Mutational Tolerance

Growth/Fitness

Fitness

Unitless

Deep Mutational Scanning

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Escherichia coli SNO301 (ampD1, ampA1, ampC8, pyrB, recA, and rpsL) cells harboring the comprehensive codon mutagenesis library CCM2 were plated on LB-agar plates supplemented with 13 different Amp concentrations (2-fold increments ranging from 0.25mg/ml to 1,024 mg/ml) to partition the library into 13 partially over lapping sub-libraries based on relative Amp resistance using a synthetic gene circuit that functions as a tunable band-pass genetic selection for Amp resistance.

Barcoded PCR amplicons were prepared from each plate and subjected to 454 deep-sequencing. The 1,325,979 sequencing reads were filtered for quality and for reads that only contained one codon substitution. We tabulated the number of sequencing counts for each allele at each Amp concentration and determined the fitness relative to TEM-1 from the statistics.

The distribution of growth as a function of Amp is roughly symmetric when plotted as the log2(Amp concentration). Normalized fitness is written as w_i = 2^(f_i) / 2^(f_wildtype).