Figure 3.

Screening of A24X and T27X libraries. (a) A24X and T27X mutant libraries were constructed and 90 colonies were seeded into 96-well plates. Biological triplicates of AMP were produced and applied (as 10% v/v) to 10⁶ CFU/mL E. faecium 8. Pathogen cell density (OD₆₀₀) after 9 h of AMP exposure is plotted as average ± standard error. Seventeen derivative AMPs were selected for additional testing based on their activity compared to the parental enterocin A. (b) The 17 functional clones were retested to identify false positives, and eighteen random ineffective AMPs were retested to identify false negatives. Average and standard error (n = 3) are shown for both the initial (solid) and the verification (empty) assays. The mutant amino acid and codon (if different—otherwise Supplementary Table S3) are indicated. ^† denotes a mutation in the amino acid sequence in either the secretion peptide or immunity gene for entA.