Journal: eLife

Article Title: Altered expression of a quality control protease in E. coli reshapes the in vivo mutational landscape of a model enzyme

doi: 10.7554/elife.53476

Figure Lengend Snippet: Figure 1. E. coli DHFR deep mutational scanning uncovers many advantageous mutations. (A) Turbidostat schematic. Reoccurring dilutions with fresh medium keep the culture optical density (OD600) below 0.075. (B) The selection coefficient for each mutant is the slope of the linear regression of allele frequency over time. The wild-type (squares) value is normalized to zero. Advantageous (red) mutations increase and disadvantageous (blue) mutations decrease in frequency. (C) Selection coefficients from deep mutational scanning as a function of enzymatic velocity for purified DHFR point mutants measured in vitro. Velocities at 20 mM DHF were calculated from Michalis-Menten parameters. Error bars reflect the standard deviation from three biological replicates. (D) Histogram of selection coefficients. The wild-type value is indicated with a vertical black line. The median standard deviation over all mutations is the cut-off for WT-like behavior (Materials and methods, Figure 1—figure supplement 3, Figure 1—figure supplement 4) and is indicated with dashed lines. Mutation are colored as advantageous (red), disadvantageous (blue), WT-like (white), or null (grey). (E) Structural model of DHFR (PDB ID: 3QL3) with cross-section slices (a–e) indicated. The DHF substrate (green) and the NADPH cofactor (purple) are represented by spheres (yellow carbons and heteroatom coloring). An arrow indicates the perspective for each slice. (a–e) five cross-section slices. Color scale indicates numbers of advantageous mutations at each position. Crosshatching indicates residues with >20% solvent accessible surface area. The online version of this article includes the following source data and figure supplement(s) for figure 1:

Article Snippet: The pooled and cleaned ligation products were transformed into E. coli Top10 cells by electroporation (BioRad GenePulser Xcell, 1 mm path length cuvette (cat# 165–2089), 1.8 kV, time constant ~5 ms) using ~5 mL to obtain a minimum of 107 transformants as measured by dilution plating on LB-agar plates with 35 mg/mL chloramphenicol.

Techniques: Selection, Mutagenesis, Purification, In Vitro, Standard Deviation, Solvent