Fig. 4. Characterization of synthetic GRNs driving pneumococcal capsule production in traits associated with virulence.

a Pneumococcal biofilm formation was measured by growing strains in microtiter plates at 34 °C. After 6 h, biofilm formation to the wells was quantified using crystal violet staining (see Methods). The amount of biofilm formed by each strain was compared to wild type S. pneumoniae D39V using a two-tailed Wilcoxon signed rank test (n = 9). b The ability of the engineered CAPSUlator strains to adhere to human nasopharyngeal epithelial Detroit-562 cells was tested by infecting a monolayer of cells at an MOI of 5. After 1 h of incubation at 37 °C the non-adherent and adherent bacteria were enumerated by plating (see Methods). The ratio of adherent vs non-adherent bacteria is shown and compared to wild type D39V using a Kruskal–Wallis test (n > 4). c Bacterial survival during starvation was tested by resuspending exponentially growing cells in 1 x PBS followed by incubation at 25 °C. Viable bacteria were quantified by plating and colony counting (see Methods). After 24 h of starvation, all synthetic GRNs except for the CAPSUlator-ON strain showed significantly reduced survival compared to wild type D39V (two-tailed Mann–Whitney test). *p < 0.05, **p < 0.01 (n > 3). Data are presented as mean values +/− SD. Each symbol represents a biologically independent replicate. Source data are provided as a Source Data file. a.u. arbitrary units.