Composite

Part:BBa_K3738024:Design

Designed by: Mark Lea   Group: iGEM21_Lethbridge   (2021-10-20)


Lbu-Cas13a with an N-terminal Anionic Tag and C-Terminal Histidine Tag (Composite Part)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1162
    Illegal PstI site found at 2017
    Illegal PstI site found at 2956
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1162
    Illegal PstI site found at 2017
    Illegal PstI site found at 2956
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 443
    Illegal BglII site found at 1379
    Illegal BglII site found at 1589
    Illegal BglII site found at 1643
    Illegal BglII site found at 1874
    Illegal BglII site found at 2147
    Illegal BglII site found at 2633
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1162
    Illegal PstI site found at 2017
    Illegal PstI site found at 2956
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1162
    Illegal PstI site found at 2017
    Illegal PstI site found at 2956
    Illegal NgoMIV site found at 2095
    Illegal NgoMIV site found at 2731
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part has been codon optimized for use in E. coli. The anionic tag is present to facilitate uptake into our delivery particle MS2 whereas the Histidine tag was added for the purpose of Nickel-Affinity chromatography in obtaining the purified protein.

This composite part includes the IPTG-inducible T7 promoter (BBa_J64997), RBS BBa_B0034, Lbu-Cas13a with an N-terminal Anionic Tag and C-Terminal 6xHistidine Tag coding region(BBa_K3738021), and double terminator BBa_B0015.



Source

The Cas13a protein comes from the Gram-negative bacteria Leptotrichia buccalis (Lbu).

References

Glasgow, J., Capehart, S., Francis, M., and Tullman, D (2012) Osmolyte-Mediated Encapsulation of Proteins inside MS2 Viral Capsids. ACS Nano. 10, 8658-8664.

Huynh, N., Depner, N., Larson, R. et al. A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila. Genome Biol 21, 279 (2020). https://doi.org/10.1186/s13059-020-02193-y

McDade Joel. CRISPR 101: Targeting RNA with Cas13a (C2c2). Retrieved from https://blog.addgene.org/crispr-101-targeting-rna-with-cas1