Fig. 1: Cloning, expression, and purification of the L. delbrueckii subsp. bulgaricus Csm complex in E. coli. | Cell Discovery

Fig. 1: Cloning, expression, and purification of the L. delbrueckii subsp. bulgaricus Csm complex in E. coli.

From: Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase

Fig. 1

a Schematic of the LdCsm system. LdCsm genes and the adjacent CRISPR assay are indicated with filled large arrows and small rectangles, respectively. Line with an arrowhead denotes the promoter of the csm gene cassette and the direction of transcription. b Strategy for reconstitution of the LdCsm effector in E. coli. LdCsm genes (cas6 + csm1–5 genes) were cloned into p15AIE, yielding p15AIE-cas (Supplementary Fig. S2). A CRISPR array carrying 10 copies of S1 spacer was generated and inserted into pUCE, giving pUCE-S1 (Supplementary Fig. S2). LdCsm2 was cloned into pET30a, giving pET30a-Csm2 that yields the His-tagged Csm2 upon plasmid-born gene expression in the cell. All three plasmids were introduced into E. coli BL21(DE3) by electroporation. c UV spectrum of SEC purification of LdCsm effector complex. E. coli cell extracts were employed for Nickel-His tag affinity purification of LdCsm2 by which LdCsm effector complexes were copurified. The resulting protein samples were further purified by SEC. Blue: UV absorbance at 280 nm; red: UV absorbance at 254 nm. d SDS-PAGE analysis of SEC samples collected in the peak region. M: protein mass marker; Input: proteins purified by nickel Csm2-His affinity chromatography. e Denaturing gel electrophoresis of 5ʹ-labeled RNAs from LdCsm samples. RNAs were extracted from the SEC-purified LdCsm samples. M: RNA size ladder.

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