Coding

Part:BBa_K1638014:Design

Designed by: Jens Sivkr Pettersen   Group: iGEM15_SDU-Denmark   (2015-08-12)

hTrx scaffold for peptide aptamers with 3xFLAG-tag


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 100
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Important note: This part cannot be used in the standard backbones pSB1C3 or pSB1K3, as they include a xhoI restriction site. Instead we recommend using the pSB1A2 backbone

Sometimes the scaffold needs to be fused to other proteins, e.g. when using a two-hybrids screening method, as adenylate cyclase-based bacterial two-hybrid system. As protein fusions cannot be accomplished with the RFC[10] standard assembly method (stop codon is included in the scar site), we reccomend doing the following:

  • 1) Design primers for amplification of the hTrx-scaffold with a DNA sequence that includes a linker in the overhang of the forward primer.
  • 1.2) Forward primer: 5'-CGCTTCTAGAGGGATCCGAAAATTTGTATTTTCAATCTGGTNNN...NNN-3' - (XbaI res. site and linker(with BamHI res. site) included)
  • 1.3) Reverse primer: 5'-ATATCTGCAGCGGCCGCTACTAGTANNN...NNN-3' (SpeI and PstI res. site included)
  • 2) Run PCR with designed primers.
  • 3) Digest PCR-products and a standard backbone with XbaI and PstI
  • 4) Ligate backbone and PCR product


Addition of linker_hTrxscaffold to a N-terminal fusion protein (e.g. the T18 and T25 domain):

  • 5) Design primers for amplification of N-terminal domain that includes a 3'-end BamHI res. site.
  • 5.1) Forward: 5'-CGTCTGGAATTCGCGGCCGCTTCTAGAGNNN...NNN-3' (includes EcoRI and XbaI res. site)
  • 5.2) Reverse: 5'-ATATGGATCCNNN...NNN-3' (includes BamHI res. site)
  • 6) Run PCR with designed primers.
  • 7) Digest PCR-products and plasmid containing linker_hTrxscaffold with EcoRI and BamHI.
  • 8) Ligate backbone and PCR product.

BE AWARE: When using the T18 and T25 domain submitted by the SDU Denmark 2015 team, the two domains already include a BamHI res. site. If this is the case, you can skip step 5 and 6.


For designing the random DNA library fitted for this scaffold we recommend doing the following:

  • Choose the size of your library. Typically the length of the variable peptide loop is between 10 and 20 amino acids.
  • Dependent on the size your variable peptide loop, you can for example contruct the library as following: ATATGCGCTCGAG(NNK)xGAGCTCATTACCG (where N represent A, T, G or C and K represent G or C). The NNK codon minimizes the number of stop codons. The XhoI restriction included allows insertion of library into scaffold.
  • Order the library as duplex-DNA
  • When delivered, digest the library and scaffold with XhoI restriction enzyme and ligate.

Source

GenBank: AY004872.1

References

[1] Borghouts C, Kunz C, Delis N, Groner B. Monomeric Recombinant Peptide Aptamers Are Required for Efficient Intracellular Uptake and Target Inhibition. Molecular Cancer Research. 2008;6(2):267-81.