Dual luciferase assay

Frameshifting was assayed by dual luciferase assay using MG, the A, the B and ΔAB strains in ΔlacIΔlacZ background transformed with pHB plasmids expressing Rluc-Fluc construct variants (Table ​(Table1).1). Bacterial cells were grown in M9 medium supplemented with 0.4% sorbitol (weight/ volume) and chloramphenicol (final concentration 10 μg/ml) at 30 °C overnight. Overnight cultures were diluted to A₆₀₀ 0.1 in the fresh M9 medium without chloramphenicol (final volume 3 ml) and grown at 30 °C to mid-logarithmic phase (A₆₀₀ 0.4). Samples (1 ml) were taken, cells were collected by centrifugation (4,000 rpm, 4 min, 4 °C), flash-frozen in liquid nitrogen and kept under − 80 °C until bioluminescent measurements with Dual-Luciferase Reporter (DLR) Assay System. The expression of Rluc–Fluc fusion protein was induced by adding 10 μl arabinose (final concentration 0.2%) to 1 ml of the remaining culture. After 1 h of induction at 30 °C cells were processed as described above for pre-induction samples. Frozen cells were resuspended in 400 μl Passive Lysis Buffer of Dual-Luciferase Reporter Assay System and incubated on ice for 10 min. Next, 40 μl of the extract was assayed for Firefly and Renilla luciferase as described in Lilleorg et al. 2017. Fluc activity (relative units) was calculated as the ratio of the Fluc to Rluc activity. Statistical significance was determined by the unpaired two sample Student's t test at a significance level of 0.05.