Composite

Part:BBa_K3738023:Design

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


Lbu-Cas13a with an N-terminal 6xHistidine Tag and C-Terminal Anionic Tag (Composite Part)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1134
    Illegal PstI site found at 1989
    Illegal PstI site found at 2928
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1134
    Illegal PstI site found at 1989
    Illegal PstI site found at 2928
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 415
    Illegal BglII site found at 1351
    Illegal BglII site found at 1561
    Illegal BglII site found at 1615
    Illegal BglII site found at 1846
    Illegal BglII site found at 2119
    Illegal BglII site found at 2605
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1134
    Illegal PstI site found at 1989
    Illegal PstI site found at 2928
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1134
    Illegal PstI site found at 1989
    Illegal PstI site found at 2928
    Illegal NgoMIV site found at 2067
    Illegal NgoMIV site found at 2703
  • 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 part is an improved part, based off of the existing part BBa_K3001003 from the 2019 Lethbridge High School iGEM Team.



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