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− | <a class="show-menu" data-toggle="collapse" data-target=".nav-collapse"><span class="show-menu-bar"></span> | + | @media (min-width:1024px){ |
− | </a> | + | #sidebar{position:relative;top:120px;max-width:200px;}} |
− | <div id="logo" style="max-width:170px"><a class="" href="https://2016.igem.org/Team:Peking"></a></div> | + | @media (max-width: 1023px){ |
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| | | |
− | <div class="nav-collapse collapse"> | + | if(scrollTop < height){ obj.style.position = 'relative'; |
− | <ul class="nav">
| + | }else{ |
− | <li class="menu-1"><a class="colapse-menu1" href="https://2016.igem.org/Team:Peking" >Home</a></li>
| + | obj.style.position = 'fixed'; |
− | <li class="dropdown menu-2"><a class="dropdown-toggle" data-toggle="dropdown" href="#" > Achievements</a>
| + | } |
− | <ul class="dropdown-menu">
| + | } |
− | <li><a href="https://2016.igem.org/Team:Peking/Demonstrate" >Results</a></li>
| + | </script> |
− | <li><a href="https://2016.igem.org/Team:Peking/Basic_Part" >Parts</a></li>
| + | |
− | <li><a href="https://2016.igem.org/Team:Peking/Collaborations" >Collaborations</a></li>
| + | |
− | </ul>
| + | |
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| + | |
− | <li class="dropdown menu-3"><a class="dropdown-toggle" data-toggle="dropdown" href="#">Project</a>
| + | |
− | <ul class="dropdown-menu">
| + | |
− | <li><a href="https://2016.igem.org/Team:Peking/Description" >Overview</a></li>
| + | |
− | <li><a href="https://2016.igem.org/Team:Peking/Design" >Design</a></li>
| + | |
− | <li><a href="https://2016.igem.org/Team:Peking/Crosslinking" >Crosslinking</a></li>
| + | |
− | <li><a href="https://2016.igem.org/Team:Peking/Uranyl-adsorption" >Uranyl adsorption</a></li>
| + | |
− | <li><a href="https://2016.igem.org/Team:Peking/Recovery" >Recovery</a></li>
| + | |
− | <li><a href="https://2016.igem.org/Team:Peking/Secretion" >Secretion</a></li>
| + | |
− | <li><a href="https://2016.igem.org/Team:Peking/Proof" >Proof and speculation</a></li>
| + | |
− | </ul>
| + | |
− | </li>
| + | |
− | <li class="dropdown menu-4"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Modeling</a>
| + | |
− | <ul class="dropdown-menu">
| + | |
− | <li><a href="https://2016.igem.org/Team:Peking/Model" >Protein polymerization</a></li>
| + | |
− | <li><a href="https://2016.igem.org/Team:Peking/Software" >Software</a></li>
| + | |
− | </ul>
| + | |
− | </li>
| + | |
− | <li class="dropdown menu-5"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Practices</a>
| + | |
− | <ul class="dropdown-menu">
| + | |
− | <li><a href="https://2016.igem.org/Team:Peking/HP/Gold" >Overview</a></li>
| + | |
− | <li><a href="https://2016.igem.org/Team:Peking/HP/311" >Field research</a></li>
| + | |
− | <li><a href="https://2016.igem.org/Team:Peking/HP/questionnaire" >Questionnaire</a></li>
| + | |
− | <li><a href="https://2016.igem.org/Team:Peking/HP/consulting" >Consulting</a></li>
| + | |
− | <li><a href="https://2016.igem.org/Team:Peking/HP/otherHP" >Other work</a></li>
| + | |
− | </ul>
| + | |
− | </li>
| + | |
− | <li class="menu-6"><a class="colapse-menu1" href="https://2016.igem.org/Team:Peking/Safety" >Safety</a>
| + | |
− | <li class="dropdown menu-7"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Lab</a>
| + | |
− | <ul class="dropdown-menu">
| + | |
− | <li><a class="" href="https://2016.igem.org/Team:Peking/Team" >Team</a></li>
| + | |
− | <li><a class="" href="https://2016.igem.org/Team:Peking/Attributions" >Attribution</a></li>
| + | |
− | <li><a class="" href="https://2016.igem.org/Team:Peking/Notebook" >Notebook</a></li>
| + | |
− | </ul>
| + | |
− | </li>
| + | |
− | <li class="menu-8"><a class="colapse-menu1" href="https://2016.igem.org/Team:Peking/Interlab" >Interlab</a>
| + | |
− | </li>
| + | |
− | </div> | + | |
− | </div> | + | |
− | </div>
| + | |
− | </div>
| + | |
− | <!--/Navigation --> | + | |
| | | |
− | <!-- Page Title======================================================================== --> | + | <script type="text/javascript"> |
− | <div id="page-title"> | + | window.onload = function(){ |
− | <div class="row">
| + | menuFixed('sidebar'); |
− | <div class="twelve columns centered text-center">
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− | <h1>Secretion<span>.</span></h1> | + | </script> |
− | <p class="title1" style="text-align:center"></p> | + | <script> |
− | </div> | + | function naver(id){ |
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| + | return window.scrollTo(0,oPos+250); |
| + | } |
| + | </script> |
| + | <!--sidebar 引用 end ==============================================================================--> |
| + | <!--panel 引用==================================================================================--> |
| + | <style type="text/css"> |
| + | .panel-default .panel-heading a{ |
| + | text-decoration: none; |
| + | display:block; |
| + | padding:10px; |
| + | } |
| + | .panel-heading.panel-title{ |
| + | text-decoration: none; |
| + | padding-top:0px; |
| + | padding-bottom:0px; |
| + | padding-left:0px; |
| + | padding-right:0px; |
| + | text-align:center; |
| + | font-size:19px; |
| + | |
| + | } |
| + | a[aria-expanded="true"] { |
| + | background-color:rgba(70, 73, 76, 0.95); |
| + | text-decoration: none; |
| + | color:white; |
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| | | |
| + | .panel-default .panel-heading a[aria-expanded="false"]{ |
| + | -o-transition: background-color 1s linear; |
| + | -moz-transition: background-color 1s linear; |
| + | -khtml-transition: background-color 1s linear; |
| + | -webkit-transition: background-color 1s linear; |
| + | -ms-transition: background-color 1s linear; |
| + | transition: background-color 1s linear; |
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| + | .panel-default .panel-heading a[aria-expanded="false"]:hover{ |
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| + | .panel-default .panel-heading a[aria-expanded="true"]:hover{ |
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| + | </style> |
| | | |
− | <div id="page-content" class="row page"> | + | <script type="text/javascript"> |
− | <div id="primary" class="twelve columns">
| + | $(document).ready(function(){ |
− | <section> | + | $("#button1").click(function(){ |
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| + | $(".panel-collapse").collapse("hide"); |
| + | }); |
| + | }); |
| + | $("#notebook").addClass("navbar-active"); |
| + | </script> |
| + | <!--panel 引用 end ==================--> |
| + | |
| + | <!-- Navigation --> |
| + | <div id="navigation" class="navbar navbar-fixed-top"> |
| + | <div class="navbar-inner "> |
| + | <div class="container no-padding"> |
| + | <a class="show-menu" data-toggle="collapse" data-target=".nav-collapse"><span class="show-menu-bar"></span> |
| + | </a> |
| + | <div id="logo" style="max-width:170px"><a class="" href="https://2016.igem.org/Team:Peking"></a></div> |
| + | |
| + | <div class="nav-collapse collapse"> |
| + | <ul class="nav"> |
| + | <li class="menu-1"><a class="colapse-menu1" href="https://2016.igem.org/Team:Peking" >Home</a></li> |
| + | <li class="dropdown menu-2"><a class="dropdown-toggle" data-toggle="dropdown" href="#" > Achievements</a> |
| + | <ul class="dropdown-menu"> |
| + | <li><a href="https://2016.igem.org/Team:Peking/Results" >Results</a></li> |
| + | <li><a href="https://2016.igem.org/Team:Peking/Basic_Part" >Parts</a></li> |
| + | <li><a href="https://2016.igem.org/Team:Peking/Collaborations" >Collaborations</a></li> |
| + | </ul> |
| + | </li> |
| + | <li class="dropdown menu-3"><a class="dropdown-toggle" data-toggle="dropdown" href="#">Project</a> |
| + | <ul class="dropdown-menu"> |
| + | <li><a href="https://2016.igem.org/Team:Peking/Description" >Overview</a></li> |
| + | <li><a href="https://2016.igem.org/Team:Peking/Design" >Design</a></li> |
| + | <li><a href="https://2016.igem.org/Team:Peking/Crosslinking" >Crosslinking</a></li> |
| + | <li><a href="https://2016.igem.org/Team:Peking/Uranyl-adsorption" >Uranyl adsorption</a></li> |
| + | <li><a href="https://2016.igem.org/Team:Peking/Clearance" >Clearance</a></li> |
| + | <li><a href="https://2016.igem.org/Team:Peking/Secretion" >Secretion</a></li> |
| + | <li><a href="https://2016.igem.org/Team:Peking/Demonstrate" >Final Performance</a></li> |
| + | </ul> |
| + | </li> |
| + | <li class="dropdown menu-4"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Modeling</a> |
| + | <ul class="dropdown-menu"> |
| + | <li><a href="https://2016.igem.org/Team:Peking/Model/GelPoint" > Model of Gel Point </a></li> |
| + | <li><a href="https://2016.igem.org/Team:Peking/Model/MassDistribution" > Model of Mass Distribution</a></li> |
| + | <li><a href="https://2016.igem.org/Team:Peking/Software" >Software</a></li> |
| + | </ul> |
| + | </li> |
| + | <li class="dropdown menu-5"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Practices</a> |
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| + | <li><a href="https://2016.igem.org/Team:Peking/HP/Gold" >Overview</a></li> |
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| + | <li><a href="https://2016.igem.org/Team:Peking/HP/consulting" >Consulting</a></li> |
| + | <li><a href="https://2016.igem.org/Team:Peking/HP/otherHP" >Education & Other</a></li> |
| + | </ul> |
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| + | <li class="menu-6"><a class="colapse-menu1" href="https://2016.igem.org/Team:Peking/Safety" >Safety</a> |
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| + | <li><a class="" href="https://2016.igem.org/Team:Peking/Team" >Team</a></li> |
| + | <li><a class="" href="https://2016.igem.org/Team:Peking/Attributions" >Attribution</a></li> |
| + | <li><a class="" href="https://2016.igem.org/Team:Peking/Notebook" >Notebook</a></li> |
| + | </ul> |
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| + | <li class="menu-8"><a class="colapse-menu1" href="https://2016.igem.org/Team:Peking/Interlab" >Interlab</a> |
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| + | </div> |
| + | <!--/Navigation --> |
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| + | <!-- Page Title======================================================================== --> |
| + | <div id="page-title"> |
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| + | <h1>Secretion</h1> |
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| + | <p class="title1" style="text-align:center"></p> |
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− | <div class="three columns"> | + | |
− | <div id="page-wrap">
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− | <div id="sidebar" style="color:#000000"> | + | |
− | <h4><a href="javascript:void(0);" onclick="naver('Background')">Background</a></h4>
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− | <ul>
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− | <li><a href="javascript:void(0);" onclick="naver('BE')"><i>E. coli</i></a></li>
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− | <li><a href="javascript:void(0);" onclick="naver('BB')"><i>B. subtilis</i></a></li>
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− | </ul>
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− | <h4><a href="javascript:void(0);" onclick="naver('Design')">Design</a></h4>
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− | <ul>
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− | <li><a href="javascript:void(0);" onclick="naver('Library')">Signal Peptide Library</a></li>
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− | <li><a href="javascript:void(0);" onclick="naver('Assays')">Secretion Assays</a></li>
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− | <li><a href="javascript:void(0);" onclick="naver('Construcion')">Construction</a></li>
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− | </ul>
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− | <h4><a href="javascript:void(0);" onclick="naver('Results')">Results<a></h4>
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− | <ul>
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− | <li><a href="javascript:void(0);" onclick="naver('RE')"><i>E. coli</i></a></li>
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− | <li><a href="javascript:void(0);" onclick="naver('RB')"><i>B. subtilis</i></a></li>
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− | <li><a href="javascript:void(0);" onclick="naver('Quantitative')">Quantitative Measurements</a></li>
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− | </ul>
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− | <h4><a href="javascript:void(0);" onclick="naver('references')">References</a></h4>
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− | </div>
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− | </div>
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| </div> | | </div> |
| + | </div> |
| + | </div><!-- Page Title End--> |
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| + | <div id="page-content" class="row page"> |
| + | <div id="primary" class="twelve columns"> |
| + | <section> |
| + | <div class="row"> |
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| + | <div id="page-wrap"> |
| + | <div id="sidebar" style="color:#000000"> |
| + | <h4><a href="javascript:void(0);" onclick="naver('Background')">Background</a></h4> |
| + | <ul> |
| + | <li><a href="javascript:void(0);" onclick="naver('BE')"><i>E. coli</i></a></li> |
| + | <li><a href="javascript:void(0);" onclick="naver('BB')"><i>B. subtilis</i></a></li> |
| + | </ul> |
| + | <h4><a href="javascript:void(0);" onclick="naver('Design')">Design</a></h4> |
| + | <ul> |
| + | <li><a href="javascript:void(0);" onclick="naver('Library')">Signal Peptide Library</a></li> |
| + | <li><a href="javascript:void(0);" onclick="naver('Assays')">Secretion Assays</a></li> |
| + | <li><a href="javascript:void(0);" onclick="naver('Construcion')">Construction</a></li> |
| + | </ul> |
| + | <h4><a href="javascript:void(0);" onclick="naver('Results')">Results<a></h4> |
| + | <ul> |
| + | <li><a href="javascript:void(0);" onclick="naver('RE')"><i>E. coli</i></a></li> |
| + | <li><a href="javascript:void(0);" onclick="naver('RB')"><i>B. subtilis</i></a></li> |
| + | <li><a href="javascript:void(0);" onclick="naver('Quantitative')">Quantitative Measurements</a></li> |
| + | |
| + | </ul> |
| + | <h4><a href="javascript:void(0);" onclick="naver('references')">References</a></h4> |
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− | <p class="lead add-bottom" style="color:#5E5656">The cell lysis step needed for the extraction of intracellular protein hydrogel can be quite tedious, so we wondered whether we can make the bacteria produce the protein hydrogel autonomously, that is, secrete the recombinant proteins continuously without lysis. To do so, we constructed a signal peptide library and carried out a series of experiments to find an optimal secretion strategy.</p> | + | <p class="lead add-bottom" style="color:#5E5656">The cell lysis step needed for the extraction of intracellular protein polymer network could be quite tedious, so we wondered whether we could make the bacteria produce the protein polymer network autonomously, that is, secrete the recombinant proteins continuously without lysis. To do so, we constructed a signal peptide library and carried out a series of experiments to find an optimal secretion strategy.</p> |
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− | <a id="Background"></a>
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| <div class="texttitle">Background</div> | | <div class="texttitle">Background</div> |
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| <div class="panel panel-default"> | | <div class="panel panel-default"> |
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| <p class="lead add-bottom" style="color:#5E5656"><i>E. coli</i>, the classical workhorse organism of biotechnology, is a mature and versatile host for the production of various proteins. Due to the high obtainable expression levels and the ease of genetic manipulation, <i>E. coli</i> was chosen as the first secretion vector.</p> | | <p class="lead add-bottom" style="color:#5E5656"><i>E. coli</i>, the classical workhorse organism of biotechnology, is a mature and versatile host for the production of various proteins. Due to the high obtainable expression levels and the ease of genetic manipulation, <i>E. coli</i> was chosen as the first secretion vector.</p> |
| + | <p>For <i>E. coli</i>, we chose pET28a with its intrinsic T7 promoter and RBS as the vector backbone.</p> |
| <h3 class="classic-title"> Basic Construction </h3> | | <h3 class="classic-title"> Basic Construction </h3> |
− | <p>For <i>E. coli</i>, we chose pET28a with its intrinsic T7 promoter and RBS as the vector backbone.</p>
| |
| <p>Applying PCR and the Golden Gate Assembly method, we first cloned the proteins of interest into pET28a. Subsequently, we used the resulting expression vectors to transform <i>E. coli</i> TOP10, which is an ideal cloning strain. Following sequence confirmation and vector expansion in TOP10, the vectors were transferred into <i>E. coli</i> BL21 for expression, which promised satisfactory secretion levels.</p> | | <p>Applying PCR and the Golden Gate Assembly method, we first cloned the proteins of interest into pET28a. Subsequently, we used the resulting expression vectors to transform <i>E. coli</i> TOP10, which is an ideal cloning strain. Following sequence confirmation and vector expansion in TOP10, the vectors were transferred into <i>E. coli</i> BL21 for expression, which promised satisfactory secretion levels.</p> |
| <br/> | | <br/> |
| <h3 class="classic-title"> Improved Construction </h3> | | <h3 class="classic-title"> Improved Construction </h3> |
| <p>Although <i>E. coli</i> is desirable for achieving maximal protein production within the cytoplasm, targeting the proteins to extracellular compartments may be difficult in this host, since <i>E. coli</i> has an outer membrane.</p> | | <p>Although <i>E. coli</i> is desirable for achieving maximal protein production within the cytoplasm, targeting the proteins to extracellular compartments may be difficult in this host, since <i>E. coli</i> has an outer membrane.</p> |
− | <p>To avoid this obstacle and increase the secretion efficiency, we adopted a gene named kil. Kil is a bacteriocin release protein (BRP) which leads to a permeabilization of the outer membrane and can thus be used for the secretion of proteins of interest into the culture medium<sup>1</sup>.</p> | + | <p>To avoid this obstacle and increase the secretion efficiency, we adopted a gene named kil. Kil is a bacteriocin release protein (BRP) which leads to a permeabilization of the outer membrane and could thus be used for the secretion of proteins of interest into the culture medium<sup>1</sup>.</p> |
| <p>However, high-level induction of kil results in cell lysis and death. To solve this problem, we selected a medium-strength promoter, J23117, from the iGEM PARTS library and fused it to the 5’ end of the kil coding sequence. We also included a lac operator between J23117 and kil, which resulted in a simple inducible synthetic kil expression gene. Consequently, kil was only expressed in the presence of IPTG, which ensured the survival of the bacteria under normal conditions.</p> | | <p>However, high-level induction of kil results in cell lysis and death. To solve this problem, we selected a medium-strength promoter, J23117, from the iGEM PARTS library and fused it to the 5’ end of the kil coding sequence. We also included a lac operator between J23117 and kil, which resulted in a simple inducible synthetic kil expression gene. Consequently, kil was only expressed in the presence of IPTG, which ensured the survival of the bacteria under normal conditions.</p> |
| <figure> | | <figure> |
− | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/c/c8/T--Peking--images_secretion_fig1.png" style="width:100%;" alt=""/> | + | <p style="text-align:center;"><img style="width:70% ;" src="https://static.igem.org/mediawiki/2016/7/76/Fig1_CXY_SECRETION.png" alt=""/></p> |
− | <figcaption>Fig. 1. Schematic representation of a simple inducible kil expression cassette. | + | <figcaption style="text-align:center;"> |
| + | Fig. 1. Schematic representation of a simple inducible kil expression cassette. |
| </figcaption> | | </figcaption> |
| </figure> | | </figure> |
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| <p class="lead add-bottom" style="color:#5E5656"><i>B. subtilis</i> is a Gram-positive bacterium and as such does not have an outer membrane, which enables it to secrete proteins directly into the medium. As a result, <i>B. subtilis</i> may offer higher product yields for secreted proteins, which makes it an attractive alternative to <i>E. coli</i>.</p> | | <p class="lead add-bottom" style="color:#5E5656"><i>B. subtilis</i> is a Gram-positive bacterium and as such does not have an outer membrane, which enables it to secrete proteins directly into the medium. As a result, <i>B. subtilis</i> may offer higher product yields for secreted proteins, which makes it an attractive alternative to <i>E. coli</i>.</p> |
| <h3 class="classic-title"> Basic Construction </h3> | | <h3 class="classic-title"> Basic Construction </h3> |
− | <p>We chose the shuttle plasmid pBES as vector backbone. PBES can be manipulated easily and multiplies quickly in <i>E. coli</i>, so we conducted all molecular cloning procedures in <i>E. coli</i>. Once successfully constructed, the plasmids were extracted and used to transform <i>B. subtilis</i> for subsequent expression.</p> | + | <p>We chose the shuttle plasmid pBES as vector backbone. PBES could be manipulated easily and multiplies quickly in <i>E. coli</i>, so we conducted all molecular cloning procedures in <i>E. coli</i>. Once successfully constructed, the plasmids were extracted and used to transform <i>B. subtilis</i> for subsequent expression.</p> |
| <h3 class="classic-title"> Improved Construction </h3> | | <h3 class="classic-title"> Improved Construction </h3> |
| <p>To increase the expression and secretion levels, we examined two promoters - PaprE and P43. These two promoters are widely used for heterologous protein production in <i>B. subtilis</i><sup>2</sup>. By fusing either PaprE or P43 to the coding sequence, we hoped that <i>B. subtilis</i> would achieve a high secretion efficiency.</p> | | <p>To increase the expression and secretion levels, we examined two promoters - PaprE and P43. These two promoters are widely used for heterologous protein production in <i>B. subtilis</i><sup>2</sup>. By fusing either PaprE or P43 to the coding sequence, we hoped that <i>B. subtilis</i> would achieve a high secretion efficiency.</p> |
| <figure> | | <figure> |
− | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/a/af/T--Peking--images_secretion_fig2.png" style="width:100%;" alt=""/> | + | <p style="text-align:center;"><img style="width:70% ;" src="https://static.igem.org/mediawiki/2016/c/c9/Fig2_CXY_SECRETION.png" alt=""/></p> |
− | <figcaption> | + | <figcaption style="text-align:center;"> |
− | Fig. 2. Schematic representation of expression cassettes used in <i>B. subtilis</i> | + | Fig. 2. Schematic representation of expression cassettes used in <i>B. subtilis</i> |
| </figcaption> | | </figcaption> |
| </figure> | | </figure> |
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| <div class="texttitle anchor" >Design</div> | | <div class="texttitle anchor" >Design</div> |
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| <div class="panel panel-default"> | | <div class="panel panel-default"> |
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| <p>Based on previous studies on their secretion performance, 4 different signal peptides, derived from LTIIb, PhoA, PelB and OmpA, were chosen for <i>E. coli</i> and 7 signal peptides, derived from Epr, Bpr, LipA, YjfA, SacB, NprE and ImdA, were chosen for <i>B. subtilis</i><sup>3</sup>.</p> | | <p>Based on previous studies on their secretion performance, 4 different signal peptides, derived from LTIIb, PhoA, PelB and OmpA, were chosen for <i>E. coli</i> and 7 signal peptides, derived from Epr, Bpr, LipA, YjfA, SacB, NprE and ImdA, were chosen for <i>B. subtilis</i><sup>3</sup>.</p> |
− | <p>To test the overall system, we first chose OmpA and ImdA, from <i>E. coli</i> and <i>B. subtilis</i> respectively, to determine whether well-established signal peptides can guide the target proteins out of the cytoplasm. This was done in order to exclude that emergent features of the complex protein folding of our multi-module monomers somehow generally inhibited secretion. Applying the Golden Gate Assembly method, we cloned these two signal peptides into the pSB1C3 vector backbone, amplified the resulting construct by PCR, and constructed the corresponding expression vectors by ligating the signal peptides along with the other expression cassette elements into either pET28a or pBES.</p> | + | <p>To test the overall system, we first chose OmpA and ImdA, from <i>E. coli</i> and <i>B. subtilis</i> respectively, to determine whether well-established signal peptides could guide the target proteins out of the cytoplasm. This was done in order to exclude that emergent features of the complex protein folding of our multi-module monomers somehow generally inhibited secretion. Applying the Golden Gate Assembly method, we cloned these two signal peptides into the pSB1C3 vector backbone, amplified the resulting construct by PCR, and constructed the corresponding expression vectors by ligating the signal peptides along with the other expression cassette elements into either pET28a or pBES.</p> |
− | <p>After we had succeeded in detecting the target proteins in culture supernatants, we kept on constructing vectors with other signal peptides. To accomplish this task, we first amplified the backbones with the signal peptides we had already constructed, and subsequently combined them with other signal peptides (Fig. 3).</p> | + | <p>After we had succeeded in detecting the target proteins in culture supernatants, we kept on constructing vectors with other signal peptides. To accomplish this task, we first amplified the backbones with the signal peptides we had already constructed, and subsequently combined them with other signal peptides (Fig. 3.).</p> |
| + | <p>Note that essentially all constructs contained modules such as 3A, His-tag and FlAsH-tag, we omitted them in the name of these constructs. From example, the construct OmpA-Histag-3A-SUP-FlAsHtag was simplified as OmpA-SUP. The same is true for other constructs.</p> |
| <figure> | | <figure> |
| <img class="featurette-image" src=" | | <img class="featurette-image" src=" |
− | https://static.igem.org/mediawiki/2016/0/01/T--Peking--images_secretion_fig3.png | + | https://static.igem.org/mediawiki/2016/c/ce/Fig3_CXY_SECRETION.png |
− | " style="width:100%;" alt=""/> | + | " style="width:100%;" alt=""/> |
| <figcaption> | | <figcaption> |
− | Fig. 3. Schematic diagram of the design and construction procedure used for secretion vectors | + | Fig. 3. Schematic diagram of the design and construction procedure used for secretion vectors |
| </figcaption> | | </figcaption> |
| </figure> | | </figure> |
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− | <a id="Assays"></a>
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| <div class="panel-body"> | | <div class="panel-body"> |
− | <p class="lead add-bottom" style="color:#5E5656">Each of the proteins of interest has its specific biochemical properties, such as SUP’s affinity to uranyl ions or SpyTag’s irreversible binding to SpyCatcher. Unfortunately, none of these intrinsic properties could be used to directly assay for protein secretion. Although SDS-PAGE can be used as a universal method to separate proteins with different molecular weights, it is not specific enough to verify the results of secretion experiments. Thus, in order to accurately and efficiently assay for protein secretion, we modified the proteins of interest with two effective molecular labels — a His-tag and a FlAsH-tag.</p> | + | <p class="lead add-bottom" style="color:#5E5656">Each of the proteins of interest has its specific biochemical properties, such as SUP’s affinity to uranyl ions or SpyTag’s irreversible binding to SpyCatcher. Unfortunately, none of these intrinsic properties could be used to directly assay for protein secretion. Although SDS-PAGE could be used as a universal method to separate proteins with different molecular weights, it is not specific enough to verify the results of secretion experiments. Thus, in order to accurately and efficiently assay for protein secretion, we modified the proteins of interest with two effective molecular labels — a His-tag and a FlAsH-tag.</p> |
| <h3 class="classic-title"> His-tag </h3> | | <h3 class="classic-title"> His-tag </h3> |
| <p>The His-tag is an artificial amino acid motif that contains at least six histidine residues and is widely used for recombinant protein purification because of its affinity for resin-immobilized bivalent nickel or cobalt ions<sup>3</sup>.</p> | | <p>The His-tag is an artificial amino acid motif that contains at least six histidine residues and is widely used for recombinant protein purification because of its affinity for resin-immobilized bivalent nickel or cobalt ions<sup>3</sup>.</p> |
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| <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/d/d8/T--Peking--images_secretion_fig4.png" style="width:100%;" alt=""/> | | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/d/d8/T--Peking--images_secretion_fig4.png" style="width:100%;" alt=""/> |
| <figcaption> | | <figcaption> |
− | Fig. 4. Schematic representation of the FlAsH-based genetic screen for protein secretion<sup>4</sup>. | + | Fig. 4. Schematic representation of the FlAsH-based genetic screen for protein secretion<sup>4</sup>. |
| </figcaption> | | </figcaption> |
| </figure> | | </figure> |
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− | <a id="Construcion"></a>
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| <div class="panel-body"> | | <div class="panel-body"> |
− | <p>We added the His-tag sequence between the signal peptide and the coding sequence of the protein of interest, so that it would be exposed at the N-terminus of the secreted protein after the signal peptide was removed during the Sec-pathway secretion process. A His-tag-reactive antibody was commercially available, enabling us to conduct further assays using western-blot analysis.</p> | + | <p class="lead add-bottom" style="color:#5E5656">We added the His-tag sequence between the signal peptide and the coding sequence of the protein of interest, so that it would be exposed at the N-terminus of the secreted protein after the signal peptide was removed during the Sec-pathway secretion process. A His-tag-reactive antibody was commercially available, enabling us to conduct further assays using western-blot analysis.</p> |
− | <p>We additionally fused the FlAsH-tag to the C-terminus of the protein of interest. Because the outer membrane of <i>E. coli</i> is naturally impermeable to FlAsH-EDT2<sup>4</sup>, undesired off-target labeling of intracellular proteins is avoided, rendering the assay highly selective for secreted proteins.</p> | + | <p class="lead add-bottom" style="color:#5E5656">We additionally fused the FlAsH-tag to the C-terminus of the protein of interest. Because the outer membrane of <i>E. coli</i> is naturally impermeable to FlAsH-EDT2<sup>4</sup>, undesired off-target labeling of intracellular proteins is avoided, rendering the assay highly selective for secreted proteins.</p> |
| + | |
| <figure> | | <figure> |
− | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/2/27/T--Peking--images_secretion_fig6.png" style="width:100%;" alt=""/> | + | <p style="text-align:center;"><img style="width:82% ;" src="https://static.igem.org/mediawiki/2016/8/83/Fig5_CXY_SECRETION.png" alt=""/></p> |
− | <figcaption> | + | <figcaption style="text-align:center;"> |
− | Fig. 5. Schematic representation of the final construct used for the assay for protein secretion | + | Fig. 5. Schematic representation of the final construct used for the assay for protein secretion |
| </figcaption> | | </figcaption> |
| </figure> | | </figure> |
− | <p>A schematic illustrating all the components used in the library design is shown in Fig. 6.</p> | + | <p>A scheme illustrating all the components used in the library design is shown in Fig. 6.</p> |
| <figure> | | <figure> |
− | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/4/47/T--Peking--images_secretion_fig6new.png" style="width:100%;" alt=""/> | + | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/0/07/Fig6_CXY_SECRETION.png" style="width:100%;" alt=""/> |
| <figcaption> | | <figcaption> |
− | Fig. 6 Blowup schematic of all the elements used in the combinatorial approach for the construction of protein secretion signal libraries. | + | Fig. 6. Blowup schematic of all the elements used in the combinatorial approach for the construction of protein secretion signal libraries. |
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− | <p>We expressed the recombinant proteins in <i>E. coli</i> strain BL21. The bacteria were cultured in M9 minimum medium for 14 hours. We subsequently separated the cytoplasmic and periplasmic components from the culture supernatants and examined them for the presence of recombinant proteins using western blot analysis. An overview of the results is shown in Table.1.</p> | + | <p>We expressed the recombinant proteins in <i>E. coli</i> strain BL21. The bacteria were cultured in M9 minimum medium for 14 hours. We subsequently separated the cytoplasmic and periplasmic components from the culture supernatants and examined them for the presence of recombinant proteins using western blot analysis. An overview of the results is shown in Table. 1. </p> |
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− | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/d/d4/T--Peking--images_secretion_table1.png" style="width:100%;" alt=""/> | + | <p style="text-align:center;"><img style="width:80% ;" src="https://static.igem.org/mediawiki/2016/d/d4/T--Peking--images_secretion_table1.png" alt=""/></p> |
− | <figcaption> | + | <figcaption style="text-align:center;"> |
− | Table. 1. Secretion efficiency of recombinant proteins with different signal peptides | + | Table. 1. Secretion efficiency of recombinant proteins with different signal peptides |
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− | <img class="featurette-image" src="" style="width:100%;" alt=""/> | + | <p style="text-align:center;"><img style="width:80% ;" src="https://static.igem.org/mediawiki/2016/f/fb/Secretion_results_3_cxy_-_%E5%89%AF%E6%9C%AC.jpg" alt=""/></p> |
− | <figcaption> | + | <figcaption style="text-align:center;"> |
− | Fig. 7. Western blot analysis of protein secretion of constructs with different signal peptides. A, B, C, D and E show the results for 3B, 3A-SUP, 3A-mSA, 3A-SUP and 3A-mRFP, respectively. The control groups contain the expression constructs without signal peptides. M: Medium (culture supernatant); C: Cytoplasmic components; P: Periplasmic components; IC: Insoluble cytoplasmic components; SC: Soluble cytoplasmic components.
| + | Fig.7. Western blot results of recombinant proteins (A)Secretion of spycatcher(3B). (B)(D)Secretion of 3A-SUP. (C)Secretion of 3A-mSA. (D)Secretion of 3A-mRFP. Control groups are proteins without signal peptides. M: Medium components C: Cytoplasmic components P: Periplasmic components IC: Insoluble cytoplasmic components SC: Soluble cytoplasmic components. |
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− | <p>We intended to quantitatively measure the secretion of recombinant proteins using the FlAsH-tetracysteine assay. The FlAsH-EDT2 molecule only shows strong fluorescence when bound to a tetracysteine motif, so that the intensity of fluorescence in the samples can be expected to be directly proportional to the amount of recombinant protein bearing a tetracysteine-tag.</p> | + | <p>We intended to quantitatively measure the secretion of recombinant proteins using the FlAsH-tetracysteine assay. The FlAsH-EDT2 molecule only shows strong fluorescence when bound to a tetracysteine motif, so that the intensity of fluorescence in the samples could be expected to be directly proportional to the amount of recombinant protein bearing a tetracysteine-tag.</p> |
− | <p>Therefore, we purified 3A-SUP tagged with the tetracysteine motif, and directly used it to calibrate the fluorescence intensity of the FlAsH-EDT2 probe based on target protein concentration. The fluorescence intensity did change with the magnitude of protein concentration, as expected. However, we did not find a good linear relationship between the target protein concentration and probe fluorescence (Table 4). Hence, only qualitative results could be obtained using this assay.</p> | + | <p>Therefore, we purified 3A-SUP tagged with the tetracysteine motif, and directly used it to calibrate the fluorescence intensity of the FlAsH-EDT2 probe based on target protein concentration. The fluorescence intensity did change with the magnitude of protein concentration, as expected. However, we did not find a good linear relationship between the target protein concentration and probe fluorescence (Table. 2.). Hence, only qualitative results could be obtained using this assay.</p> |
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− | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/9/95/T--Peking--images_secretion_table2.png" style="width:100%;" alt=""/> | + | <img class="featurette-image" src="" style="width:100%;" alt=""/> |
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− | Table 4. Fluorescence data of the FlAsH-tetracysteine assay | + | |
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| + | <p style="text-align:center;"><img style="width:90% ;" src="https://static.igem.org/mediawiki/2016/9/95/T--Peking--images_secretion_table2.png" alt=""/></p> |
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| + | Table. 2. Fluorescence data of the FlAsH-tetracysteine assay |
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− | <h3>References:</h3>
| + | <h3>References:</h3> |
− | <p>[1] Usama, B. et al., Increasing the secretion ability of the kil gene for recombinant proteins in Escherichia coli by using a strong stationary-phase promoter. Biotechnical Letter 29, 1893–1901 (2007).</p>
| + | <p>[1] Usama, B. et al., Increasing the secretion ability of the kil gene for recombinant proteins in Escherichia coli by using a strong stationary-phase promoter. <i>Biotechnical Letter</i> ,<b>29</b>, 1893–1901 (2007).</p> |
− | <p>[2] Kim et al., Comparison of PaprE , PamyE and P43 promoter strength for β-galactosidase and staphylokinase expression in Bacillus subtilis. Biotechnology and bioprocess engineering 13, 313-318 (2008).</p>
| + | <p>[2] Kim et al., Comparison of PaprE , PamyE and P43 promoter strength for β-galactosidase and staphylokinase expression in Bacillus subtilis. <i>Biotechnology and bioprocess engineering 13</i>,<b> 313-318</b> (2008).</p> |
− | <p>[3] Hengen, P.N., Purification of His-Tag fusion proteins from Escherichia coli. Trends in Biochemical Sciences 20(7), 285-286 (1995).</p>
| + | <p>[3] Hengen, P.N., Purification of His-Tag fusion proteins from Escherichia coli.<i> Trends in Biochemical Sciences</i>,<b> 20(7)</b>, 285-286 (1995).</p> |
− | <p>[4] Haitjema, C.H., et al., Universal Genetic Assay for Engineering Extracellular Protein Expression. ACS Synthetic Biology 3(2), 74-82 (2014).</p>
| + | <p>[4] Haitjema, C.H., et al., Universal Genetic Assay for Engineering Extracellular Protein Expression. <i>ACS Synthetic Biology</i>, <b>3(2), 74-82</b> (2014).</p> |
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