Type III-A CRISPR-Cas effector ribonuclease - Csm6 with Histag and TEV cut site
Csm6 is a non-specific endoribonuclease that cleaves single-stranded RNA upon activation by cyclic adenylates or linear adenine homopolymers terminated with a 2'3'-cyclic phosphate.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NotI site found at 501
- 21Illegal BglII site found at 461
Illegal BglII site found at 1403
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC
Cloning and characterization
From IDT, gene blocks with a 6x-His-tag, tobacco etch virus protease cut site, and restriction enzyme cut sites on the 5' and 3' end for NcoI and BlpI, respectively was oredered. After digesting the gene segments with restriction enzymes, they were ligated to the pET-52b expression vector. The assembled plasmid was transformed into a C41 pLysS cell line. Colonies were grown on selective media to screen the cells containing transformed plasmids containing Csm6 gene. Colony PCR was performed to make sure that the transformed plasmids were correct in insertion and orientation.
As the results of the colony PCR for TtCsm6 is a bit unclear, both the teams decided to run a PCR amplification of the isolated plasmid from the positive colonies with the primers same as that of the colony PCR. This would confirm the presence of insert in the isolated plasmids.
As we can see, both the isolated plasmids have the intended lengths of insert reflecting successful cloning and plasmid isolation. UCSC Igem team 2021 used those colonies to purify protein by stimulating expression overnight with IPTG, followed by cell lysis and sonication, but the final results were not available before the wiki freeze.