Heterogeneous Mutant Cell Isolation

The genomic region of approximately 300–600 kb centered on the position of the mutation was amplified by PCR with PrimeSTAR HS DNA Polymerase (TaKaRa Bio, Japan) and the corresponding primers (Supplementary Table 7). Amplicons were purified using a MinElute PCR Purification Kit (Qiagen, United States), and Sanger sequencing was conducted by Eurofins Genomics K. K. (Tokyo, Japan). The resulting electropherogram was analyzed using Sequence Scanner Software v2.0 (Thermo Fisher Scientific, United States), and the ratio of the mutants within the cell population was calculated according to the peak values, as described previously (Kishimoto et al., 2015 (link)). Stored cell cultures with an interval of ∼100 generations were analyzed to identify the heterogeneity of the cell population. Single-colony isolation was performed from the heterogeneous population to isolate the homogeneous mutants. The cell culture was spread on LB agar plates, and 10∼30 single colonies per plate were subjected to Sanger sequencing. The colonies of the homogeneous mutant were stored for the fitness assay as described above.