Team:Heidelberg/Toolbox Guide

From 2014.igem.org

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</div>
</div>
</div>
</div>
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<div class="col-lg-12">
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<div class="panel panel-default">
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<div class="panel-heading">
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<h3 class="panel-title">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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</div>
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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 class="radio">
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<label>
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<input type="radio" data-bind="checked: data.hasBsaI, checkedValue: false, click: data.q0A.bind(null, true)" />
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There is no BsaI site.
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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.hasBsaI, checkedValue: true, click: data.q0A.bind(null, true)" />
 +
There is a BsaI site.
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</label>
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</div>
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</div>
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<div class="panel panel-default" data-bind="if: data.q0A">
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<div class="panel-heading">
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<h3 class="panel-title">2. Do you want to use split inteins or sortase to circularize your protein?</h3>
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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.useSortase, checkedValue: false, click: data.q1A.bind(null, true)" />
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NpuDnaE intein RFC <span data-bind="value: RFCnumber"></span> circularization construct
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</label>
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<ul>
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<li>Successfully used in our project</li>
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<li>High efficiency</li>
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<li>In vivo circularization</li>
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</ul>
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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.useSortase, checkedValue: true, click: data.q1A.bind(null, true)" />
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Sortase A circularization construct
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</label>
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<ul>
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<li>In vitro only</li>
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<li>Well-purified protein required</li>
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<li>Not successfully tested yet</li>
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</ul>
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</div>
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</div>
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<div class="panel panel-default" data-bind="if: data.q1A">
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<div class="panel-heading">
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<h3 class="panel-title">3. Can your protein be easily expressed in <i>E. coli</i>?</h3>
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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.needsSmt3, checkedValue: false, click: data.q2A.bind(null, true)" />
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Yes, no problem.
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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.needsSmt3, checkedValue: true, click: data.q2A.bind(null, true)" />
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No, I will have to add <a href="http://parts.igem.org/Part:BBa_K1362429">Smt3</a>.
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</label>
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</div>
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</div>
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<div class="panel panel-default" data-bind="if: data.q2A() &amp;&amp; !data.useSortase()">
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<div class="panel-heading">
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<h3 class="panel-title">4. Which exteins do you want to use? They will remain as scars in your circular protein.</h3>
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</div>
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<!-- ko foreach: availableExteins -->
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<div class="radio">
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<label>
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<input type="radio" data-bind="checked: $parent.data.exteins, checkedValue: $data, click: $parent.data.q3A.bind(null, true)" />
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<span data-bind="text: N"></span>/<span data-bind="text: C"></span>
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<!-- ko if: comment -->
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(<span data-bind="text: comment"></span>)
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<!-- /ko -->
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<ul data-bind="foreach: notes">
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<li data-bind="text: $data"></li>
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</ul>
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</label>
</div>
</div>
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<!-- /ko -->
</div>
</div>
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<div class="panel panel-default" data-bind="if: data.q3A() || (data.q2A() &amp;&amp; data.useSortase())">
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<div class="panel-heading">
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<h3 class="panel-title"><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>
 +
</div>
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<div class="radio">
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<label>
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<input type="radio" data-bind="checked: data.endsAreClose, checkedValue: true, click: data.q4A.bind(null, true)" />
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They are close together.
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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.endsAreClose, checkedValue: false, click: data.q4A.bind(null, true)" />
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I want/have to use <span data-bind="text: softwareName"></span>.
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</label>
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</div>
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</div>
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<div data-bind="if: data.q4A() &amp;&amp; !data.endsAreClose()">
 +
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.
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NILS – hier könnte dein instruction-file-ersatz stehen
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 +
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NILS – hier auch
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 +
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NILS – hier immernoch
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 +
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NILS – hier ebnfalls und auch gerne noch umfangreicher
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Please save this link:<br />
 +
<code>https://2014.igem.org/{{PAGENAME}}#<span data-bind="text: comeBackHash"></span></code>
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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 class="panel panel-default" data-bind="if: data.comingBack">
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<div class="panel-heading">
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<h3 class="panel-title"><span data-bind="text: data.useSortase() ? 5 : 6"></span>. Hello again. What is the result of <span data-bind="text: softwareName"></span>?</h3>
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</div>
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<div class="radio">
 +
<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 class="panel panel-default" data-bind="if: data.q5A">
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<div class="panel-heading">
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<h3 class="panel-title"><span data-bind="text: data.useSortase() ? 6 : 7"></span>. Have you decided to use one linker or try different linkers?</h3>
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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: 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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</div>
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<script type="text/javascript">
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function CircularizationViewModel(hash) {
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var self = this;
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self.RFCnumber = '[???]';
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self.softwareName = '[hoffentlich nicht LinkIT]';
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self.availableExteins = [
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{ N: 'RGK', C: 'CWE', comment: '', notes: ['Efficient, tested by us', 'Large scar'] },
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{ N: 'RGT', C: 'CWE', comment: '', notes: ['Efficient, tested by us', 'Large scar'] },
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{ N: 'AEY', C: 'CFN' , comment: '', notes: ['Efficient, native extein', 'Large scar'] },
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{ N: 'XXX', C: 'CXX', comment: 'X = native amino acids of the protein', notes: ['Probably less efficient (depends on the protein)', 'Small scar'] },
 +
];
 +
 +
var data = hash && $.parseJSON($.base64.decode(hash.substr(1)));
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self.data = {
 +
comingBack: data ? data.comingBack : false,
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q0A: ko.observable(data ? data.q0A : false),
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hasBsaI: ko.observable(data && data.hasBsaI),
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q1A: ko.observable(data ? data.q1A : false),
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useSortase: ko.observable(data && data.useSortase),
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q2A: ko.observable(data ? data.q2A : false),
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needsSmt3: ko.observable(data && data.needsSmt3),
 +
q3A: ko.observable(data ? data.q3A : false),
 +
exteins: ko.observable(data && data.exteins),
 +
q4A: ko.observable(data ? data.q4A : false),
 +
endsAreClose: ko.observable(data && data.endsAreClose),
 +
q5A: ko.observable(data ? data.q5A : false),
 +
linkerLength: ko.observable(data && data.linkerLength),
 +
q6A: ko.observable(data ? data.q6A : false),
 +
testSeveral: ko.observable(data && data.testSeveral),
 +
};
 +
 +
self.hash = ko.computed(function () {
 +
self.data.q0A();
 +
self.data.q1A();
 +
self.data.q2A();
 +
self.data.q3A();
 +
self.data.q4A();
 +
self.data.q5A();
 +
self.data.q6A();
 +
self.data.hasBsaI();
 +
self.data.useSortase();
 +
self.data.needsSmt3();
 +
self.data.exteins();
 +
self.data.endsAreClose();
 +
self.data.linkerLength();
 +
self.data.testSeveral();
 +
 +
window.location.hash = $.base64.encode(ko.toJSON(self.data));
 +
console.log(window.location.hash, ko.toJSON(self.data));
 +
return window.location.hash;
 +
 +
if (self.q0A()) {
 +
window.location.hash = '#' + (self.hasBsaI() ? '1': '0');
 +
 +
if (self.q1A()) {
 +
window.location.hash += self.useSortase() ? '1': '0';
 +
 +
if (self.q2A()) {
 +
window.location.hash += self.needsSmt3() ? '1': '0';
 +
 +
if (self.q3A() && !self.useSortase())
 +
window.location.hash += self.exteins().N;
 +
 +
if (self.q4A()) {
 +
window.location.hash += self.endsAreClose() ? '1': '0';
 +
 +
if (self.q5A()) {
 +
window.location.hash += self.linkerLength();
 +
 +
if (self.q6A()) {
 +
window.location.hash += self.testSeveral() ? '1': '0';
 +
}
 +
}
 +
}
 +
}
 +
}
 +
} else {
 +
window.location.hash = '';
 +
}
 +
 +
return window.location.hash;
 +
});
 +
 +
self.comeBackHash = ko.computed(function () {
 +
var tmp = self.data.comingBack;
 +
self.data.comingBack = true;
 +
var hash = $.base64.encode(ko.toJSON(self.data));
 +
self.data.comingBack = tmp;
 +
return hash;
 +
});
 +
}
 +
 +
vm = new CircularizationViewModel(window.location.hash);
 +
ko.applyBindings(vm);
 +
 +
/*function parseHash(hash) {
 +
var data = hash.substring(1);
 +
for(i = 0; i< data.length; i++) {
 +
switch(i){
 +
case 1:
 +
vm.hasBsaI(data[i]);
 +
break;
 +
case 2:
 +
vm.
 +
}
 +
}
 +
}*/
 +
</script>
</html>
</html>

Revision as of 22:03, 14 October 2014

use the intein
TOOLBOX to
CIRCULARIZE
OLIGOMERIZE
FUSE & TAG
PURIFY
ACTIVATE &
DEACTIVATE

1. Please go to www.rcsb.org and get a pdb file of your protein. If you cannot find one, we will not be able to assist you circularizing it.

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.

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 Please save this link:
https://2014.igem.org/{{PAGENAME}}# 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?

Retrieved from "http://2014.igem.org/Team:Heidelberg/Toolbox_Guide"