Fig. 1
From: Optimization of the antimicrobial peptide Bac7 by deep mutational scanning
Sequence-activity relationship of Bac71-23. a DMS workflow. epPCR: the Bac71-23 gene is amplified at a high error rate using an error-prone DNA polymerase in the presence of Mn2+. Cloning: the mutagenized DNA sequences are inserted into plasmids downstream of inducible promoters. Transformation: E. coli TOP10 is transformed with the generated peptide-encoding DNA library. Growth: the pooled transformants are grown in a single shaking flask (n = 3), peptide synthesis is induced and plasmids are isolated after 4 h. NGS: the abundance of each peptide-encoding DNA sequence is determined by NGS at the time of induction and 4 h later. Analysis: for each peptide-encoding DNA sequence, the log2-fold change is determined (log2 ratio of abundances at the two time points). Histogram showing the log2-fold changes of the abundance of the peptide-encoding DNA of all 601,551 variants. Ranking: peptide sequences are ranked by the degree of the observed antimicrobial effect. The more negative a log2-fold change, the higher the observed antimicrobial effect and vice versa. b Bac71-23 sequence-activity relationship displaying the magnitude of the observed antimicrobial effect. For each amino acid residue substitution (and stop codon), the enrichment in higher or lower antimicrobial peptides is determined and a z-score (z) is calculated (see the “Methods” section). z corresponds to the number of standard deviations by which the calculated enrichment lies above (positive values) or below (negative values) the mean a null distribution indicating no enrichment. z is empirically divided into four groups, corresponding to very positive (yellow; z ≥ 40), positive (green; z ≥ 4), negative (blue; z ≤ -4), or very negative (purple; z ≤ −40) effects on the antimicrobial activity. No effect on growth inhibition is detectable if the z is close to 0 (white; −4 < z < 4). Black dots are used for indication of Bac71-23 wild-type amino acid residues. The underlined positions are chosen as targets for the subsequent site-saturation mutagenesis.