Engineering kanamycin resistance of E. coli with GREACE. (A) E. coli cells transformed with pQ-lib, pUC18, and pQ-dnaQ were pre-cultivated in LB medium and then 1:100 serial diluted in gradually increased kanamycin concentrations, from Kan15 to Kan300. Error bars presented the standard deviation of growth analysis for 3 independent evolution experiments. (B) Cells carrying the above three plasmids were sampled at the end of the kanamycin resistance evolution process. Appropriate 108 cells were spread on plates with kanamycin concentration of the next level, and cultivated for colony counting. Pie charts represent the proportions of dnaQ mutants in 30 randomly selected colonies of E. coli cells carrying the pQ-lib at the end of cultivations.