Fig. 5

Phenotypic effects of single synonymous codon mutants in the background of an N-terminal hyperactive synonymous mutation, inferred through deep sequencing. A Correlations between the biological replicates. The fraction of each mutant normalised to the ‘WT’ value is estimated for all mutants in resistant (extreme left panel), sensitive (middle panel) and RelE reporter strains (extreme right panel). The (0,0) point represent the WT B In vivo activities of a few individual mutants to validate deep sequencing inferred phenotypes in a low-throughput manner, by transforming individual constructs in resistant (Top10Gyr) and sensitive (Top10) strains (left panel). These mutants are made in the background of WT K4_AAA. Mutants are tenfold serially diluted and spotted on LB Agar plates. ES´^CcdB scores for individual mutants obtained from deep sequencing are mentioned in the bottom panel. Y6_TAA and WTF’ are used as controls. Y6_TAA is a stop codon mutation at the sixth position of CcdB, thereby making it non-functional. WTF’ is the WT ccdAB operon sequence as present in the F plasmid. These two should grow equally well in both resistant and sensitive strains. A correlation of r = 0.95 (P < .001) is observed between the ES´^CcdB scores and normalised CFU count of mutants relative to WT in the sensitive (Top10) strain obtained in the spotting assay. Error bars for the duplicate experiments are also shown (right panel). C Effects of double-site synonymous mutations as a function of location in the gene. ES´^CcdB scores averaged over all mutants for each position. Black dashed line represents the WT K4_AAA score. As done previously, the entire data is divided into N-terminal (1–13), middle (14–86) and C-terminal (87–101) region