Device

Part:BBa_K1638035:Design

Designed by: Jens Sivk�r Pettersen   Group: iGEM15_SDU-Denmark   (2015-09-09)


hTrx-based scaffold fused to T18 through intein and a flexible linker


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1563
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1229
    Illegal BamHI site found at 711
    Illegal XhoI site found at 2374
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 252
    Illegal NgoMIV site found at 662
    Illegal NgoMIV site found at 2106
    Illegal AgeI site found at 468
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

A fragment of this part (linker_intein_scaffold) was synthesized by IDT as a gBlock gene fragment. This gene fragment was suffixed to the T18 domain of cyaA from Bordetella pertussis by digesting the two parts with BamHI and PstI followed by ligation.


Library

  • 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 the scaffold.
  • Order the library as duplex-DNA
  • When delivered, cut the library and scaffold with XhoI restriction enzyme and ligate.

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

Source

pUT18C
Human thioredoxin sequence: UniProt (AC: P10599)
Intein sequence: pTYB21 (IMPACT kit)

References

[1]: Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proceedings of the National Academy of Sciences of the United States of America. 1998;95(10):5752-6.
[2]: 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.
[3]: IMPACT™ Kit. 2015. New England Biolabs; [accessed 2015 Sep 11]. https://www.neb.com/products/e6901-impact-kit