Team:Heidelberg/Toolbox Guide

From 2014.igem.org

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<div class="row">
<div class="row">
<div class="col-lg-12">
<div class="col-lg-12">
-
<span class="middle">use our <span class="red-text">intein</span></span>
+
<span class="middle">use the <span class="red-text">intein</span></span>
-
</div>
+
</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 GUIDE&nbsp;</span><span class="middle" >to</span>
+
<span class="large">TOOLBOX&nbsp;</span><span class="middle" >to</span>
-
</div>
+
</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">
-
<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>
+
<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">
-
<a href="https://2014.igem.org/Team:Heidelberg/Project/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>
+
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox_Guide/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>
</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">
-
<a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Fusion_and_Tagging"><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>
+
<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>FUSE & TAG</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">
-
<a href="https://2014.igem.org/Team:Heidelberg/Project/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>
+
<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>
</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">
-
<a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#On_Off"><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>
+
<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>
</div>
</div>
-
</div>
 
-
<div class="col-lg-12" style="color:white;">
 
-
<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>
 
-
<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-body">
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.gotProteinStructure, checkedValue: true, click: data.q9A.bind(null, true)" name="gotProteinStructure">
 
-
I’ve found one.
 
-
</label>
 
-
</div>
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.gotProteinStructure, checkedValue: false, click: data.q9A.bind(null, true)" name="gotProteinStructure">
 
-
The 3D structure of my protein is unknown
 
-
</label>
 
</div>
</div>
</div>
</div>
-
</div>
 
-
<div data-bind="fadeVisible: data.q9A" style="display:none;">
 
-
<h4>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.</h4>
 
-
<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)" value="false" name="hasBasI"/>
 
-
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)" value="true"  name="hasBasI"/>
 
-
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)" name="InteinorSortase"/>
 
-
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)" name="InteinorSortase"/>
 
-
Sortase A circularization construct
 
-
</label>
 
-
<ul>
 
-
<li>In vitro more efficient</li>
 
-
<li>Reversible</li>
 
-
<li>Not successfully tested by us</li>
 
-
</ul>
 
-
</div>
 
-
</div>
 
-
</div>
 
-
</div>
 
-
 
-
<div data-bind="fadeVisible: data.q1A">
 
-
<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)" name="needsSmt3"/>
 
-
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)" name="needsSmt3"/>
 
-
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()">
 
-
<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)" name="Extein"/>
 
-
<span data-bind="text: N"></span>/<span data-bind="text: C"></span>
 
-
<!-- ko if: comment -->
 
-
(<span data-bind="text: comment"></span>)
 
-
<!-- /ko -->
 
-
<ul data-bind="foreach: notes">
 
-
<li data-bind="text: $data"></li>
 
-
</ul>
 
-
</label>
 
-
</div>
 
-
<!-- /ko -->
 
-
</div>
 
-
</div>
 
-
</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)" name="closeEnds"/>
 
-
They are close together.
 
-
</label>
 
-
</div>
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.endsAreClose, checkedValue: false, click: data.q4A.bind(null, true)" name="closeEnds"/>
 
-
I want/have to use <span data-bind="text: softwareName"></span>.
 
-
</label>
 
-
</div>
 
-
</div>
 
-
</div>
 
-
</div>
 
-
 
-
<div data-bind="fadeVisible: data.q4A() &amp;&amp; !data.endsAreClose()">
 
-
Please use <span data-bind="text: softwareName"></span> to generate a linker for your circular protein. [LINK]
 
-
 
-
This step might take up to 11 days.<br />
 
-
 
-
NILS – hier könnte dein instruction-file-ersatz stehen <br />
 
-
 
-
 
-
NILS – hier auch<br />
 
-
 
-
 
-
NILS – hier immernoch<br />
 
-
 
-
 
-
NILS – hier ebnfalls und auch gerne noch umfangreicher<br />
 
-
 
-
 
-
Please save this link:<br />
 
-
<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>
 
-
 
-
 
-
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>.
 
-
</div>
 
-
 
-
<div data-bind="fadeVisible: data.comingBack">
 
-
<h3>Hello again. What is the result of <span data-bind="text: softwareName"></span>?</h3>
 
-
<div class="panel panel-default">
 
-
<div class="panel-body">
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.linkerLength, checkedValue: 0, click: data.q5A.bind(null, true)" name="linkerScarSize"/>
 
-
I do not need a linker.
 
-
</label>
 
-
</div>
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.linkerLength, checkedValue: 1, click: data.q5A.bind(null, true)" name="linkerScarSize"/>
 
-
The linker + scars are &lt; 15 amino acids in length.
 
-
</label>
 
-
</div>
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.linkerLength, checkedValue: 2, click: data.q5A.bind(null, true)" name="linkerScarSize"/>
 
-
The linker + scars are betweeen 16 and 30 amino acids in length.
 
-
</label>
 
-
</div>
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.linkerLength, checkedValue: 3, click: data.q5A.bind(null, true)" name="linkerScarSize"/>
 
-
The linker + scars are &gt; 30 amino acids in length.
 
-
</label>
 
-
</div>
 
-
</div>
 
-
</div>
 
-
</div>
 
-
<div data-bind="fadeVisible: data.q5A">
 
-
<h3>Have you decided to use one linker or try different linkers?</h3>
 
-
<div class="panel panel-default">
 
-
<div class="panel-body">
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.testSeveral, checkedValue: false, click: data.q6A.bind(null, true)" name="testSeveral"/>
 
-
I trust <span data-bind="text: softwareName"></span>. One should be enough.
 
-
</label>
 
-
</div>
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.testSeveral, checkedValue: true, click: data.q6A.bind(null, true)" name="testSeveral"/>
 
-
I want to test several linkers.
 
-
</label>
 
-
</div>
 
-
</div>
 
-
</div>
 
-
</div>
 
-
<!-- /ko -->
 
-
 
-
<div data-bind="fadeVisible: !data.gotProteinStructure() &amp;&amp; (data.q3A() || data.q2A() &amp;&amp; data.useSortase())">
 
-
<h3>How large is your protein?</h3>
 
-
<div class="panel panel-default">
 
-
<div class="panel-body">
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.proteinSize, checkedValue: 0, click: data.q7A.bind(null, true)" name="porteinSize" />
 
-
Less than 50 amino acids
 
-
</label>
 
-
</div>
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.proteinSize, checkedValue: 1, click: data.q7A.bind(null, true)" name="porteinSize" />
 
-
50-150 amino acids
 
-
</label>
 
-
</div>
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.proteinSize, checkedValue: 2, click: data.q7A.bind(null, true)" name="porteinSize" />
 
-
151-300 amino acids
 
-
</label>
 
-
</div>
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.proteinSize, checkedValue: 3, click: data.q7A.bind(null, true)" name="porteinSize" />
 
-
301-500 amino acids
 
-
</label>
 
-
</div>
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.proteinSize, checkedValue: 4, click: data.q7A.bind(null, true)" name="porteinSize" />
 
-
501-700 amino acids
 
-
</label>
 
-
</div>
 
-
<div class="radio">
 
-
<label>
 
-
<input type="radio" data-bind="checked: data.proteinSize, checkedValue: 5, click: data.q7A.bind(null, true)" name="porteinSize" />
 
-
More than 700 amino acids
 
-
</label>
 
-
</div>
 
-
</div>
 
-
</div>
 
-
</div>
 
</div>
</div>
-
 
-
<div class="col-lg-12">
 
-
<div data-bind="fadeVisible: (data.q4A() &amp;&amp; data.endsAreClose()) || (data.q5A() &amp;&amp; data.linkerLength() == 0) || data.q6A() || data.q7A()">
 
-
 
-
<h3>Protocol:</h3>
 
-
<!--Module 1  -->
 
-
<p>
 
-
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.
 
-
</p>
 
-
 
-
<!-- Module 2 -->
 
-
<p>Get the DNA of the protein you want to circularize.</p>
 
-
 
-
<!-- Module 3 -->
 
-
<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:
 
-
<ul>
 
-
<li>Balanced GC-content</li>
 
-
<li>Restriction sites (especially PstI)</li>
 
-
<li>Codon usage</li>
 
-
<li>Avoid self annealing</li>
 
-
</ul>
 
-
 
-
<!-- Module 4 -->
 
-
<p>Design and order primers for your <span data-bind="if: data.linkerLength() == 2 || data.linkerLength() == 3">protein</span> insert. </p>
 
-
<ol>
 
-
<li><p>In 5'-3' direction, the forward primer should have the following sequence:</p>
 
-
<p>CT GGTCTCA <span data-bind="if: !data.useSortase()">CAAC</span><span data-bind="if: data.useSortase()">GGGT</span><span data-bind="if: data.exteins().N == 'RGK' || data.exteins().N == 'RGT'">TGCTGGGAA</span><span data-bind="if: data.exteins().N == 'AEY'">TGCTTCAAC</span><span data-bind="if: data.exteins().N == 'XXX'">TGC</span> + binding part
 
-
(=additional random nucleotides, BsaI site, <span data-bind="if: !data.useSortase()">RFC <span data-bind="text: RFCnumber"></span> E</span> <span data-bind="if: data.useSortase()">overhang</span><span data-bind="if: data.exteins().N == 'RGK' || data.exteins().N == 'RGT'">, CWE</span><span data-bind="if: data.exteins().N == 'AEY'">, CFN</span><span data-bind="if: data.exteins().N == 'XXX'">, Cys</span>)
 
-
</p>
 
-
</li>
 
-
<li><p>In 5'-3' direction, the reverse primer should have the following sequence:</p>
 
-
<p><span data-bind="if: data.linkerLength() == 2 || data.linkerLength() == 3 || data.proteinSize() == 3 || data.proteinSize() == 4 || data.proteinSize() == 5">CT GGTCTCT + binding part
 
-
(=additional random nucleotides, BsaI site)</span>
 
-
<span data-bind="if: !(data.linkerLength() == 2 || data.linkerLength() == 3 || data.proteinSize() == 3 || data.proteinSize() == 4 || data.proteinSize() == 5)">CT GGTCTCT <span data-bind="if: !data.useSortase()">AGCA</span><span data-bind="if: data.useSortase()">GAAG</span> <span data-bind="if: data.exteins().N == 'RGK'">TTTACCACG</span><span data-bind="if: data.exteins().N == 'RGT'"> GGTACCACG</span><span data-bind="if: data.exteins().N == 'AEY'">ATATTCCGC</span> <span data-bind="if: data.linkerLength() == 1 || data.proteinSize() == 0 || data.proteinSize() == 1 || data.proteinSize() == 2">+ linker sequence (reverse complement)</span> + binding part
 
-
(=additional random nucleotides, BsaI site, <span data-bind="if: !data.useSortase()">RFC <span data-bind="text: RFCnumber"></span> B</span><span data-bind="if: data.useSortase()">overhang</span> <span data-bind="if: data.exteins().N == 'RGK'">, RGK</span><span data-bind="if: data.exteins().N == 'RGT'">, RGT</span><span data-bind="if: data.exteins().N == 'AEY'">, AEY</span><span data-bind="if: data.exteins().N == 'RGK' || data.exteins().N == 'RGT' || data.exteins().N == 'AEY'"> reverse complement</span>)</span>
 
-
</p>
 
-
</li>
 
-
<li data-bind="style: { display: ((data.linkerLength() == 1 &amp;&amp; data.testSeveral()) || data.proteinSize() == 0 || data.proteinSize() == 1 || data.proteinSize() == 2) ? 'inline' : 'none'}">
 
-
<p>For any additional linker you want to test, you just need one additional reverse primer with the corresponding reverse complement linker DNA sequence.</p>
 
-
</li>
 
-
</ol>
 
-
<p>The Tm values of the binding parts should be between 60 °C and 70 °C and as similar as possible.</p>
 
-
<span data-bind="if: data.linkerLength() == 2 || data.linkerLength() == 3 || data.proteinSize() == 3 || data.proteinSize() == 4 || data.proteinSize() == 5">
 
-
Design and order oligos for your linker insert<span data-bind="if: data.testSeveral() || !data.gotProteinStructure()">s</span>.
 
-
<ol>
 
-
<li>
 
-
<p>
 
-
In 5'-3' direction, the forward <span data-bind="if: !data.linkerLength() == 3">oligo</span><span data-bind="if: data.linkerLength() == 3">strand</span> should have the following sequence:
 
-
</p>
 
-
<p>
 
-
Last 4 nucleotides of your protein + linker sequence + <span data-bind="if: data.exteins().N == 'RGK'">CGTGGTAAA (=RGK)</span><span data-bind="if: data.exteins().N == 'RGT'">CGTGGTACC (=RGT)</span><span data-bind="if: data.exteins().N == 'AEY'">GCGGAATAT (=AEY)</span>
 
-
</p>
 
-
</li>
 
-
<li>
 
-
<p>
 
-
In 5'-3' direction, the reverse <span data-bind="if: !data.linkerLength() == 3">oligo</span><span data-bind="if: data.linkerLength() == 3">strand</span> should have the following sequence:</p>
 
-
<p><span data-bind="if: !data.useSortase()">AGCA</span><span data-bind="if: data.useSortase()">GAAG</span> <span data-bind="if: data.exteins().N == 'RGK'">TTTACCACG</span><span data-bind="if: data.exteins().N == 'RGT'">GGTACCACG</span><span data-bind="if: data.exteins().N == 'AEY'">ATATTCCGC</span> + linker sequence (reverse complement)
 
-
(=<span data-bind="if: !data.useSortase()">RFC <span data-bind="text: RFCnumber"></span> B</span> <span data-bind="if: data.useSortase()">overhang</span> <span data-bind="if: data.exteins().N == 'RGK'">, RGK)</span><span data-bind="if: data.exteins().N == 'RGT'">, RGT</span><span data-bind="if: data.exteins().N == 'AEY'">, AEY</span><span data-bind="if: data.exteins().N == 'RGK' || data.exteins().N == 'RGT' || data.exteins().N == 'AEY'">reverse complement</span>)
 
-
 
-
</p>
 
-
</li>
 
-
</ol>
 
-
</span> <!-- Strange span ... check with max-->
 
-
 
-
<span data-bind="if: data.linkerLength() == 3">Probably these oligos would be longer than 90 nucleotides. In this case we recommend you to split the insert into several smaller inserts and order them as pairs of oligos. Use overhangs of 4-6 nucleotides and be careful that every overhang remains unique.
 
-
The largest linker we have successfully cloned this way was composed of 65 amino acids. ]
 
-
<span data-bind="if: data.testSeveral() || !data.gotProteinStructure()">For any additional linker you want to test, you just need another linker insert with the corresponding linker sequence.</span>
 
-
</span>
 
-
<!-- Module 5 -->
 
-
<p data-bind="if: data.linkerLength() == 2 || data.linkerLength() == 3 || data.proteinSize() == 3 || data.proteinSize() == 4 || data.proteinSize() == 5">Anneal your linker insert<span data-bind="if: data.testSeveral() || !data.gotProteinStructure()">s</span> as described here -LINK ZU MAT/MET ANNEALING-. </p>
 
-
 
-
<!-- Module 6 -->
 
-
<p>Use a Touchdown-Twostep PCR -LINK ZU MAT/MET TOUCHDOWNTWOSTEPPCR- to create your <span data-bind="if: data.linkerLength() == 2 || data.linkerLength() == 3 || data.proteinSize() == 3 || data.proteinSize() == 4 || data.proteinSize() == 5">protein</span> insert and purify it.</p>
 
-
 
-
<!-- Module 7 -->
 
-
Use Golden Gate assembly –LINK ZU MAT/MET GOLDEN GATE ASSEMBLY (CYCLING) to replace the mRFP selection marker in the circularization construct with <!-- Klammernfehler? --> <span data-bind="if: !(data.linkerLength() == 2 || data.linkerLength() == 3 || data.proteinSize() == 3 || data.proteinSize() == 4 || data.proteinSize() == 5)">your insert.</span><span data-bind="if: data.linkerLength() == 2 || data.linkerLength() == 3 || data.proteinSize() == 3 || data.proteinSize() == 4 || data.proteinSize() == 5">both your protein insert and your linker insert</span><span data-bind="if: data.testSeveral() || !data.gotProteinStructure()">s</span>.
 
-
<p data-bind="if: data.linkerLength() == 3">Because your annealed oligos are not 5'-phosphorylated, you have to add T4-polynucleotide kinase to the Golden Gate reaction.</p>
 
-
<p data-bind="if: data.hasBsaI()">Since there is a BsaI site in your protein, you have to religate – LINK ZU MAT/MET RELIGATION after the Golden Gate reaction.</p>
 
-
<!-- Module 8 -->
 
-
<p>
 
-
Add an inducible promotor by standard biobrick cloning or use one of our pSBX vectors –LINK ZU PSBX VECTOR-TEXT.
 
-
</p>
 
-
<!-- Module 9 -->
 
-
<p>Express your circular protein. If you are using our pSBX vectors, you can follow this protocol –LINK ZU PROTOKOLL EXPRESSION</p>
 
-
<!-- Module 10 -->
 
-
<span data-bind="if: data.useSortase()">SILVAN SCHREIBT HIER NOCH WAS ZU SORTASE</span>
 
-
 
-
</div>
 
-
</div>
 
-
</div>
 
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</div>
 
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Revision as of 01:33, 18 October 2014

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