Team:Heidelberg/Toolbox Guide

From 2014.igem.org

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<div class="col-lg-12" style="color:white;">
<div class="col-lg-12" style="color:white;">
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<h3>1. Please go to <a href="http://www.rcsb.org/">www.rcsb.org</a> and get a pdb file of your protein. If you cannot find one, we will not be able to assist you circularizing it.</h3>
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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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<h4>If the 3D structure is unknown, we will help you to find a set of potentially suitable linkers. </h4>
<div class="panel panel-default">
<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: true, 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>
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</div>
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</div>
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<div data-bind="fadeVisible: data.q9A" style="display: none;" class="panel panel-default">
<div class="panel-body">
<div class="panel-body">
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.
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.
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</div>
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<!-- ko if: data.gotProteinStructure -->
<div data-bind="fadeVisible: data.q3A() || (data.q2A() &amp;&amp; data.useSortase())">
<div data-bind="fadeVisible: data.q3A() || (data.q2A() &amp;&amp; data.useSortase())">
<h3><span data-bind="text: data.useSortase() ? 4 : 5"></span>. 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>
<h3><span data-bind="text: data.useSortase() ? 4 : 5"></span>. 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>
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</div>
</div>
</div>
</div>
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<!-- /ko -->
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</div>
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<div data-bind="if: data.q9A() &amp;&amp; !data.gotProteinStructure()">
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<div class="col-lg-12">
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<h3>How large is your protein?</h3>
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<div data-bind="if: (data.q4A() &amp;&amp; data.endsAreClose()) || (data.q5A() &amp;&amp; data.linkerLength() == 0) || data.q6A()">
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<h3>Protocol:</h3>
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<div class="panel panel-default">
<div class="panel panel-default">
<div class="panel-body">
<div class="panel-body">
<div class="radio">
<div class="radio">
<label>
<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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<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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I trust <span data-bind="text: softwareName"></span>. One should be enough.
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Less than 50 amino acids
</label>
</label>
</div>
</div>
<div class="radio">
<div class="radio">
<label>
<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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<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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I want to test several linkers.  
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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">
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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
</label>
</label>
</div>
</div>
</div>
</div>
</div>
</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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</div>
</div>
</div>
</div>

Revision as of 22:13, 15 October 2014

use the intein
TOOLBOX to

Please go to www.rcsb.org 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.

If the 3D structure is unknown, we will help you to find a set of potentially suitable linkers.

2. Do you want to use split inteins or sortase to circularize your protein?

  • Successfully used in our project
  • High efficiency
  • In vivo circularization
  • In vitro only
  • Well-purified protein required
  • Not successfully tested yet

3. Can your protein be easily expressed in E. coli?

4. Which exteins do you want to use? They will remain as scars in your circular protein.

. If you want to save time, check manually whether the ends are close together (approx.  Å or closer). For example, you can use the Swiss-PdbViewer or PyMOL.

Please use to generate a linker for your circular protein. [LINK] This step might take up to 11 days.
NILS – hier könnte dein instruction-file-ersatz stehen
NILS – hier auch
NILS – hier immernoch
NILS – hier ebnfalls und auch gerne noch umfangreicher
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In order to generate your linker, needs to know the scar amino acid sequence that is caused by circularization. In your case, it is .

. Hello again. What is the result of ?

. Have you decided to use one linker or try different linkers?

How large is your protein?

Protocol: