Team:Heidelberg/Toolbox Guide

From 2014.igem.org

(Difference between revisions)
Line 240: Line 240:
<h3><span data-bind="text: data.useSortase() ? 5 : 6"></span>. Hello again. What is the result of <span data-bind="text: softwareName"></span>?</h3>
<h3><span data-bind="text: data.useSortase() ? 5 : 6"></span>. Hello again. What is the result of <span data-bind="text: softwareName"></span>?</h3>
<div class="panel panel-default">
<div class="panel panel-default">
-
<div class="radio">
+
<div class="panel-body">
-
<label>
+
<div class="radio">
-
<input type="radio" data-bind="checked: data.linkerLength, checkedValue: 0, click: data.q5A.bind(null, true)" />
+
<label>
-
I do not need a linker.
+
<input type="radio" data-bind="checked: data.linkerLength, checkedValue: 0, click: data.q5A.bind(null, true)" />
-
</label>
+
I do not need a linker.
-
</div>
+
</label>
-
<div class="radio">
+
</div>
-
<label>
+
<div class="radio">
-
<input type="radio" data-bind="checked: data.linkerLength, checkedValue: 1, click: data.q5A.bind(null, true)" />
+
<label>
-
The linker + scars are &lt; 15 amino acids in length.
+
<input type="radio" data-bind="checked: data.linkerLength, checkedValue: 1, click: data.q5A.bind(null, true)" />
-
</label>
+
The linker + scars are &lt; 15 amino acids in length.
-
</div>
+
</label>
-
<div class="radio">
+
</div>
-
<label>
+
<div class="radio">
-
<input type="radio" data-bind="checked: data.linkerLength, checkedValue: 2, click: data.q5A.bind(null, true)" />
+
<label>
-
The linker + scars are betweeen 16 and 30 amino acids in length.
+
<input type="radio" data-bind="checked: data.linkerLength, checkedValue: 2, click: data.q5A.bind(null, true)" />
-
</label>
+
The linker + scars are betweeen 16 and 30 amino acids in length.
-
</div>
+
</label>
-
<div class="radio">
+
</div>
-
<label>
+
<div class="radio">
-
<input type="radio" data-bind="checked: data.linkerLength, checkedValue: 3, click: data.q5A.bind(null, true)" />
+
<label>
-
The linker + scars are &gt; 30 amino acids in length.
+
<input type="radio" data-bind="checked: data.linkerLength, checkedValue: 3, click: data.q5A.bind(null, true)" />
-
</label>
+
The linker + scars are &gt; 30 amino acids in length.
 +
</label>
 +
</div>
</div>
</div>
</div>
</div>
Line 269: Line 271:
<h3><span data-bind="text: data.useSortase() ? 6 : 7"></span>. Have you decided to use one linker or try different linkers?</h3>
<h3><span data-bind="text: data.useSortase() ? 6 : 7"></span>. Have you decided to use one linker or try different linkers?</h3>
<div class="panel panel-default">
<div class="panel panel-default">
-
<div class="radio">
+
<div class="panel-body">
-
<label>
+
<div class="radio">
-
<input type="radio" data-bind="checked: data.testSeveral, checkedValue: false, click: data.q6A.bind(null, true)" />
+
<label>
-
I trust <span data-bind="text: softwareName"></span>. One should be enough.
+
<input type="radio" data-bind="checked: data.testSeveral, checkedValue: false, click: data.q6A.bind(null, true)" />
-
</label>
+
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)" />
 +
I want to test several linkers.
 +
</label>
 +
</div>
</div>
</div>
-
<div class="radio">
+
</div>
-
<label>
+
</div>
-
<input type="radio" data-bind="checked: data.testSeveral, checkedValue: true, click: data.q6A.bind(null, true)" />
+
 
-
I want to test several linkers.  
+
</div>
-
</label>
+
<div class="col-lg-12">
 +
<div data-bind="if: (data.q4a() &amp;&amp; data.endsAreClose()) || (data.q5a() &amp;&amp; data.linkerLength() == 0) || data.q6a()">
 +
 
 +
<h3>Protocol:</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)" />
 +
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)" />
 +
I want to test several linkers.  
 +
</label>
 +
</div>
</div>
</div>
</div>
</div>

Revision as of 19:37, 15 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
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https://2014.igem.org/#
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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?

Protocol:

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