Coding

Part:BBa_K3381004:Design

Designed by: Jeriann Hsiao   Group: iGEM20_Waterloo   (2020-10-20)

FutA2-CBM2a


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1442
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The His-tag and TEV linker sequences were codon optimized for E. coli, removing all cut sites for RFc 10 and NdeI/BamHI (required for cloning into pET 11a). Then a 5-poly-AT + NdeI prefix and a BamHI + 5-poly-AT suffix were added to facilitate cloning into the pET 11a vector by standard assembly. Lastly, the prefix and suffix for RFc 10 were appended.


Source

Sources of DNA sequences, in order of occurrence in the fusion protein from N' to C':

His-tag and TEV linker sequence: Retrieved from His-tag and TEV linker that occurs in bacterial expression vector pMCSG7: https://plasmid.med.harvard.edu/PlasmidRepository/file/sequence/pMCSG7.gb

Sequence of FUTA2 (iron-binding domain) retrieved from Genbank (Synechocystis species PCC 6803): https://www.ncbi.nlm.nih.gov/nuccore/BA000022.2?from=291626&to=292708

Sequence of the CBM linker domain was back-translated from its amino acid sequence, which was retrieved from the following paper describing a natural occurrence of the linker in a protein with a cellulose-binding domain: https://www.jbc.org/content/293/34/13006.full

Sequence of CBM2a (cellulose-binding domain) retrieved from DNA sequence of BBa_K863101 (Cellulose-binding domain of Cellulomonas fimi exoglucanase): https://parts.igem.org/Part:BBa_K863101


References

Courtade, G., Forsberg, Z., Heggset, E. B., Eijsink, V. G., & Aachmann, F. L. (2018, August 24). The carbohydrate-binding module and linker of a modular lytic polysaccharide monooxygenase promote localized cellulose oxidation. Retrieved September 30, 2020, from https://www.jbc.org/content/293/34/13006.full

Koropatkin, N., Randich, A. M., Bhattacharyya-Pakrasi, M., Pakrasi, H. B., & Smith, T. J. (2007, September 14). The Structure of the Iron-binding Protein, FutA1, from Synechocystis 6803. Retrieved September 30, 2020, from https://www.jbc.org/content/282/37/27468.full

Zhou, J., Chen, J., Zhuang, N., Zhang, A., Chen, K., Xu, N., . . . Jiang, M. (2020, May 12). Immobilization and Purification of Enzymes With the Novel Affinity Tag ChBD-AB From Chitinolyticbacter meiyuanensis SYBC-H1. Retrieved September 30, 2020, from https://www.frontiersin.org/articles/10.3389/fbioe.2020.00579/full