Team:Heidelberg/Toolbox Guide/Localization

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Latest revision as of 03:46, 18 October 2014

use the intein
TOOLBOX to

First of all, check the DNA sequence of your proteins for EcoRI, XbaI, SpeI, PstI and BsaI 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 site, the cloning will be more difficult.

Choose the tag you want to add or remove

Please select the organism in that the tag should be used

Which Tag would you like to use?

Do you want to add or remove the Tag

At which Poition do you want to addremove the tag?

Does your protein of interest contain methionin as a first amino acid?

Does your protein of interest contain a stop codon?

How do you want to induce the trans-splicing reaction?

Protocol

  • Get BBa_K1362105 (RBS + NpuDnaE C-intein RFC [] assembly construct) from the registry of standard biological parts.
  • Get BBa_K1362104 (RBS +NpuDnaE N-intein RFC [] assembly construct) from the registry registry of standard biological parts.
  • Get your tag from the registry of standard biological parts.
  • Get the DNA of your protein of interest.
  • Use Golden Gate Assembly to replace the mRFP selection marker in BBa_K1362105 (RBS + NpuDnaE C-intein RFC [] assembly construct) with the C-terminal tag. Use Golden Gate Assembly to replace the mRFP selection marker in BBa_K1362104 (RBS + NpuDnaE N-intein RFC [] assembly construct) with the N-terminal tag.
  • Design and order primers

    In 5'-3 direction, the forward primer should have the following sequence:

    NNNNNNNNGGTCTCCCAACTGCTGGGAA + binding part NNNNNNNNGGTCTCAGATG + binding part NNNNNNNNGGTCTCAGATG + binding part

    In 5'-3' direction, the reverse primer should have the following sequence:

    binding part + NNNNNNNNGGTCTCTATTA binding part + NNNNNNNNGGTCTCGAGCACTTGCCCCT
    Be careful not to include stop codons of your protein of interest into the binding part.

  • Use PCR to create your insert and purify it.

  • Use Golden Gate Assembly to replace the mRFP selection marker in BBa_K1362105 (RBS + SspDnaE C-intein RFC [] assembly construct) with your C-terminal tag BBa_K1362104 (RBS + SspDnaE N-intein RFC [] assembly construct). With your N-terminal tag.

  • Backtranslate the amino acid sequence of your protein of interest into a nucleotide

  • Design and order primers:

    In 5'-3' direction, the forward primer should have the following sequence:

    NNNNNNGGTCTCAGATG + binding part NNNNNNGGTCTCAG + binding part

    In 5'-3' direction, the reverse primer should have the following sequence:

    NNNNNNNNGGTCTCGAGCACTTGCCCCT + binding part

    Be careful not to include stop codons of your protein of interest into the binding part.

    In 5'-3' direction, the forward primer should have the following sequence:

    NNNNNNGGTCTCCCAACTGCTGGGAA + binding part

    In 5'-3' direction, the reverse primer should have the following sequence:

    NNNNNNGGTCTCTATTA + binding part

  • Use PCR to create your insert and purify it.

  • Use Golden Gate assembly to to exchange the mRFP selection marker in BBa_K1362104 ( SspDnaE N-intein RFC [] assembly construct) with your insert. BBa_K1362105 ( SspDnaE C-intein RFC [] assembly construct) with your insert.

    Since there is a BsaI site in your protein, you have to religate after the Golden Gate reaction.

  • Clone both assembly constructs into compatible expression vectors.

    Please use: LOV to induce splicing