Fig. 4: Study of the long-term performance of the three biosafety systems in clonal M. pneumoniae strains.

a Growth kinetics (growth index corresponding to the ratio Abs430 nm/Abs560 nm) of the clonal strains C5 (I), C20 (II) and C26 (III) analysed at an early (p2) and late passage (p15) at different induction conditions. We compare the growth of strains C5 and C20 (with one and two copies of the kill-switch KS1, respectively) when uninduced at the early passage p2 (0 mM IPTG, blue lines) or IPTG-induced that triggers the kill-switch at the passage 2 (1 mM IPTG; p2, red lines) or passage 15 (1 mM IPTG; p15, orange line). We analysed the growth of strain C26 (with KS1 and TKS2) in theophylline-medium (0.5 mM Theophylline, permissive growth conditions with both circuits inactive, blue line), or theophylline-free medium (0 mM Theophylline, slow activation of TKS2, light brown line) in the early passage p2. The growth is also analysed when both circuits are activated simultaneously in ATc and IPTG medium at passage 2 (1 mM IPTG and 50 ng/mL ATc; p2, red line) or passage 15 (1 mM IPTG and 50 ng/mL ATc; p15, orange line). The black line is a negative control of the growth index reads from an empty medium and shows the baseline of these long experiments, as the Abs560 reads have a drift (SP4, black line). Mean values from three bio-replicates. Error bars indicate standard deviation. b Escape frequency (EF) for strain C5 at several strain passages, determined as the ratio between IPTG-resistant CFUs and the total CFUs. The dashed line shows the detection limit calculated in the analysis, considering the total CFUs plated. Source data are provided as a Source Data file.