Ampicillin

The SARS-CoV-2 M^pro-encoding sequence were cloned into pGEX-4T1 vector (Genscript) with N-terminal self-cleavage site (SAVLQ/SGFRK) and at the C-terminus, the construct codes for the human rhinovirus 3 C PreScission protease cleavage site (SGVTFQ ↓ GP) connected to a His6 tag. The plasmid constructs were transformed into BL21 Star™ (DE3) cells (Thermo Fisher Scientific). The cultures were grown in Terrific Broth media supplemented with ampicillin (Quality Biological, Gaithersburg, MD). Protein expression was induced by adding 1 mM iso-propyl beta-D-thiogalactopyranoside at an optical density of 0.8 at 600 nm and the cultures were maintained at 20 °C overnight. SARS-CoV2 M^pro were purified first by affinity chromatography using TALON™ cobalt-based affinity Resin (Takara Bio). The authentic N-terminus is generated by 3CL M^pro autoprocessing during expression, whereas the authentic C-terminus is generated by the treatment with PreScission protease and the resulting authentic 306 amino acid M^pro were further purified by SEC using a HiLoad Superdex 200 pg column (GE Healthcare) in 20 mM Tris, pH 7.5, 150 mM NaCl, and 2 mM DTT. Finally, the purified and concentrated SARS-CoV M^pro (6.6–8.15 mg/mL) was stored in 200 mM ammonium acetate (pH 6.7).

Total mRNA was extracted from freshly dissected anal fins of 80 anesthetized glass catfish using the FastTrack 2.0 mRNA Isolation kit (Life Technologies). The cDNA library was constructed in pDONR222 using the CloneMiner II cDNA Library Construction kit (Life Technologies). The final cDNA library was cloned into pcDNA-DEST40 by LR recombination, transformed into One Shot TOP10 Chemically Competent cells (Life Technologies), and stored as glycerol stocks in 500 µL aliquots at −80 °C. The cDNA sub-libraries were constructed by replica plating. A 500 µL glycerol stock of the total cDNA library was added to 5 mL LB (Quality Biological) containing 100 mg/mL Ampicillin (Sigma-Aldrich) and shaken at 225 rpm, 37 °C for 1 hour. The total volume was evenly plated on ten 10-cm Ampicillin plates (Quality Biological) and incubated overnight at 37 °C. The following day, the ten plates were replica plated to a second set of ten plates using nitrocellulose membranes and each nitrocellulose membrane was submerged in a flask containing 50 mL LB. The plates and flasks were incubated overnight at 37 °C, cDNA sub-libraries were purified from the flasks’ inoculums, and glycerol stocks were prepared.