Part:BBa_K3738024
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 6xHistidine Tag and C-Terminal Anionic Tag coding region (BBa_K3738020), and double terminator BBa_B0015.The part is codon-optimized for use in E. coli It is improved from the Lethbridge High School iGEM 2019's Parts BBa_K3001003, BBa_K3001000 and BBa_K3001002 by introducing the anionic MS2 phage-like-particle uptake anionic tag as well as the transcriptional and translational regulators for optimal overexpression and 6XHistidine tag required for nickel affinity chromatography purification.
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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1162
Illegal PstI site found at 2017
Illegal PstI site found at 2956 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1162
Illegal PstI site found at 2017
Illegal PstI site found at 2956 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1162
Illegal PstI site found at 2017
Illegal PstI site found at 2956 - 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]
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