Bacteria play critical roles in many environmental processes, in human health and disease, and in biotechnology. Therefore, it is important to understand the processes by which bacteria change over time, and to measure the actual rates of change in their genes, physiological functions, and ecological performance. The two research teams will propagate and analyze 12 populations of Escherichia coli as they evolve in a controlled laboratory environment for thousands of bacterial generations. To measure adaptation, the researchers will place the bacteria from later generations in competition with the ancestral strain, which has been stored in a deep freeze, and which will be revived for these assays. To quantify genetic change, the researchers will sequence the genomes of bacteria from the evolving populations at different time points and compare them to the ancestor?s DNA sequence. These analyses will provide unique information about the rates, mechanisms, effects, and predictability of bacterial evolution. The knowledge gained will benefit science and society because bacteria are essential to the environment, health, and biotechnology, and because their on-going evolution can impact these vital systems. This long-running study has attracted substantial public interest, and the investigators will continue to communicate their findings with the public as well as with other scientists. <br/><br/>This long-term research project provides a unique study of bacterial evolution by generating an exceptionally long time-series of samples from 12 replicate populations, all of them founded from the same ancestral strain of E. coli and maintained for decades in identical environments. The research teams will propagate the populations by transferring them daily into fresh medium; every 500 generations, samples will be stored frozen, where they remain viable and available for study. During the next five years, the experiment should surpass 85,000 generations. The frozen samples will be used to analyze the dynamics of adaptation by natural selection, the coupling of phenotypic and genomic evolution, and the repeatability of evolutionary changes. Among the questions to be addressed: Will the fitness of the bacteria relative to their common ancestor continue to increase? Do the trajectories for relative fitness and genomic evolution have similar curvatures, or are they discordant? How repeatable are the fitness gains and genomic changes across the replicate populations? The biological samples and datasets generated in this project will be shared with the scientific community.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.