Ni nta

Cas13a was purified as referred to in previous studies (East-Seletsky et al., 2016 (link), 2017 (link); Su et al., 2020 (link)). Briefly, the Cas13a gene in plasmid pC019-LwCas13a from Leptotrichia wadei (Addgene, United States) was subcloned into the psmarti vector (xhoI restriction site), and the correct plasmid was transformed into BL21(DE3) (General Biosystem, China). The expression of the target protein was induced at different temperatures (15 and 37°C) and different concentrations of IPTG (0.2 and 1.0 mM) (Amresco, United States). The Cas13a protein was purified by Ni-NTA (Smart-Lifesciences, China) using the 6 × His Tag antibody and horseradish peroxidase conjugate (Invitrogen, United States). After the addition of SUMO Protease (General Biosystem, China) to remove the fusion SUMO label, and the purified target protein was dialyzed into protein buffer [50 mM Tris, pH 7.5, 600 mM NaCl, 5% (vol/vol) glycerol, and 2 mM DTT].

The gene of the sensor NS‐Goji 1.3 was constructed in a pET51b (+) vector for recombinant expression in E. coli (DE3). The E. coli (DE3) was cultured 1 L LB medium containing 50 μg/mL ampicillin at 37°C with a shaking rate of 220 rpm to reach an optical density of 0.6–0.8 at 600 nm. Then 0.5 mM isopropyl β‐D‐thiogalactopyranoside (IPTG) was added into the medium to induce sensor expression at 16°C for 16 h. The E. coli (DE3) was collected by centrifugation at 8000 rpm for 15 min and the resulting pellet was suspended in 25 mM Tris–HCl buffer containing 500 mM NaCl and 20 mM imidazole at pH 8.0 for lysis using high‐pressure homogenizer at 4°C. The lysate was centrifuged at 13,000 g for 30 min at 4°C and the resulting supernatant was filtrated by 0.22 μm syringe filters for purification by Ni‐NTA (Smart Lifesciences Lnc., China) and Strep‐Tactin (Smart Lifesciences Lnc., China) columns. The purified protein was desalted and exchanged into 50 mM HEPES buffer containing 50 mM NaCl (pH 7.2) with an Amicon Ultra‐15 centrifugal filter (10 kDa MWCO, Merk Millipore Inc.). Protein concentration was measured by Bradford assay. The obtained protein solution was added into an equal volume of glycerol for long‐term storage at −80°C.