Isopropyl β d 1 thiogalactopyranoside (iptg)

For bacmid recombination [51 ], chemically competent DH10EmBacY cells were transformed with the expression construct of interest and incubated overnight in the shaker at 37°C in 2 mL of Luria-Bertani (LB) medium. Culture was plated on LB agar plates containing 50 μg/mL kanamycin (NZYTech), 10 μg/mL tetracycline (AppliChem), 10 μg/mL gentamycin (Sigma-Aldrich), 0.5 mM IPTG (NZYTech) and 40 μg/mL X-Gal (NZYTech), and incubated at 37°C. White colonies containing recombined bacmid were picked and cultured overnight in LB medium supplemented with kanamycin, tetracycline, and gentamycin in the shaker at 37°C. Bacmid DNA was isolated by alkaline lysis followed by isopropanol precipitation. For virus production [51 ], Sf21 cells in 6-well plates (10⁶ cells/well) were transfected with bacmid DNA using X-tremeGene HP DNA Transfection Reagent (Roche). 50 hr after transfection, the supernatant containing initial virus V0 was collected and used to infect a 25-mL culture (SFM4 medium, Hyclone) of Sf21 cells (0.5 × 10⁶ cells/mL) to produce V1 virus. Culture density was kept at 1 × 10⁶ cells/mL (diluted with fresh medium when needed) until cell proliferation ceased. 48 hr after proliferation arrest, V1 was harvested by centrifuging the cell suspension (5 min at 800 × g) and collecting the supernatant. V1 stock was stored at 4°C in the dark.