Inducible Protein Expression in S2060 Cells

S2060 cells were transformed with the AP(s) and CP(s) of interest as described above. Overnight cultures of single colonies grown in 2xYT media supplemented with maintenance antibiotics were diluted 500-fold into DRM media^{17 (link)} with maintenance antibiotics in a 96-well deep well plate (Axygen). The plate was sealed with a porous sealing film and grown at 37˚C with shaking at 230 RPM for 4 h, whereupon the culture reached OD₆₀₀ ~ 0.4–0.6. The cells were then induced with 100 ng/mL anhydrotetracycline (aTc; Fluka) and 5 μM arabinose (Gold Biotechnology) before incubation for an additional 1 h at 37˚C with shaking at 230 RPM. 120 μL of cells was transferred to a 96-well black-walled clear-bottomed plate (Costar), then 600 nm absorbance and luminescence were read using an Infinite M1000 Pro microplate reader (Tecan). OD600-normalized luminescence values were obtained by dividing raw luminescence by background-subtracted 600 nm absorbance. The background value was set to the 600 nm absorbance of wells containing DRM only.