In vitro Translation of NanoLuc Luciferase

In vitro translation of NanoLuc was performed using a PURExpress ΔRibosome kit (NEB) as previously described (11 (link)). Briefly, 2.0 μl of solution A, 0.6 μl of factor mix, and 18 ng of DNA template (containing the T7 promoter, a 5′ untranscribed region [UTR] with a Shine-Dalgarno sequence, and the NanoLuc coding sequence) were mixed with 12.5 nM purified 70S ribosomes in a total volume of 5 μl. After 1 h of incubation at 37°C, relative luminescence units (RLU) produced from the translated luciferase was measured with a Nano-Glo luciferase assay kit (Promega Inc.) according to the manufacturer’s instructions. The inhibition of translation activity of 70S ribosomes purified from designated strains was assayed by adding the designated antibiotic at specified concentrations in the reaction mixture prior to ribosome addition and measuring luciferase activity after 1 h of incubation at 37°C. A reaction mixture without the antibiotic was used as a reference to determine percent inhibition. The IC₅₀ value from a dose-response curve was obtained from nonlinear regression of log₁₀ inhibitor versus activity plot using the variable slope model of GraphPad Prism software. The 95% confidence interval for each best-fit curve, also computed by Graph-Prism software from the standard errors, represents 95% probability that the computed IC₅₀ value is in the given range.