Isopropyl β d thio galactosidase iptg

A pET28b plasmid encoding the Δ3B1-3B2-3B3-3C-His6 sequence from FMDV A₁₀61 with two mutations to enhance enzyme solubility (C95K/C142A) was obtained from Stephen Curry at Imperial College London (50 (link)). Escherichia coli BL21(DE3) pLysS was transformed with this plasmid and grown to an optical density at 600 nm of 0.4 to 0.7 absorbance units. Expression was induced by the addition of 1 mM isopropyl-β-d-thio-galactosidase (IPTG; Sigma) for 4 h at 37°C. Bacteria were harvested by centrifugation and resuspended in a lysis buffer containing 50 mM HEPES, pH 7.1, 200 mM NaCl, 1× HALT protease inhibitor cocktail (Thermo Fisher Scientific), 1% Igepal CA-630 (Sigma), 100 μg/ml lysozyme, 100 μg/ml DNase (Invitrogen) on ice for 1 h. Lysates were subjected to 12, 30-s sonication cycles at an amplitude of 10 μm (Microson XL-2000; Misonix) with 30 s on ice between each sonication. Lysates were clarified by centrifugation and His-tagged 3C^pro purified by nickel ion affinity chromatography using HisTrap columns (GE Healthcare). Elution fractions were analyzed by SDS-PAGE, and fractions with the highest concentration of purified protein were pooled and dialyzed against 50 mM HEPES, pH 7.1, 0.2 M NaCl, 1 mM EDTA, 1 mM β-mercaptoethanol, and 5% glycerol. Purified dialyzed proteins were stored at −80°C.

All cultures were grown in Lennox Luria-Bertani Broth (LB) (Sigma-Aldrich). Media was supplemented with amp (100 μg/mL, Sigma-Aldrich) to maintain a selection of sgRNA plasmids, or supplemented with cm (35 μg/mL, Sigma-Aldrich) to maintain a selection of dCas9/dCas9-ω/pEP Pol1 plasmids. Unless noted, amp and cm were always included in media. Growth of gene knockout strains was performed without supplementation of any antibiotic. Expression of dCas9/dCas9-ω during experiments was driven by supplementation of 50 ng/mL anhydrotetracycline (aTc, Sigma-Aldrich). Expression of error-prone Pol1 during experiments was driven by supplementation of 100 μM Isopropyl-β-D-thiogalactosidase (IPTG, Sigma-Aldrich). All cultures were grown at 37°C, with shaking at 225 rpm unless otherwise noted. Growth at 30°C was used during cloning of the error-prone strains in order to drive expression of wild-type Pol1.