We looked through the distribution kit, found these two parts which regulate biofilm production, and tested them. We characterize the protein sizes of OmpR234 (BBa_K342003) and CsgD (BBa_K805015), and show that expression of OmpR234 and CsgD both increase biofilm production.

Characterization of BBa_K342003 and BBa_K805015

In order to characterize these existing parts, we cloned them behind a promoter, RBS, and in front of a transcriptional terminator (figures 3-8 and 3-9). PCR checks showed that cloning was successful (figure 3-11). Sequencing results from Tri-I Biotech confirmed that both of our constructs are correct.

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Figure 3-8 CsgD expression with a strong promoter + strong RBS combination. Figure: Justin Y.

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Figure 3-9 OmpR234 expression with a strong promoter + strong RBS combination. Figure: Justin Y.


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Figure 3-11. PCR check for CsgD and OmpR full constructs. The expected size of CsgD full construct is 1100 bp (orange box) and OmpR full construct is 1200 bp (blue box). Cloning: Catherine Y., Dylan L., Justin Y.


To make the final composite, a strong RBS (BBa_B0034) was inserted in front of BBa_S05398 to make BBa_S05399. FInally, BBa_S05397 was inserted before BBa_S05399 to complete the full construct BBa_K2229300 (figure 3-13). Sequencing results from Tri-I Biotech confirmed that our final construct is correct.

SDS-PAGE Gel

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Figure 3-15 SDS-PAGE shows E. coli overexpressing CsgD or OmpR234. Predicted sizes of the curli proteins are listed on the right, and E. coli expressing GFP was used as a positive control. Protein Gel & Figure: Justin Y.

SDS-PAGE was performed using transformed and lysed E. coli cultures. Bacteria overexpressing CsgD and OmpR234 showed darker bands at 25 kDa and 27 kDa, respectively, compared to controls that only contain the respective ORFs.

Congo Red Assay

After confirming protein expression, we wanted to test if our constructs actually lead to faster and more robust biofilm production. We used Congo Red (CR), a dye commonly used to measure biofilm production (Reinke & Gestwicki 2011). CR solution mixed with bacterial liquid cultures were transferred to 12-well plates with glass coverslips and incubated at 37˚C for one day. The samples were then washed with PBS and dried. Any stained biofilm on the glass coverslips was solubilized in ethanol, and absorbance was measured at 500 nm (figures 3-19). If biofilms were present, the solution would appear red, which could be quantified by an absorbance value.

To test if the overexpression of CsgD or OmpR234 actually lead to faster and more robust biofilm production, we used Congo Red (CR), a dye commonly used to measure biofilm production (Reinke & Gestwicki 2011). CR solution mixed with bacterial liquid cultures were grown in 12-well plates with glass coverslips, and incubated at 37˚C for one day. The samples were then washed with PBS and dried. Any stained biofilm on the glass coverslips was solubilized in ethanol, and absorbance was measured at 500 nm (figures 3-16 and 3-17). If biofilms were present, the solution would appear red, which could be quantified by an absorbance value.

We find that overexpressing either CsgD or OmpR234 increases biofilm production, as we hypothesized (figure 3-16 and 3-17). In our experiments, overexpression of CsgD leads to about twice the amount of biofilm compared to BBa_K850015 (figure 3-16), whereas overexpression of OmpR234 leads to about 8 times more biofilm compared to BBa_K342003 (figure 3-17). When bacteria expressing OmpR234 was grown in petri dishes, biofilms appeared thicker compared to controls (figure 3-18).

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Figure 3-16: Overexpression of CsgD (BBa_K2229100) doubles biofilm production A) Congo red assay stains biofilm (red). B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm. Experiment & Figure: Yvonne W.

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Figure 3-17 Overexpression of OmpR234 (BBa_K2229200) leads to ~8 times more biofilm production than control (BBa. A) Congo red assay stains biofilm (red). B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm. Experiment & Figure: Yvonne W.

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Figure 3-18 Overexpression of OmpR234 (BBa_K2229200) produces more biofilm compared to BBa_K342003, which contains just the ORF. Experiment & Figure: Yvonne W.