Difference between revisions of "Team:SHSBNU China/Improve"

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{{SHSBNU_China}}
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<html lang="zh-CN">
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<html>
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<head>
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    <title>Improvements</title>
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<body>
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    <link id="Sec_CSS" rel="stylesheet" type="text/css" href="https://2018.igem.org/Template:SHSBNU_China/Sample_css?action=raw&ctype=text/css" />
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    <div id="main_main">
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<section id="main">
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<div onclick="window.scrollTo(0,0);" id="Top_Button">
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<img style="width:5vw;height:5vw" src="https://static.igem.org/mediawiki/2018/e/e2/T--SHSBNU_China--Back_To_Top.png"/>
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</div>
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<header id="page_header">
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<div id="header_classification">
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<div id="home_button">
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<a style="height:100%; width:100%;" href="https://2018.igem.org/Team:SHSBNU_China">
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<img style="height:100%; width:100%;" src="https://static.igem.org/mediawiki/2018/4/48/T--SHSBNU_China--Home_Button.png"/>
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</a>
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<div class="column full_size judges-will-not-evaluate">
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<div style="height:5vw;" class="first_classfication" onmouseover="fcova()" onmouseout="fcoua()" id="fc_Project"><!--Project-->
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<h3>★  ALERT! </h3>
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<div class="fc_box">
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<p>This page is used by the judges to evaluate your team for the <a href="https://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
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<a class="fc_link" style="height:100%; width:100%;" href="https://2018.igem.org/Team:SHSBNU_China/Project">
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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<h4 class="fc_content">Project</h4>
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</div>
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</a>
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</div>
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</div>
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<div style="height:5vw;" class="first_classfication" onmouseover="fcovb()" onmouseout="fcoub()" id="fc_Experiment"><!--Experiment-->
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<div class="fc_box">
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<a class="fc_link" style="height:100%; width:100%;" href="https://2018.igem.org/Team:SHSBNU_China/Experiments">
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<h4 class="fc_content">Experiment</h4>
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</a>
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</div>
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</div>
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<div style="height:5vw;" class="first_classfication" onmouseover="fcovc()" onmouseout="fcouc()" id="fc_Model"><!--Model-->
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<div class="fc_box">
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<a class="fc_link" style="height:100%; width:100%;" href="https://2018.igem.org/Team:SHSBNU_China/Model">
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<h4 class="fc_content">Model</h4>
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</a>
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</div>
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</div>
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<div style="height:5vw;" class="first_classfication" onmouseover="fcovd()" onmouseout="fcoud()" id="fc_Human Practice"><!--Human Practice-->
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<div class="fc_box">
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<a class="fc_link" style="height:100%; width:100%;" href="https://2018.igem.org/Team:SHSBNU_China/Human Practices">
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<h4 class="fc_content">Human Practice</h4>
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</a>
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</div>
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</div>
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<div style="height:5vw;" class="first_classfication" onmouseover="fcove()" onmouseout="fcoue()" id="fc_Demonstrate"><!--Demonstrate-->
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<div class="fc_box">
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<a class="fc_link" style="height:100%; width:100%;" href="https://2018.igem.org/Team:SHSBNU_China/Demonstrate">
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<h4 class="fc_content">Demonstrate</h4>
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</a>
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</div>
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</div>
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<div style="height:5vw;" class="first_classfication" onmouseover="fcovf()" onmouseout="fcouf()" id="fc_Safety"><!--Safety-->
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<div class="fc_box">
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<a class="fc_link" style="height:100%; width:100%;" href="https://2018.igem.org/Team:SHSBNU_China/Safety">
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<h4 class="fc_content">Safety</h4>
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</a>
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</div>
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</div>
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<div style="height:5vw;" class="first_classfication" onmouseover="fcovg()" onmouseout="fcoug()" id="fc_Attribution"><!--Attribution-->
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<div class="fc_box">
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<a class="fc_link" style="height:100%; width:100%;" href="https://2018.igem.org/Team:SHSBNU_China/Attributions">
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<h4 class="fc_content">Attribution</h4>
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</a>
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</div>
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</div>
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<div style="height:5vw; padding: 0vw; align-self: center; vertical-align: middle; display: table; line-height: 3vw;" class="first_classfication" onmouseover="fcovh()" onmouseout="fcouh()" id="fc_Team"><!--Team-->
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<div style="height: 2.8vw; display: table-cell; vertical-align: middle">
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<a class="fc_link" style="height:100%; width:100%;" href="https://2018.igem.org/Team:SHSBNU_China/Team">
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<h4 style="padding-right: 0.30vw">Team</h4>
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</a>
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</div>
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</div>
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</div>
  
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<div id="header_title">
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<a href="https://2018.igem.org/Team:SHSBNU_China">
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<img id="title_pic" src="https://static.igem.org/mediawiki/2018/2/23/T--SHSBNU_China--Title.jpeg"/>
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</a>
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</div>
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</header>
  
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<div class="clear"></div>
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<div id="menu">
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<h6 id="menu_intro">Improve</h6>
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<div class="second_classfication">
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#I">Demonstration</a>
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</div>
  
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<div class="second_classfication">
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#VI">Using sfGFP-SpyCatcher</a>
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            </div>
  
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<div class="column full_size">
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<div class="second_classfication">
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<h1>Improve</h1>
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#VII">Verification - Using SpyCatcher-CotA</a>
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<p>For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Registry. Please include a link to your improved part on this page.</p>
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            </div>
  
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<h3>Gold Medal Criterion #2</h3>
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<div class="second_classfication">
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<p><b>Standard Tracks:</b> Create a new part that has a functional improvement upon an existing BioBrick part. The sequences of the new and existing parts must be different. You must perform experiments with both parts to demonstrate this improvement.  Document the experimental characterization on the Part's Main Page on the Registry for both the existing and new parts. Both the new and existing Main Page of each Part’s Registry entry must reference each other. Submit a sample of the new part to the Registry.
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#C">Conclusion</a>
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            </div>
  
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The existing part must NOT be from your 2018 part number range and must be different from the part documented in bronze #4.
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<div class="second_classfication">
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Project">Project</a>
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            </div>
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<div id="menu_blank">
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</div>
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</div>
  
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<br><br>
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        <div id="articles">
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<b>Special Tracks:</b> Improve the function of an existing iGEM project (that your current team did not originally create) and display your achievement on your wiki.</p>
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<div id="sec_title">
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<h1>Improve</h1>
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</div>
  
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<div id="sec_content">
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<h2 id="I">I. Overview</h2>
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<div class="content">
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<div style="width: 46vw" class="content_pic_left">
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<img class="pictures" id = "21000" src="https://static.igem.org/mediawiki/2018/b/b9/T--SHSBNU_China--21000.png"/>
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<p class="pic_text">Fig1.1: CsgA-SpyTag</p>
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</div>
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<p class="text">
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    CsgA is a amyloid protein which is a dominant component of the biofilms of E. coli. We fused a functional peptide domain SpyTag with gene CsgA (previous part: BBa_K1583000), forming CsgA-SpyTag (Improved Part: BBa_K2684006). By utilizing the steady isopeptide bond combination ability of the SpyTag-SpyCatcher system , it is possible to fix different SpyChatcher-fusing proteins, like enzymes or receptors, to the biofilm to introduce a variety of artificial functions.
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</p>
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<p class="text">
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(Team: Peking, 2016)
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</p>
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<div style="width: 46vw" class="content_pic_left">
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<img class="pictures" id = "Improve_1" src="https://static.igem.org/mediawiki/2018/b/bb/T--SHSBNU_China--Improve_1.jpg"/>
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<p class="pic_text">Fig1.2: SpyTag and SpyCatcher System</p>
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</div>
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</div>
  
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</div>
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<h2 id="VI">II. Verification - Using sfGFP-SpyCatcher</h2>
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<div class="content">
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<p class="text">
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We used sfGFP-SpyCatcher as indicator to test whether the addition of SpyTag peptide could confer the artificial functions to the biofilm, such as covalent immobilization of SpyCatcher-fusion proteins. </p>
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<p class="text">
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<a href="https://2018.igem.org/Team:SHSBNU_China/Protocol#SSS">
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Protocol for SpyTag-SpyCatcher system verification
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</a>
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</p>
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<p class="text">
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We tested our improved part BBa_K2684006 and the previous part BBa_K1583000 with the same protein expression system settings (E. coli BL21, pET28a vector, and the same induction and expression protocol).</p>
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<div style="width: 46vw" class="content_pic_left">
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<img class="pictures" id = "Improve_2" src="https://static.igem.org/mediawiki/2018/9/90/T--SHSBNU_China--Improve_2.png"/>
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<p class="pic_text"></p>
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</div>
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<p class="text">
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The decrease of florescence of the supernatant indicates the immobilization of sfGFP-SpyCatcher, and there’s a significant difference between the two groups.</p>
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</div>
  
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<h2 id="VII">III. Verification - Using SpyCatcher-CotA</h2>
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<div class="content">
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<div style="width: 20vw" class="content_pic_left">
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<img class="pictures" id = "Improve_3" src="https://static.igem.org/mediawiki/2018/7/7c/T--SHSBNU_China--Improve_3.png"/>
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<p class="pic_text"></p>
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</div>
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<p class="text">
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The immobilization of SpyCatcher-CotA fusion protein to the CsgA-SpyTag biofilm was tested, and a commercial laccase activity assay kits was used to measure the laccase activity. </p>
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<p class="text">
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<a href="https://2018.igem.org/Team:SHSBNU_China/Protocol#BXL">
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Protocol for Biofilm x Laccase
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</a>
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</p>
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<p class="text">
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The difference between our improved part and the previous part is significant.  Thus, we proved that SpyCatcher-CotA can be attached to the biofilm containing functional peptide SpyTag. </p>
  
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<br>
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<br>
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</div>
  
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</html>
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<h2 id="C">IV. Conclusion</h2>
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<div class="content">
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<!--
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<p class="text">
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To conclude, we introduce a new ability to the previous CsgA part, which is capturing all fusion protein with SpyCatcher domain, by fusing SpyTag peptide to CsgA coding sequence. We verified the protein immobilization function of our improved part using sfGFP-SpyCatcher and SpyCatcher-CotA, and proved that SpyCatcher fusion protein is still functional after fixed to the biofilm.
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</p>
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<p style="text-indent:0" class="text">
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<strong>Reference:</strong>
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</p>
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<p class="text">
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“Uranium Reaper.” <i>Uranium Reaper - Team: Peking</i>, 2016.igem.org/Team:Peking.
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</p>
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</div>
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</div>
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<div id=footer>
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<div style="display:table-cell;vertical-align:middle;">
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<p style="font-size:1vw;font-family:'Times New Roman';font-style:italic;color:#666666">SHSBNU_China 2018, Created by Azir</p>
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</div>
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</div>
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</div>
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</section>
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</div>
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</body>

Latest revision as of 03:55, 18 October 2018

Improvements

Improve

I. Overview

Fig1.1: CsgA-SpyTag

CsgA is a amyloid protein which is a dominant component of the biofilms of E. coli. We fused a functional peptide domain SpyTag with gene CsgA (previous part: BBa_K1583000), forming CsgA-SpyTag (Improved Part: BBa_K2684006). By utilizing the steady isopeptide bond combination ability of the SpyTag-SpyCatcher system , it is possible to fix different SpyChatcher-fusing proteins, like enzymes or receptors, to the biofilm to introduce a variety of artificial functions.

(Team: Peking, 2016)

Fig1.2: SpyTag and SpyCatcher System

II. Verification - Using sfGFP-SpyCatcher

We used sfGFP-SpyCatcher as indicator to test whether the addition of SpyTag peptide could confer the artificial functions to the biofilm, such as covalent immobilization of SpyCatcher-fusion proteins.

Protocol for SpyTag-SpyCatcher system verification

We tested our improved part BBa_K2684006 and the previous part BBa_K1583000 with the same protein expression system settings (E. coli BL21, pET28a vector, and the same induction and expression protocol).

The decrease of florescence of the supernatant indicates the immobilization of sfGFP-SpyCatcher, and there’s a significant difference between the two groups.

III. Verification - Using SpyCatcher-CotA

The immobilization of SpyCatcher-CotA fusion protein to the CsgA-SpyTag biofilm was tested, and a commercial laccase activity assay kits was used to measure the laccase activity.

Protocol for Biofilm x Laccase

The difference between our improved part and the previous part is significant. Thus, we proved that SpyCatcher-CotA can be attached to the biofilm containing functional peptide SpyTag.



IV. Conclusion

To conclude, we introduce a new ability to the previous CsgA part, which is capturing all fusion protein with SpyCatcher domain, by fusing SpyTag peptide to CsgA coding sequence. We verified the protein immobilization function of our improved part using sfGFP-SpyCatcher and SpyCatcher-CotA, and proved that SpyCatcher fusion protein is still functional after fixed to the biofilm.

Reference:

“Uranium Reaper.” Uranium Reaper - Team: Peking, 2016.igem.org/Team:Peking.