Team:Heidelberg/Toolbox Guide

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<div class="col-lg-12">
<div class="col-lg-12">
<span class="middle">use the <span class="red-text">intein</span></span>
<span class="middle">use the <span class="red-text">intein</span></span>
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</div>
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</div>
</div>
</div>
<div class="row">
<div class="row">
<div class="col-lg-12" style="position: relative; top: -20px;">
<div class="col-lg-12" style="position: relative; top: -20px;">
<span class="large">TOOLBOX&nbsp;</span><span class="middle" >to</span>
<span class="large">TOOLBOX&nbsp;</span><span class="middle" >to</span>
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</div>
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</div>
</div>
</div>
<div class="row toolbox-icons-bar">
<div class="row toolbox-icons-bar">
<div class="col-lg-2 col-lg-offset-1 col-md-offset-1 col-md-2 col-sm-4 col-xs-6">
<div class="col-lg-2 col-lg-offset-1 col-md-offset-1 col-md-2 col-sm-4 col-xs-6">
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<a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Circularization"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/5/58/Heidelberg_Toolbox_Circularization.png" class="img-responsive" /></span>CIRCULARIZE</a>
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<a href="https://2014.igem.org/Team:Heidelberg/Toolbox_Guide/Circularization"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/5/58/Heidelberg_Toolbox_Circularization.png" class="img-responsive" /></span>CIRCULARIZE</a>
</div>
</div>
<div class="col-lg-2 col-md-2 col-sm-4 col-xs-6">
<div class="col-lg-2 col-md-2 col-sm-4 col-xs-6">
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<a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Oligomerization"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/4/40/Oligomerization.png" class="img-responsive" /></span>OLIGOMERIZE</a>
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<a href="https://2014.igem.org/Team:Heidelberg/Toolbox_Guide/Localization"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/3/3a/Heidelberg_Localize_button.png" class="img-responsive" /></span>LOCALIZE</a>
</div>
</div>
<div class="col-lg-2 col-md-2 col-sm-4 col-xs-6">
<div class="col-lg-2 col-md-2 col-sm-4 col-xs-6">
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<a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Fusion"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/8/87/Heidelberg_Toolbox_Fusion.png" class="img-responsive" /></span>FUSE & TAG</a>
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<a href="https://2014.igem.org/Team:Heidelberg/Toolbox_Guide/Fusion"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/8/87/Heidelberg_Toolbox_Fusion.png" class="img-responsive" /></span>ATTACH</a>
</div>
</div>
<div class="col-lg-2 col-md-2 col-sm-4 col-xs-6">
<div class="col-lg-2 col-md-2 col-sm-4 col-xs-6">
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<a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Purification"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/0/04/Heidelberg_Toolbox_Purification.png" class="img-responsive" /></span>PURIFY</a>
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<a href="https://2014.igem.org/Team:Heidelberg/Toolbox_Guide/Purification"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/0/04/Heidelberg_Toolbox_Purification.png" class="img-responsive" /></span>PURIFY</a>
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</div>
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<div class="col-lg-2 col-md-2 col-sm-4 col-xs-6">
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<a href="https://2014.igem.org/Team:Heidelberg/Toolbox/OnOff"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/c/c2/Heidelberg_Toolbox_On-Off.png" class="img-responsive" /></span>ACTIVATE &<br />DEACTIVATE</a>
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</div>
</div>
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</div>
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<!--<div class="col-lg-2 col-md-2 col-sm-4 col-xs-6">
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<div class="col-lg-12" style="color:white;">
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<a href="https://2014.igem.org/Team:Heidelberg/Toolbox_Guide/OnOff"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/c/c2/Heidelberg_Toolbox_On-Off.png" class="img-responsive" /></span>ACTIVATE &<br />DEACTIVATE</a>
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<h3>Please go to <a href="http://www.rcsb.org/">www.rcsb.org</a> and get a pdb file of your protein. If the 3D structure of your protein is known, we will be able to provide you an appropriate linker.</h3>
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</div>-->
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<h4>If the 3D structure is unknown, we will help you to find a set of potentially suitable linkers. </h4>
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<div class="panel panel-default">
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<div class="panel-body">
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<div class="radio">
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<label>
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<input type="radio" data-bind="checked: data.gotProteinStructure, checkedValue: true, click: data.q9A.bind(null, true)" name="ko_unique_1" value="true">
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I’ve found one.
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</label>
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</div>
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<div class="radio">
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<label>
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<input type="radio" data-bind="checked: data.gotProteinStructure, checkedValue: false, click: data.q9A.bind(null, true)" name="ko_unique_2" value="false">
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The 3D structure of my protein is unknown
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</label>
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</div>
</div>
</div>
</div>
-
</div>
 
-
<div data-bind="fadeVisible: data.q9A" style="display:none;">
 
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<h3>Additionally, check the DNA sequence of your proteins for EcoRI, XbaI, SpeI, PstI and BsaI recognition sites. If there are E/X/S/P sites, you might have problems to change your backbone or add a promotor. If there is a BsaI recognition site, the cloning will be more difficult.</h3>
 
-
<div class="panel panel-default">
 
-
<div class="panel-body">
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.hasBsaI, checkedValue: false, click: data.q0A.bind(null, true)" />
 
-
There is no BsaI site.
 
-
</label>
 
-
</div>
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.hasBsaI, checkedValue: true, click: data.q0A.bind(null, true)" />
 
-
There is a BsaI site.
 
-
</label>
 
-
</div>
 
-
</div>
 
-
</div>
 
-
</div>
 
-
<div data-bind="fadeVisible: data.q0A">
 
-
<h3>Do you want to use split inteins or sortase to circularize your protein?</h3>
 
-
<div class="panel panel-default">
 
-
<div class="panel-body">
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.useSortase, checkedValue: false, click: data.q1A.bind(null, true)" />
 
-
NpuDnaE intein RFC <span data-bind="value: RFCnumber"></span> circularization construct
 
-
</label>
 
-
<ul>
 
-
<li>Successfully used in our project</li>
 
-
<li>High efficiency</li>
 
-
<li>In vivo circularization</li>
 
-
</ul>
 
-
</div>
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.useSortase, checkedValue: true, click: data.q1A.bind(null, true)" />
 
-
Sortase A circularization construct
 
-
</label>
 
-
<ul>
 
-
<li>In vitro only</li>
 
-
<li>Well-purified protein required</li>
 
-
<li>Not successfully tested yet</li>
 
-
</ul>
 
-
</div>
 
-
</div>
 
-
</div>
 
-
</div>
 
-
 
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<div data-bind="fadeVisible: data.q1A">
 
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<h3>Can your protein be easily expressed in <i>E. coli</i>?</h3>
 
-
<div class="panel panel-default">
 
-
<div class="panel-body">
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.needsSmt3, checkedValue: false, click: data.q2A.bind(null, true)" />
 
-
Yes, no problem.
 
-
</label>
 
-
</div>
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.needsSmt3, checkedValue: true, click: data.q2A.bind(null, true)" />
 
-
No, I will have to add <a href="http://parts.igem.org/Part:BBa_K1362429">Smt3</a>.
 
-
</label>
 
-
</div>
 
-
</div>
 
-
</div>
 
-
</div>
 
-
<div data-bind="fadeVisible: data.q2A() &amp;&amp; !data.useSortase()">
 
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<h3>Which exteins do you want to use? They will remain as scars in your circular protein.</h3>
 
-
<div class="panel panel-default">
 
-
<div class="panel-body">
 
-
<!-- ko foreach: availableExteins -->
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: $parent.data.exteins, checkedValue: $data, click: $parent.data.q3A.bind(null, true)" />
 
-
<span data-bind="text: N"></span>/<span data-bind="text: C"></span>
 
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<!-- ko if: comment -->
 
-
(<span data-bind="text: comment"></span>)
 
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<!-- /ko -->
 
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<ul data-bind="foreach: notes">
 
-
<li data-bind="text: $data"></li>
 
-
</ul>
 
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</label>
 
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</div>
 
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<!-- /ko -->
 
-
</div>
 
-
</div>
 
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</div>
 
-
<!-- ko if: data.gotProteinStructure -->
 
-
<div data-bind="fadeVisible: data.q3A() || (data.q2A() &amp;&amp; data.useSortase())">
 
-
<h3>If you want to save time, check manually whether the ends are close together (approx. <span data-bind="text: !data.useSortase() &amp;&amp; data.exteins().N == 'XXX' ? 5 : 15"></span>&thinsp;&Aring; or closer). For example, you can use the <a href="http://spdbv.vital-it.ch/">Swiss-PdbViewer</a> or <a href="http://www.pymol.org/">PyMOL</a>.</h3>
 
-
<div class="panel panel-default" >
 
-
<div class="panel-body">
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.endsAreClose, checkedValue: true, click: data.q4A.bind(null, true)" />
 
-
They are close together.
 
-
</label>
 
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</div>
 
-
<div class="radio">
 
-
<label>
 
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<input type="radio" data-bind="checked: data.endsAreClose, checkedValue: false, click: data.q4A.bind(null, true)" />
 
-
I want/have to use <span data-bind="text: softwareName"></span>.
 
-
</label>
 
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</div>
 
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</div>
 
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</div>
 
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</div>
 
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<div data-bind="fadeVisible: data.q4A() &amp;&amp; !data.endsAreClose()">
 
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Please use <span data-bind="text: softwareName"></span> to generate a linker for your circular protein. [LINK]
 
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This step might take up to 11 days.<br />
 
-
 
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NILS – hier könnte dein instruction-file-ersatz stehen <br />
 
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-
 
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NILS – hier auch<br />
 
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NILS – hier immernoch<br />
 
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NILS – hier ebnfalls und auch gerne noch umfangreicher<br />
 
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Please save this link:<br />
 
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<code id="comebackurl" style="display:inline-block; width:80%; overflow-y:hidden; vertical-align: middle;">https://2014.igem.org/<span id="pagename"></span>#<span data-bind="text: comeBackHash"></span></code><div style="display:inline-block; width: 20%; color:#de4230; vertical-align: middle;" id="copy-to-clip">&nbsp;<img src="/wiki/images/c/c6/Heidelberg_Clipboard.png" alt="Clipboard icon" />&nbsp;Copy to clipboard</div>
 
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In order to generate your linker, <span data-bind="text: softwareName"></span> needs to know the scar amino acid sequence that is caused by circularization. In your case, it is <span data-bind="text: data.useSortase() ? 'LPETG' : data.exteins().N == 'XXX' ? 'just C' : (data.exteins().N + data.exteins().C)"></span>.
 
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</div>
 
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<div data-bind="fadeVisible: data.comingBack">
 
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<h3>Hello again. What is the result of <span data-bind="text: softwareName"></span>?</h3>
 
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<div class="panel panel-default">
 
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<div class="panel-body">
 
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<div class="radio">
 
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<label>
 
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<input type="radio" data-bind="checked: data.linkerLength, checkedValue: 0, click: data.q5A.bind(null, true)" />
 
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I do not need a linker.
 
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</label>
 
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</div>
 
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<div class="radio">
 
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<label>
 
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<input type="radio" data-bind="checked: data.linkerLength, checkedValue: 1, click: data.q5A.bind(null, true)" />
 
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The linker + scars are &lt; 15 amino acids in length.
 
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</label>
 
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</div>
 
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<div class="radio">
 
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<label>
 
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<input type="radio" data-bind="checked: data.linkerLength, checkedValue: 2, click: data.q5A.bind(null, true)" />
 
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The linker + scars are betweeen 16 and 30 amino acids in length.
 
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</label>
 
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</div>
 
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<div class="radio">
 
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<label>
 
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<input type="radio" data-bind="checked: data.linkerLength, checkedValue: 3, click: data.q5A.bind(null, true)" />
 
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The linker + scars are &gt; 30 amino acids in length.
 
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</label>
 
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</div>
 
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</div>
 
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</div>
 
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</div>
 
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<div data-bind="fadeVisible: data.q5A">
 
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<h3>Have you decided to use one linker or try different linkers?</h3>
 
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<div class="panel panel-default">
 
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<div class="panel-body">
 
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<div class="radio">
 
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<label>
 
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<input type="radio" data-bind="checked: data.testSeveral, checkedValue: false, click: data.q6A.bind(null, true)" />
 
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I trust <span data-bind="text: softwareName"></span>. One should be enough.
 
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</label>
 
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</div>
 
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<div class="radio">
 
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<label>
 
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<input type="radio" data-bind="checked: data.testSeveral, checkedValue: true, click: data.q6A.bind(null, true)" />
 
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I want to test several linkers.
 
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</label>
 
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</div>
 
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</div>
 
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</div>
 
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</div>
 
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<!-- /ko -->
 
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<div data-bind="fadeVisible: !data.gotProteinStructure() &amp;&amp; (data.q3A() || data.q2A() &amp;&amp; data.useSortase())">
 
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<h3>How large is your protein?</h3>
 
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<div class="panel panel-default">
 
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<div class="panel-body">
 
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<div class="radio">
 
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<label>
 
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<input type="radio" data-bind="checked: data.proteinSize, checkedValue: 0, click: data.q7A.bind(null, true)" name="ko_unique_17" value="0">
 
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Less than 50 amino acids
 
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</label>
 
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</div>
 
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<div class="radio">
 
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<label>
 
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<input type="radio" data-bind="checked: data.proteinSize, checkedValue: 1, click: data.q7A.bind(null, true)" name="ko_unique_18" value="1">
 
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50-150 amino acids
 
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</label>
 
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</div>
 
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<div class="radio">
 
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<label>
 
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<input type="radio" data-bind="checked: data.proteinSize, checkedValue: 2, click: data.q7A.bind(null, true)" name="ko_unique_18" value="2">
 
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151-300 amino acids
 
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</label>
 
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</div>
 
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<div class="radio">
 
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<label>
 
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<input type="radio" data-bind="checked: data.proteinSize, checkedValue: 3, click: data.q7A.bind(null, true)" name="ko_unique_18" value="3">
 
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301-500 amino acids
 
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</label>
 
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</div>
 
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<div class="radio">
 
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<label>
 
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<input type="radio" data-bind="checked: data.proteinSize, checkedValue: 4, click: data.q7A.bind(null, true)" name="ko_unique_18" value="4">
 
-
501-700 amino acids
 
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</label>
 
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</div>
 
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<div class="radio">
 
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<label>
 
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<input type="radio" data-bind="checked: data.proteinSize, checkedValue: 5, click: data.q7A.bind(null, true)" name="ko_unique_18" value="5">
 
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More than 700 amino acids
 
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</label>
 
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</div>
 
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</div>
 
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</div>
 
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</div>
 
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</div>
 
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<div class="col-lg-12">
 
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<div data-bind="if: (data.q4A() &amp;&amp; data.endsAreClose()) || (data.q5A() &amp;&amp; data.linkerLength() == 0) || data.q6A() || data.q7A()">
 
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<h3>Protocol:</h3>
 
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<!--Module 1  -->
 
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<p>
 
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Get BBa_K136200<span data-bind="if: !data.useSortase()&amp;&amp;!data.needsSmt3()">0</span><span data-bind="if: !data.useSortase()&amp;&amp;data.needsSmt3()">1</span><span data-bind="if: data.useSortase()&amp;&amp;!data.needsSmt3()">2</span><span data-bind="if: data.useSortase()&amp;&amp;data.needsSmt3()">3</span> <span data-bind="if: !data.useSortase()">NpuDnaE intein RFC <span data-bind="text: RFCnumber"></span></span><span data-bind="if: data.useSortase()">Sortase A</span> circularization construct <span data-bind="if: !data.useSortase()&amp;&amp;data.needsSmt3()">(with FLAG and Smt3)</span><span data-bind="if: data.useSortase()&amp;&amp;!data.needsSmt3()">(with His6)</span><span data-bind="if: data.useSortase()&amp;&amp;data.needsSmt3()">(with Smt3 and His6)</span> from the registry.
 
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</p>
 
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<!-- Module 2 -->
 
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<p>Get the DNA of the protein you want to circularize.</p>
 
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<!-- Module 3 -->
 
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<p data-bind="if: !data.gotProteinStructure()">We recommend you to try circularization without a linker and with flexible GS-linkers of different lenghts up to <span data-bind="if: data.proteinSize() == 0">8</span><span data-bind="if: data.proteinSize() == 1">10</span><span data-bind="if: data.proteinSize() == 2">15</span><span data-bind="if: data.proteinSize() == 3">20</span><span data-bind="if: data.proteinSize() == 4">25</span><span data-bind="if: data.proteinSize() == 5">30</span> amino acids (including exteins).</p>
 
-
Backtranslate the amino acid sequence of your linker<span data-bind="if: data.testSeveral() || !data.gotProteinStructure()">s</span> to a nucleic acid sequence. Be aware of:
 
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<ul>
 
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<li>Balanced GC-content</li>
 
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<li>Restriction sites (especially PstI)</li>
 
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<li>Codon usage</li>
 
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<li>Avoid self annealing</li>
 
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</ul>
 
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</div>
 
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</div>
 
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</div>
 
</div>
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Latest revision as of 03:43, 18 October 2014