Fig. 4

BLA in vivo evolution system and droplet microfluidic screening of BLA mutants with increased activity. a Scheme of the iterative mutagenic process. b Growth of the experimental strain and the control strains cultured for 30 h in liquid enrichment medium containing 2% starch as the sole carbon source after mutagenesis, and the initial OD₆₀₀ was unified to 0.2. No BLA(MutS60), MutS60 strain harboring BLAdel-ori plasmid and Pol I* plasmid; BLA(BL21), BL21(DE3) strain harboring BLA-ori plasmid and no Pol I plasmid; EBLA(MutS60), MutS60 strain harboring BLA-ori plasmid and Pol I* plasmid. c Growth of the mutagenic cultures in the enrichment medium for 24, 19, and 16 h, respectively. d Relative enzyme activity of wild type (grey column) and variants (green columns) sorted by microfluidic devices. e Enzyme activity of the retransformed strains carrying mutant BLA (N473Y) and wild-type BLA after fermentation for 10 h in shake flasks