Part:BBa_K3738026
Lbu-Cas13a with an N-Terminal 6xHistidine Tag and C-Terminal Anionic Tag
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).
Lbu-crRNA (Lbu signifying Leptotrichia buccalis, the source of the Cas13a protein and crRNA) is an RNA structure containing a direct-repeat stem loop, a recognition element for binding with the Lbu-Cas13a protein. The crRNA also contains a downstream complementary region, designed to base-pair with a target ssRNA sequence. Complex formation of crRNA-Cas13a occurs, and when the target sequence is perfectly paired with the crRNA, activation of the enzyme occurs and subsequent non-discriminate cleavage of collateral ssRNAs (O’Connell., 2019).
This crRNA was designed to bind with an important protein in the synthesis of harmful toxins called microcystins produced by blue-green algae (cyanobacteria) blooms. McyH is a gene in the Mcy gene cluster of Microcystic Aeruginosa and codes for a transporter protein. The protein is comprised of two homodimers, each with a hydrophobic N-terminus domain and C-terminus containing an ATPase domain. Pearson et al., 2004 experimentally examined the impacts of impairing expression of this gene, and combined with bioinformatic data, found that McyH is likely a vital exporter of harmful microcystins and essential in their biosynthetic pathway. For this reason, we have designed crRNA to target the 5’-end of McyH in order to inhibit production of microcystins.
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, followed by a second T7 promoter (BBa_J64997), McyH complementary Lbu-crRNA (BBa_K3738025), and a second BBa_B0015 terminator. 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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1089
Illegal PstI site found at 1944
Illegal PstI site found at 2883 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1089
Illegal PstI site found at 1944
Illegal PstI site found at 2883 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 370
Illegal BglII site found at 1306
Illegal BglII site found at 1516
Illegal BglII site found at 1570
Illegal BglII site found at 1801
Illegal BglII site found at 2074
Illegal BglII site found at 2560 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1089
Illegal PstI site found at 1944
Illegal PstI site found at 2883 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1089
Illegal PstI site found at 1944
Illegal PstI site found at 2883
Illegal NgoMIV site found at 2022
Illegal NgoMIV site found at 2658 - 1000COMPATIBLE WITH RFC[1000]
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