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Part:BBa_K215002

Designed by: Jeff Nivala   Group: iGEM09_Washington   (2009-09-30)
Revision as of 05:47, 1 October 2009 by Siegeljb (Talk | contribs)

pLac+RBS+Secretion Signal and Streptavidin Binding Tags

Any favorite protein (afp) can be inserted into a composite tag BBa_K215001. The composite tag has an NheI (compatible with XbaI and SpeI, but make sure the resulting product stays in frame, described below) which places the afp in between a series of tags which can be used with the University of Washington 2009 Ideal Protein Purification system (IPP). The series of tags are a Nano tag (binds to streptavidin), a 6x His tag (for IMAC purification), a TEV cute site (for removal of N-terminal tags), the NheI site (insertion of afp), TEV, 6x His tag, the 181 C-terminal amino acids of prtB (secretion tag recognized by the secretion system: BBa_K215107). In order for the secretion tag to signal the secretion of afp the BioBrick BBa_K215107 must be present as well.

Cutting a BioBrick coding sequence at X and S and pasting into the NheI site of this construct will result in a stop codon at the resulting scar site. Therefore, to insert a gene a PCR product must be generated that keeps the gene of interest in frame with the rest of the sequence. To do this:

  1. Design a primer that complements the coding sequence of interest starting at the second amino acid and ending in frame before the stop codon.
    1. Forward Primer: <8 random bp's> - <NheI/XbaI/SpeI> - <15-21bp complementing your gene of interest, starting at the second codon>
    2. Reverse Primer: <15-21bp complementing your gene of interest, ending BEFORE the stop codon (make sure it is a muliple of three to stay in frame)> - <NheI/XbaI/SpeI> - <8 random bp's>
  1. Amplify the gene of interest
  2. Clone into the NheI site of this construct.
  3. Screen colonies with either the Forward Primer+VR or the VF2+Reverse primer for inserts in the appropriate direction.
  4. Your fusion protein is tagged and ready for secretion and streptavidin binding!


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 196
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
n/apLac+RBS+Secretion Signal and Streptavidin Binding Tags