E coli krx cells

Recombinant Pum and Nos for EMSAs were expressed in KRX E. coli cells (Promega) in 2xYT media with 25 µg/mL Kanamycin and 2 mM MgSO₄ at 37°C to OD₆₀₀ of 0.7–0.9, at which point protein expression was induced with 0.1% (w/v) rhamnose for 3 hr. Cell pellets were washed with 50 mM Tris-HCl, pH 8.0, 10% [w/v] sucrose and pelleted again. Pellets were suspended in 25 mL of 50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 2 mM MgCl₂, 150 mM NaCl, 1 mM DTT, 0.05% (v/v) Igepal CA-630, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml pepstatin, and 10 µg/ml leupeptin. To lyse cells, lysozyme was added to a final concentration of 0.5 mg/mL and cells were incubated at 4°C for 30 min with gentle rocking. MgCl₂ was increased to 7 mM and DNase I (Roche) was added to 10 µg/mL followed by incubation for 20 min. Lysates were cleared at 50,000xg for 30 min at 4°C. Halo-tag containing proteins were purified using HaloLink Resin (Promega) at 4°C. Beads were washed 3 times with 50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 2 mM MgCl₂, 1 M NaCl, 1 mM DTT, 0.5% [v/v] Igepal CA-630) and 3 times with Elution Buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM DTT, 20% [v/v] glycerol). After washing, beads were resuspended in Elution Buffer with 30 U of AcTEV protease (Invitrogen), cleavage proceeded for 24 hr at 4°C, and beads were removed by centrifugation through a micro-spin column (Bio-Rad).

The DNA sequence corresponding to the CyanoQ homologue of T. elongatus (tll2057) without the sequence encoding the predicted signal peptide and lipid-binding Cys24 residue was cloned into a pRSET-A vector modified as described in Bialek et al. (2013 (link)). The corresponding PCR fragment was amplified from T. elongatus genomic DNA using Phusion polymerase (NEB, UK) and primers CyanoQ-XhoI-F (5′-TATATACTCGAGGGCGGCCCCAGTGCCACCACTCCACCCCCACCCACCTA-3′) and CyanoQ-EcoRI-R (5′-TATATAGAATTCTTACTAGGACAACTCAGGCAAGCTGTTGAGAT-3′) introducing underlined restriction sites, double digested with XhoI and EcoRI and ligated (Quick Ligation Kit, NEB, UK) into the modified and XhoI/EcoRI linearised pRSET-A. The vector was then transformed into KRX E. coli cells (Promega, UK).