Fig. 4. Platform for high-throughput screening of BAM inhibitors.

a Scheme for upscaling of BAM-OMV production. BAM-OMV expression is carried out in a 100 L fermentation culture in LB medium. After expression, LB cultures are passed through a continuous-flow centrifugation step for harvesting. The supernatant is then filtered to eliminate remaining cell debris and intact cells, and concentrated 25-fold by cross-flow microfiltration with a 30 kDa cut-off. The concentrate is then ultracentrifuged to collect BAM-OMVs which are further washed, flash-frozen in liquid nitrogen and stored at −80 °C. b Dose response curve of BAM-mediated OmpT folding in BAM-OMVs at variable darobactin concentration. The IC₅₀ of darobactin is 0.23 μM. c Slopes of neutral (blue) and 100% inhibition control (green) reactions carried out in an Aurora 1536 well plate, representing a robust sensitivity window with a z’ factor of 0.7. d Schematic work flow for high-throughput screening for BAM inhibitors. An end-point measurement is done for all compounds, then a dose-response curve is recorded for initial hits and inhibition constants are calculated. The data underlying panels (b–c) are provided as Source Data. Figure 4a was created with BioRender.com.