Figure 2. Experimental Design.

Holder and Bull [22] used a clone of the bacteriophage G4 to found a population that was propagated through 50 serial transfers at 41.5 °C, followed by 50 serial transfers at 44 °C. In each serial transfer 1 × 10⁶ phages were added to a 10 ml culture containing 1 × 10⁹ exponentially growing E. coli hosts, incubated for approximately 45 min at the specified temperature, and then treated with chloroform to kill any remaining hosts. During the 45-min incubation, the phage population size increased to approximately 1 × 10¹⁰ phages, and 1 × 10⁶ of these phages were used to initiate the next serial transfer. The only exception occurred after the 50^th serial transfer, when the population was forced through a single phage bottleneck to homogenize the population. In the current study, we isolated single phage from this evolving population at transfers 20, 50, and 100 (designated G4₂₀, G4₅₀, and G4₁₀₀). We characterized the performance of these phages and the ancestral G4 (designated G4₀) across a range of temperatures.