2.4. In vitro Translation Assay

The inhibition of cell free GFP synthesis by Onc18 and its mutants was evaluated with an E. coli lysate-based transcription-translation coupled assay (RTS100, 5Prime) as previously described.²⁸ The GFP translation inhibition assay is intended to focus on oncocin’s inhibition of the ribosome in contrast to the overall growth inhibition assay. Briefly, transcription-translation reactions with or without oncocin variants were mixed following the manufacturer’s instructions with the exception that the final reaction volume was reduced from 50 μL to 6 μL. Each reaction contained 1.44 μL E. coli lysate, 1.2 μL reaction mix, 1.44 μL amino acids, 0.12 μL methionine, 0.6 μL reconstitution buffer, 0.6 μL GFPmut3 vector,²⁹ and 0.6 μL peptides or control buffer. Reactions were incubated for 4 h at 30 °C in a CFX Connect Real-Time PCR Detection System instrument. GFP expression was quantified through the rate of production in the first 30 min (λ_Exc. = 450-490 nm, λ_Em = 515-530 nm). The production rate was calculated by differentiation of fluorescent signals at different time points. All assays were conducted in triplicate.