Figure 2.

Improvement of thermal stability through directed evolution. (a) Multiple rounds of this procedure leads to increased resistance to thermal degradation. Incubation of T3 phages at 60 ${}^{\circ }$C for 1 h resulted in active phage survival of 6.6%, 36.0%, 59.1%, and 69.9% after 0, 10, 20, and 30 rounds of directed evolution, respectively. Relative concentrations were calculated using plaque-counting assays and linear regressions over four 40:140 $\mathrm{\mu }$L serial dilutions (n = 4); error bars representing ± SD. (b) The effect of temperature on degradation rate. The percent survival of wild type and mutant bacteriophages over the course of a 70 min incubation at 60, 62, and 64 ${}^{\circ }$C, quantified using a linear regression over serial dilutions (n = 3). Curves were fit to Eq. (1). Error bars represent the difference between theoretical curve values and measured titer for a given time point. (c) Mutant phages display improved tolerance to acidic conditions. Percent survival after 30 min incubation under acidic conditions, relative to neutral control conditions. (d) Half life values calculated for wild type and mutant phages at 25, 60, 62, and 64 ${}^{\circ }$C; error bars are ± SD. Relative concentrations were calculated using plaque-counting assays and linear regressions, with error bars representing ± SD. Theoretical curves were fit to experimental data using a fractional-order kinetic model and the Arrhenius law.