Fig. 2.

Mutant clone screening process. The nspS mutant library was transformed into the V. cholerae ∆nspS strain and colonies were individually grown in separate wells of a 96-well deep-well plate (step 1). For each mutant clone, crystal-violet staining was performed to assess biofilm formation for one biological replicate (step 2). Biofilm formation using crystal-violet stain was used again to assess three biological replicates for mutant clones with low biofilm formation in the initial assay (step 3). Mutant clones that produced low biofilms were analysed via Western blot for protein expression (step 4). For clones that had protein expression, a final biofilm assay was performed that measured the optical density of the biofilm to confirm the low biofilm phenotype (step 5). Plasmids from mutant clones that had low biofilm formation and protein expression that was detected via Western blot were purified and sent for sequencing (step 6).