Cas13a characterization
Protein cloning, expression and purification
We decided to compare 3 versions of Cas13a that were previously characterized in the literature [Gootenberg, Doudna]: Lbu, Lsh, and Lwa. We ordered the Lbu and Lsh plasmids from Addgene, and we cloned Lwa using Golden Gate assembly (sequence was taken from Gootenberg). Lbu and Lsh were expressed in E.coli Rosetta2, as the sequences were not codon-optimized, and Lwa was expressed in E.coli BL21 (DE3) star. We created three Biobricks from the Lwa sequence: BBa_K2323000 (containing the Lwa coding sequence and a Tphi terminator), BBa_K2323001 (where a 6xHis/Twin strep tag and a SUMO tag are added to the N-terminal end of BBa_K2323000), and BBa_K2323004 (where BBa_K2323001 is preceded by the T7 promoter and the Elowitz RBS). We improved the TEV-protease BBa_K1319008 by tagging it with a 6xHis tag, purified it and successfully used it for the TEV cleavage of our Cas13a proteins.
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