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− | <div class="navbar-inner ">
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− | <div class="container no-padding"> | + | var obj = document.getElementById(id); |
− | <a class="show-menu" data-toggle="collapse" data-target=".nav-collapse"><span class="show-menu-bar"></span> | + | var _getHeight = obj.offsetTop; |
− | </a> | + | |
− | <div id="logo" style="max-width:170px"><a class="" href="https://2016.igem.org/Team:Peking"></a></div> | + | window.onscroll = function(){ |
| + | changePos(id,_getHeight); |
| + | } |
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| + | function changePos(id,height){ |
| + | var obj = document.getElementById(id);var windowBottom = $(window).scrollTop() + $(window).innerHeight(); |
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− | <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>
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| + | } |
− | <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>
| + | <script type="text/javascript"> |
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| + | </script> |
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− | <li><a href="https://2016.igem.org/Team:Peking/Clearance" >Clearance</a></li>
| + | return window.scrollTo(0,oPos+250); |
− | <li><a href="https://2016.igem.org/Team:Peking/Secretion" >Secretion</a></li>
| + | } |
− | <li><a href="https://2016.igem.org/Team:Peking/Proof" >Final Performance</a></li>
| + | </script> |
− | </ul>
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− | </li>
| + | <!--panel 引用==================================================================================--> |
− | <li class="dropdown menu-4"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Modeling</a>
| + | <style type="text/css"> |
− | <ul class="dropdown-menu">
| + | .panel-default .panel-heading a{ |
− | <li><a href="https://2016.igem.org/Team:Peking/Model" >Protein polymerization</a></li>
| + | text-decoration: none; |
− | <li><a href="https://2016.igem.org/Team:Peking/Software" >Software</a></li>
| + | display:block; |
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| + | padding:10px; |
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| + | } |
− | <li class="dropdown menu-5"><a class="dropdown-toggle" data-toggle="dropdown" href="#" >Practices</a>
| + | .panel-heading.panel-title{ |
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| + | text-decoration: none; |
− | <li><a href="https://2016.igem.org/Team:Peking/HP/Gold" >Overview</a></li>
| + | padding-top:0px; |
− | <li><a href="https://2016.igem.org/Team:Peking/HP/311" >Field research</a></li>
| + | padding-bottom:0px; |
− | <li><a href="https://2016.igem.org/Team:Peking/HP/questionnaire" >Questionnaire</a></li>
| + | padding-left:0px; |
− | <li><a href="https://2016.igem.org/Team:Peking/HP/consulting" >Consulting</a></li>
| + | padding-right:0px; |
− | <li><a href="https://2016.igem.org/Team:Peking/HP/otherHP" >Other work</a></li>
| + | text-align:center; |
− | </ul>
| + | font-size:19px; |
− | </li>
| + | |
− | <li class="menu-6"><a class="colapse-menu1" href="https://2016.igem.org/Team:Peking/Safety" >Safety</a>
| + | } |
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| + | a[aria-expanded="true"] { |
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− | <li><a class="" href="https://2016.igem.org/Team:Peking/Team" >Team</a></li>
| + | text-decoration: none; |
− | <li><a class="" href="https://2016.igem.org/Team:Peking/Attributions" >Attribution</a></li>
| + | color:white; |
− | <li><a class="" href="https://2016.igem.org/Team:Peking/Notebook" >Notebook</a></li>
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| + | |
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| + | transition: background-color 1s linear; |
| + | } |
| + | .panel-default .panel-heading a[aria-expanded="false"]:hover{ |
| + | background-color:rgba(70, 73, 76, 0.95); |
| + | text-decoration: none; |
| + | color:white; |
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| + | -o-transition: opacity 1s linear; |
| + | -moz-transition: opacity 1s linear; |
| + | -khtml-transition: opacity 1s linear; |
| + | -webkit-transition: opacity 1s linear; |
| + | -ms-transition: opacity 1s linear; |
| + | transition: opacity 0.7s linear; |
| + | } |
| + | .panel-default .panel-heading a[aria-expanded="true"]:hover{ |
| + | opacity:0.7; |
| + | } |
| + | </style> |
| + | |
| + | <script type="text/javascript"> |
| + | $(document).ready(function(){ |
| + | $("#button1").click(function(){ |
| + | $(".panel-collapse").collapse("show"); |
| + | }); |
| + | }); |
| + | $(document).ready(function(){ |
| + | $("#button2").click(function(){ |
| + | $(".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/Demonstrate" >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/Proof" >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" >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> |
| </div> | | </div> |
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− | <a id="Background"></a><div>
| + | <a id="Background"></a><div> |
− | <div class="texttitle">Background</div>
| + | <div class="texttitle">Background</div> |
− | <br/><a id="Necessity"></a>
| + | <br/><a id="Necessity"></a> |
− | <h3 class="classic-title"><span>The Necessity of Crosslinking</span> </h3> <p>Compared to monomers, polymers formed by covalent crosslinking possess some special properties. For example, polymers typically offer greater mechanical strength and a larger contact area. They are often times also easier to handle. We thus considered crosslinking the monomers to form polymers, and take the latter as the basic functional units.</p>
| + | <h3 class="classic-title"><span>The Necessity of Crosslinking</span> </h3> <p>Compared to monomers, polymers formed by covalent crosslinking possess some special properties. For example, polymers typically offer greater mechanical strength and a larger contact area. They are often times also easier to handle. We thus considered crosslinking the monomers to form polymers, and take the latter as the basic functional units.</p> |
− | <br/> <a id="Characteristics"></a>
| + | <br/> <a id="Characteristics"></a> |
− | <h3 class="classic-title"><span>Characteristics</span> </h3> <p>Firstly, the monomers need to be able to crosslink under complex environmental conditions and show high preference for the reaction with each other. Faster and more selective reactions would be highly helpful for practical applications.</p>
| + | <h3 class="classic-title"><span>Characteristics</span> </h3> <p>Firstly, the monomers need to be able to crosslink under complex environmental conditions and show high preference for the reaction with each other. Faster and more selective reactions would be highly helpful for practical applications.</p> |
| <p>Secondly, since functional proteins are to be fused to the monomers to achieve the final goals, their orthogonality must be considered. If there are no covalent interactions between monomer backbones and the attached functional elements, the target proteins could be used to functionalize a polymerized hydrogel, thus adequately taking advantage of the superiority of polymers.</p> | | <p>Secondly, since functional proteins are to be fused to the monomers to achieve the final goals, their orthogonality must be considered. If there are no covalent interactions between monomer backbones and the attached functional elements, the target proteins could be used to functionalize a polymerized hydrogel, thus adequately taking advantage of the superiority of polymers.</p> |
| <p>What’s more, polymers are comparatively easy to recover and could thus be eco-friendly. Since the aim is to construct a brand new material, it is extremely important to consider its potential effects on the environment from the onset of the project.</p> | | <p>What’s more, polymers are comparatively easy to recover and could thus be eco-friendly. Since the aim is to construct a brand new material, it is extremely important to consider its potential effects on the environment from the onset of the project.</p> |
| | | |
− | <br/><a id="SpyTag-SpyCatcher"></a>
| + | <br/><a id="SpyTag-SpyCatcher"></a> |
− | <h3 class="classic-title" style="font-size:20px;"><span>Implementation of Crosslinking - the SpyTag-SpyCatcher System</span> </h3>
| + | <h3 class="classic-title" style="font-size:20px;"><span>Implementation of Crosslinking - the SpyTag-SpyCatcher System</span> </h3> |
| <p>Inspired by the autocatalytic formation of the isopeptide bond between a specific Lys and an Asp residue in Streptococcus pyogenes (Spy) fibronectin-binding protein FbaB, researchers split its autocatalytic domain, CnaB2, and obtained two peptides which they named SpyTag and SpyCatcher. The resulting peptides are able to form isopeptide bonds with each other spontaneously. SpyTag, SpyCatcher and their stable cross-linking products provided us with an ideal tool to manipulate the protein hydrogel<sup>1</sup>.</p> | | <p>Inspired by the autocatalytic formation of the isopeptide bond between a specific Lys and an Asp residue in Streptococcus pyogenes (Spy) fibronectin-binding protein FbaB, researchers split its autocatalytic domain, CnaB2, and obtained two peptides which they named SpyTag and SpyCatcher. The resulting peptides are able to form isopeptide bonds with each other spontaneously. SpyTag, SpyCatcher and their stable cross-linking products provided us with an ideal tool to manipulate the protein hydrogel<sup>1</sup>.</p> |
| <figure> | | <figure> |
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− | </div>
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− |
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− | <br/><a id="Network"></a><div>
| + | |
− | <h3 class="classic-title" style="font-size:20px;"><span>Leap Before the Practical Application - the Spy Crosslinking Network</span> </h3>
| + | |
− | <p>The SpyTag / SpyCatcher System could be applied in the field via polymerization to obtain the “Spy Crosslinking Network”. The functional domains provided by SpyTag and SpyCatcher were linked via an elastin like protein (ELP), yielding the new fusion protein monomers able to undergo three-dimensional polymerization (TDP). This kind of polymerization could yield hyper-branched products and hydrogel at low concentrations, and gels with a high degree of cross-linkage at high concentrations<sup>2,3</sup>.</p>
| + | |
− | <figure>
| + | |
− | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/9/9e/T--Peking--images_crosslinking_fig2.png" style="width:100%;" alt=""/>
| + | |
− | <figcaption>
| + | |
− | Fig. 2. The Spy Crosslinking Network. (A) Monomers containing multiple functional groups, SpyTag or SpyCatcher, react with each other both in situ and in vitro. (B) Schematic illustration of the products formed by mixing protein precursors 3A and 2B. Their mixing at high concentration (w.t. >=7.5%) leads to the formation of a covalently cross-linked gel.
| + | |
− | </figcaption>
| + | |
− | </figure>
| + | |
− | </div>
| + | |
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| | | |
− | <br/><br/><br/>
| + | <br/><a id="Network"></a><div> |
− | <a id="Design"></a><div>
| + | <h3 class="classic-title" style="font-size:20px;"><span>Leap Before the Practical Application - the Spy Crosslinking Network</span> </h3> |
− | <div class="texttitle">Design</div>
| + | <p>The SpyTag / SpyCatcher System could be applied in the field via polymerization to obtain the “Spy Crosslinking Network”. The functional domains provided by SpyTag and SpyCatcher were linked via an elastin like protein (ELP), yielding the new fusion protein monomers able to undergo three-dimensional polymerization (TDP). This kind of polymerization could yield hyper-branched products and hydrogel at low concentrations, and gels with a high degree of cross-linkage at high concentrations<sup>2,3</sup>.</p> |
− | <br/><a id="Theoretical"></a>
| + | <figure> |
− | <h3 class="classic-title" style="font-size:20px;"><span>Theoretical Deduction - Determining the optimal n count for nA-FP</span></h3>
| + | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/9/9e/T--Peking--images_crosslinking_fig2.png" style="width:100%;" alt=""/> |
− | <p>We first have to find out the optimal n count of nA-FP (functional peptides), which includes both SpyTag modules and functional proteins, and will react with another monomer, such as 3B.</p>
| + | <figcaption> |
− | <p>In order to acquire hyper-branched polymers and promote the strength and contact area of the resulting hydrogel, we decided to abandon 1A-FP and 2A-FP monomers, and instead decided to increase the weight proportion of functional proteins in the monomer. We thus chose 3A-SUP(n>=3). Although 4A-SUP and 6A-SUP performed better than 3A-SUP in crosslinking since it has more SpyTag groups available to react with 3B, other experiments showed that 4/6A-SUP performed poorer than 3A-SUP in uranyl adsorption at the same concentration (see the Uranyl adsorption part). Following the analysis presented here, we defined the optimal n count as 3.</p>
| + | Fig. 2. The Spy Crosslinking Network. (A) Monomers containing multiple functional groups, SpyTag or SpyCatcher, react with each other both in situ and in vitro. (B) Schematic illustration of the products formed by mixing protein precursors 3A and 2B. Their mixing at high concentration (w.t. >=7.5%) leads to the formation of a covalently cross-linked gel. |
− | <figure>
| + | </figcaption> |
− | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/b/b7/T--Peking--images_crosslinking_fig3.png" style="width:100%;" alt=""/>
| + | </figure> |
− | <figcaption>
| + | </div> |
− | Fig. 3. Schematic diagram of triple SpyTag-SUP (3A-SUP) and triple SpyCatcher (3B).
| + | |
− | </figcaption>
| + | |
− | </figure>
| + | <br/><br/><br/> |
| + | <a id="Design"></a><div> |
| + | <div class="texttitle">Design</div> |
| + | <br/><a id="Theoretical"></a> |
| + | <h3 class="classic-title" style="font-size:20px;"><span>Theoretical Deduction - Determining the optimal n count for nA-FP</span></h3> |
| + | <p>We first have to find out the optimal n count of nA-FP (functional peptides), which includes both SpyTag modules and functional proteins, and will react with another monomer, such as 3B.</p> |
| + | <p>In order to acquire hyper-branched polymers and promote the strength and contact area of the resulting hydrogel, we decided to abandon 1A-FP and 2A-FP monomers, and instead decided to increase the weight proportion of functional proteins in the monomer. We thus chose 3A-SUP(n>=3). Although 4A-SUP and 6A-SUP performed better than 3A-SUP in crosslinking since it has more SpyTag groups available to react with 3B, other experiments showed that 4/6A-SUP performed poorer than 3A-SUP in uranyl adsorption at the same concentration (see the Uranyl adsorption part). Following the analysis presented here, we defined the optimal n count as 3.</p> |
| + | <figure> |
| + | <p style="text-align:center;"><img style="width: 60 %;" src=" https://static.igem.org/mediawiki/2016/b/b7/T--Peking--images_crosslinking_fig3.png" alt=""/></p> |
| + | <figcaption> |
| + | Fig. 3. Schematic diagram of triple SpyTag-SUP (3A-SUP) and triple SpyCatcher (3B). |
| + | </figcaption> |
| + | </figure> |
| | | |
− | <br/><a id="Scaffold"></a>
| + | |
| + | <br/><a id="Scaffold"></a> |
| <h3 class="classic-title"><span>Setting up the Scaffold - Construction of the Monomers</span></h3> | | <h3 class="classic-title"><span>Setting up the Scaffold - Construction of the Monomers</span></h3> |
| <p>In order to use the SpyTag-SpyCatcher system as scaffold, we fused three SpyTag modules (A) spaced by (VPGVG)4, and combined them with an N-terminal 6xHis tag and another functional protein called the Super Uranyl-binding Protein(SUP) at the C-terminus. We also fused three SpyCatcher modules (B) spaced by (VPGVG)15, with an N-terminal 6xHis tag.</p> | | <p>In order to use the SpyTag-SpyCatcher system as scaffold, we fused three SpyTag modules (A) spaced by (VPGVG)4, and combined them with an N-terminal 6xHis tag and another functional protein called the Super Uranyl-binding Protein(SUP) at the C-terminus. We also fused three SpyCatcher modules (B) spaced by (VPGVG)15, with an N-terminal 6xHis tag.</p> |
| <p>We successfully assembled the constructs SpyTag-SpyTag-SpyTag-SUP (3A-SUP), SpyTag-SpyTag-SpyTag-mSA (3A-mSA), SpyCatcher-SpyCatcher-SpyCatcher (3B), together with a number of similar fusion proteins. Since these proteins were easy to obtain either by lysing the bacteria or by bacterial secretion (See the Secretion section for further information), we were able to build a brand new biomaterial that displays multi-functionality at quite a low cost.</p> | | <p>We successfully assembled the constructs SpyTag-SpyTag-SpyTag-SUP (3A-SUP), SpyTag-SpyTag-SpyTag-mSA (3A-mSA), SpyCatcher-SpyCatcher-SpyCatcher (3B), together with a number of similar fusion proteins. Since these proteins were easy to obtain either by lysing the bacteria or by bacterial secretion (See the Secretion section for further information), we were able to build a brand new biomaterial that displays multi-functionality at quite a low cost.</p> |
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− | <a id="Methods"></a>
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| <div class="texttitle">Methods</div> | | <div class="texttitle">Methods</div> |
| <p class="lead add-bottom" style="color:#5E5656">☆☆☆ To better elucidate the properties of the monomers, the concentration was kept at 1mg/mL, unless stated otherwise. This concentration is only conductive to oligomerization and did not induce hydrogel formation. ☆☆☆</p> | | <p class="lead add-bottom" style="color:#5E5656">☆☆☆ To better elucidate the properties of the monomers, the concentration was kept at 1mg/mL, unless stated otherwise. This concentration is only conductive to oligomerization and did not induce hydrogel formation. ☆☆☆</p> |
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| <div class="texttitle">Results</div> | | <div class="texttitle">Results</div> |
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| <div class="panel panel-default"> | | <div class="panel panel-default"> |
| <div class="panel-heading panel-title" role="tab" id="heading1"> | | <div class="panel-heading panel-title" role="tab" id="heading1"> |
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− | <a id="pH"></a>
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| <div class="panel panel-default"> | | <div class="panel panel-default"> |
| <div class="panel-heading panel-title" role="tab" id="heading1"> | | <div class="panel-heading panel-title" role="tab" id="heading1"> |
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| <p>The first part of the gradient experiment is concerned with pH changes. Given that the Spy Crosslinking Network would face an aqueous environment with nearly neutral pH in practical applications, and since the proteins have their own pH optima, we decided to use a pH gradient of 6.3/7.3/8.3. 3A-SUP and 3B monomers were first dissolved in TBS at certain pH values, after which they were contacted at pH values from the mentioned gradient at 25°C for 2 hours. Samples were taken every 30min in order to investigate the variation tendency of the monomers. Finally, SDS-PAGE was used to separate the proteins and the gels were scanned and analyzed using the software Lane 1D. Thus, the relationship between time and the content of 3B and 3A was measured. The experiments were done in triplicates to enable quantitative analysis.</p> | | <p>The first part of the gradient experiment is concerned with pH changes. Given that the Spy Crosslinking Network would face an aqueous environment with nearly neutral pH in practical applications, and since the proteins have their own pH optima, we decided to use a pH gradient of 6.3/7.3/8.3. 3A-SUP and 3B monomers were first dissolved in TBS at certain pH values, after which they were contacted at pH values from the mentioned gradient at 25°C for 2 hours. Samples were taken every 30min in order to investigate the variation tendency of the monomers. Finally, SDS-PAGE was used to separate the proteins and the gels were scanned and analyzed using the software Lane 1D. Thus, the relationship between time and the content of 3B and 3A was measured. The experiments were done in triplicates to enable quantitative analysis.</p> |
| <figure> | | <figure> |
− | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/4/4b/T--Peking--images_crosslinking_fig6.png" style="width:100%;" alt=""/> | + | <p style="text-align:center;"><img style="width: 90 %;" src=" https://static.igem.org/mediawiki/2016/4/4b/T--Peking--images_crosslinking_fig6.png" alt=""/></p> |
| + | <img class="featurette-image" src="" style="width:100%;" alt=""/> |
| <figcaption> | | <figcaption> |
| Fig. 6. pH Gradient Experiment. (A) The reactive extent for 3B at different pH. Experiments were repeated three times and error bars were added. (B) The mass distribution of oligomers at different time at pH7.3 (70kDa-450kDa). | | Fig. 6. pH Gradient Experiment. (A) The reactive extent for 3B at different pH. Experiments were repeated three times and error bars were added. (B) The mass distribution of oligomers at different time at pH7.3 (70kDa-450kDa). |
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− | <a id="Temperature"></a>
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| <div class="panel panel-default"> | | <div class="panel panel-default"> |
| <div class="panel-heading panel-title" role="tab" id="heading1"> | | <div class="panel-heading panel-title" role="tab" id="heading1"> |
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| <p>The second part of the gradient experiment is concerned with temperature. According to common environmental temperatures, three typical values, 16°C, 25°C, 37°C were introduced in this experiment. Similar to the pH Gradient Experiment, the monomers 3A-SUP and 3B were first dissolved in TBS at pH=7.3, after which they were contacted at the temperatures from the mentioned gradient. The reactions were conducted at pH=7.3 for 2 hours. The subsequent steps were the same as described for the pH gradient experiment.</p> | | <p>The second part of the gradient experiment is concerned with temperature. According to common environmental temperatures, three typical values, 16°C, 25°C, 37°C were introduced in this experiment. Similar to the pH Gradient Experiment, the monomers 3A-SUP and 3B were first dissolved in TBS at pH=7.3, after which they were contacted at the temperatures from the mentioned gradient. The reactions were conducted at pH=7.3 for 2 hours. The subsequent steps were the same as described for the pH gradient experiment.</p> |
| <figure> | | <figure> |
− | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/5/50/T--Peking--images_crosslinking_fig7.png" style="width:100%;" alt=""/> | + | <p style="text-align:center;"><img style="width: 90 %;" src="https://static.igem.org/mediawiki/2016/5/50/T--Peking--images_crosslinking_fig7.png " alt=""/></p> |
| <figcaption> | | <figcaption> |
| Fig. 7. Temperature Gradient Experiment. (A) The reactive extent for 3B at different temperature. Experiments were repeated three times and error bars were added. (B) The mass distribution of oligomers at different time at 16℃ (70kDa-400kDa). | | Fig. 7. Temperature Gradient Experiment. (A) The reactive extent for 3B at different temperature. Experiments were repeated three times and error bars were added. (B) The mass distribution of oligomers at different time at 16℃ (70kDa-400kDa). |
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| <div class="texttitle">Conclusion</div> | | <div class="texttitle">Conclusion</div> |
| <p>The crosslinking experiments showed that monomers containing SpyTag domains could react with triple SpyCatcher (3B) monomers, and that the crosslinking reaction may be favored at appropriately low pH (in the experiments, pH=6.3) and appropriately high temperature (in the experiments, T=37℃). Crosslinking leads to oligomerization at low monomer concentrations, and produces highly-crosslinked polymers with larger masses at high monomer concentrations. To this end, we have determined the optimal conditions for polymerization, which consisted of appropriately low pH, appropriately high temperature and comparatively higher monomer concentrations. The findings were in accordance with the universal principles of polyester condensation, as expected.</p> | | <p>The crosslinking experiments showed that monomers containing SpyTag domains could react with triple SpyCatcher (3B) monomers, and that the crosslinking reaction may be favored at appropriately low pH (in the experiments, pH=6.3) and appropriately high temperature (in the experiments, T=37℃). Crosslinking leads to oligomerization at low monomer concentrations, and produces highly-crosslinked polymers with larger masses at high monomer concentrations. To this end, we have determined the optimal conditions for polymerization, which consisted of appropriately low pH, appropriately high temperature and comparatively higher monomer concentrations. The findings were in accordance with the universal principles of polyester condensation, as expected.</p> |
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| <div class="references"> | | <div class="references"> |
| <h3>References:</h3> | | <h3>References:</h3> |