Device

Part:BBa_K3123000:Design

Designed by: Jay Yung   Group: iGEM19_Washington   (2019-10-12)


CBD Nanobody Luciferase with FKPA


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1977
    Illegal BamHI site found at 1307
    Illegal XhoI site found at 1929
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 290
    Illegal NgoMIV site found at 1404
    Illegal AgeI site found at 711
    Illegal AgeI site found at 1825
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Synthesis of this part requires it to be broken into three fragments, as there is too much repetition between the three coding regions for them to be synthesized as one gBlock. The anchor binder sequence is flanked by a BamHI cut site, the dimer binder sequence is preceded by a BamHI cut site and flanked by a BglII cut site, followed by the biobrick suffix sequence. The FKPA sequence is preceded by a BglII cut site and flanked by the biobrick suffix sequence. This design allows the user to synthesize all three fragments and build either the part described here, or another part our team submitted that does not contain the FKPA sequence. It is also important to note that for DNA synthesis, the FKPA sequence must be preceded and flanked by terminal adapters; our team had multiple synthesis failures without these adapters. The 5' end adapter is 5'CTCACTTGTAGAACGGTGATCAGCCTGTGCTCTAGAGCCTGATAGTTGAGCGATACACAC3'; the 3' end adapter is 5'acatctatggatgtactcagcgagcagatgctctc3'.


Source

This part came from a synthetic library of nanobodies. The library was created from trinucleotide mutagenesis (TRIM) on the nanobodies naturally found in camels. These nanobodies all share a common scaffold, and have variable regions that allow for a variety of binding. After an extensive screening process this nanobody was chosen because of its binding specificity to CBD. The luciferase coding sequence was identified from Oplophorus gracilirostris, a deep sea shrimp. The FKPA sequence was identified from the E. coli strain MC4100.

References

1. Shoukai Kang, Kristian Davidsen, Luis Gomez-Castillo, Huayi Jiang, Xiaonan Fu, Zengpeng Li, Yu Liang, Molly Jahn, Mahmoud Moussa, Frank DiMaio, and Liangcai Gu Journal of the American Chemical Society 2019 141 (28), 10948-10952 DOI: 10.1021/jacs.9b03522
2. Andrew S. Dixon, Marie K. Schwinn, Mary P. Hall, Kris Zimmerman, Paul Otto, Thomas H. Lubben, Braeden L. Butler, Brock F. Binkowski, Thomas Machleidt, Thomas A. Kirkland, Monika G. Wood, Christopher T. Eggers, Lance P. Encell, and Keith V. Wood ACS Chemical Biology 2016 11 (2), 400-408 DOI: 10.1021/acschembio.5b00753