Team:Heidelberg/Toolbox Guide

From 2014.igem.org

(Difference between revisions)
Line 115: Line 115:
<div class="radio">
<div class="radio">
<label>
<label>
-
<input type="radio" data-bind="checked: !data.gotProteinStructure, checkedValue: true, click: data.q9A.bind(null, true)" name="ko_unique_2" value="false">
+
<input type="radio" data-bind="checked: data.gotProteinStructure, checkedValue: false, click: data.q9A.bind(null, true)" name="ko_unique_2" value="false">
The 3D structure of my protein is unknown
The 3D structure of my protein is unknown
</label>
</label>
Line 307: Line 307:
<!-- /ko -->
<!-- /ko -->
-
<div data-bind="if: data.q9A() &amp;&amp; !data.gotProteinStructure()">
+
<div data-bind="if: data.q3A() &amp;&amp; !data.gotProteinStructure()">
<h3>How large is your protein?</h3>
<h3>How large is your protein?</h3>
<div class="panel panel-default">
<div class="panel panel-default">
Line 356: Line 356:
<h3>Protocol:</h3>
<h3>Protocol:</h3>
 +
<!--Module 1  -->
 +
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><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.
 +
<!-- Module 2 -->
 +
Get the DNA of the protein you want to circularize.
 +
 +
<!-- 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[ data.testSeveral() || !data.gotProteinStructure() |s] 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>
</div>
</div>
</div>
</div>

Revision as of 08:31, 16 October 2014

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

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
Please save this link:
https://2014.igem.org/#
 Clipboard icon Copy to clipboard
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:

Get BBa_K1362000123NpuDnaE intein RFC ???Sortase Acircularization construct (with FLAG and Smt3)(with His6)(with Smt3 and His6) from the registry. Get the DNA of the protein you want to circularize.

We recommend you to try circularization without a linker and with flexible GS-linkers of different lenghts up to 81015202530 amino acids (including exteins).

Backtranslate the amino acid sequence of your linker[ data.testSeveral() || !data.gotProteinStructure() |s] to a nucleic acid sequence. Be aware of:
  • Balanced GC-content
  • Restriction sites (especially PstI)
  • Codon usage
  • Avoid self annealing

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