Fig. 1: P. putida S16 ΔcycN provides a platform for genetic selection.
From: Directed evolution unlocks oxygen reactivity for a nicotine-degrading flavoenzyme
a, Growth of wild-type P. putida S16 and ΔcycN in liquid culture with nicotine as the sole carbon source. The mean is plotted with error bands that represent the s.d. of three biological replicates; A600, absorbance at 600 nm. b, Growth of wild-type P. putida S16 in liquid culture with 3 mM nicotine as a sole carbon source is shown in solid black. Growth of P. putida S16 ΔcycN strains transformed with the wild-type sequence of nicA2 in the same medium is shown by the dashed black, and the growth of nicA2 variants coming from the selection is shown in the colored curves with their allele numbers indicated. Note that the color scheme of the variants carries over between the growth curves and the data in c and d. The mean is plotted with error bands that represent the s.d. of three biological replicates. c, nicA2 variants isolated from different generations of the selection were purified and characterized for their encoded mutations and steady-state kinetic parameters (Supplementary Table 1 and Extended Data Fig. 1a). The steady-state kinetic assays in this study were performed under ambient conditions, meaning that the kcat values discussed here actually represent an apparent kcat for nicotine turnover at an oxygen concentration of roughly 250 µM. Note that the y axis has a logarithmic scale; WT, wild type. d, The maximum growth rate of variants transformed in P. putida S16 ΔcycN tested in b plotted against the determined kcat of each variant. The mean is plotted with error bars that represent the 95% confidence intervals of three replicates for each value; ΔA, change in absorbance.
