Team:Heidelberg/Toolbox Guide

From 2014.igem.org

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<h3>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.</h3>
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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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<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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There is no BsaI site.
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There is a BsaI site.
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There is a BsaI site.
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<h3>2. Do you want to use split inteins or sortase to circularize your protein?</h3>
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<h3>Do you want to use split inteins or sortase to circularize your protein?</h3>
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<h3>3. Can your protein be easily expressed in <i>E. coli</i>?</h3>
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<h3>Can your protein be easily expressed in <i>E. coli</i>?</h3>
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<h3>4. Which exteins do you want to use? They will remain as scars in your circular protein.</h3>
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<h3>Which exteins do you want to use? They will remain as scars in your circular protein.</h3>
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<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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<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>
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<h3><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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<h3>Hello again. What is the result of <span data-bind="text: softwareName"></span>?</h3>
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<h3><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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<h3>Have you decided to use one linker or try different linkers?</h3>
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Revision as of 08:49, 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.

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.

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

Can your protein be easily expressed in E. coli?

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:

Get BBa_K1362000123 NpuDnaE intein RFC Sortase A circularization 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 linkers 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"