Difference between revisions of "Part:BBa K3738024"

Revision as of 17:09, 20 October 2021

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

Cas13a is an enzyme originating from Leptotrichia buccalis (Lbu) which functions to cleave single-stranded RNAs (ssRNAs); particularly mRNAs. This function is achieved following protein-RNA complex formation with CRISPR RNA (crRNA) via crRNA backbone contacts with residues from the Helical-2, HEPN1, and Linker domains of Cas13a. The crRNA contains a spacer region coding for a direct repeat stem loop as well as a region complementary to target ssRNAs. Once the enzyme complex interacts with a target ssRNA, a structural conformation change occurs within the domains of the protein that permits active site formation for non-discriminate ssRNA cleavage (O’Connel et al., 2019).

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.

O'Connell MR. Molecular Mechanisms of RNA Targeting by Cas13-containing Type VI CRISPR–Cas Systems. Journal of Molecular Biology. 2019;431(1):66-87. doi: https://doi.org/10.1016/j.jmb.2018.06.029.

Sequence and Features