Project Summary A hallmark of living organisms is their ability to adapt to the world around them. Such adaptation takes place across many different scales in both space and time. On cellular time scales, molecules in individual cells enable them to regulate metabolic genes and thrive despite changing carbon sources. Over generations, cells evolve and adapt to stresses such as antibiotics. In the work proposed here, we will uncover principles of such adaptation over the functional hierarchy from molecules to cells to populations. To achieve this goal, our laboratory will use our tradition of integrating precise measurements with biophysical theory on specifically constructed biological systems. Our first research thrust is built on the recognition that despite the amazing successes of molecular biology and genome science over the last half century, we still know next to nothing about how genes are regulated for more than 65% of the genes in the commensal gut bacterium E. coli, which is arguably biology's best-understood organism. With this award we will create a first draft of the regulatory genome for the entire E. coli genome - including the binding energies and identities of every transcription factor and the genes or operons that they regulate. A second thrust focuses on a key way that living organisms respond to intracellular and environmental signals: through conformational changes in individual allosteric proteins and in assemblies of proteins, such as those found in the spindle responsible for chromosome segregation. Using a system we developed to finely control cytoskeleton-motor interactions using light, we seek to discover a predictive theoretical framework that guides and constrains how we view assembly and adaptation. The third thrust in our study of adaptation will build upon the previous two threads by providing a rigorous examination of how genomes and the proteins that interact with them evolve. The specific case studies will focus on the evolution of transcriptional regulation with special emphasis on the response of pathogenic bacteria to antibiotic drugs. The cell is the fundamental organizational unit of living organisms and the work proposed here will provide a far-reaching but detailed view of how cells adapt. The approach we adopt is ultimately biophysical; we insist that our analysis of the cell be at once quantitative and predictive, sharpening our questions and deepening our understanding in a way that can ultimately be parlayed into new strategies for improving human health.