Project Summary Advances in synthetic biology provide powerful tools to interrogate the complex relationship between network structure and function. In this study, we will combine synthetic biology with computational modeling to investigate network-mediated regulation of cell damage and deterioration, a complex biological process. As similar studies in mammals are prohibitively time- and resource-intensive, we choose to focus on Saccharomyces cerevisiae, which has proven to be a genetically tractable model for many fundamental processes in mitotic cells and has allowed identification of many conserved genes that regulate cell-fate decisions in eukaryotes. Emerging questions include how these genes interact and how the interactions change dynamically to drive multi-generational cell deterioration dynamics. We recently found two distinct phenotypes in genetically identical yeast cells as they approach cell death: one with decreased ribosomal DNA (rDNA) silencing and nucleolar decline (Mode 1) whereas the other with heme depletion and mitochondrial decline (Mode 2). We found that stochasticity plays an important role in choosing one of the two paths, but once the fate decision is made, it is almost always irreversible. We identified a core molecular circuit, consisting of the lysine deacetylase Sir2 and the heme-activated protein (HAP) transcriptional complex, that governs the decision to select one of these two paths. Based on the model, we were able to engineer cells to follow a third path with a dramatically extended period of growth and survival, free of deterioration (Mode 3). In this proposal, we will expand these efforts and systematically perturb and rewire the core circuit that controls cell fate in order to reprogram its decision-making process. In Aim 1, we will use chemically-inducible promoters to control expression of Sir2 and HAP and thereby modulate cell-fate decisions in isogenic cells. We will use microfluidics to generate distinct, dynamic patterns of Sir2 and HAP expression and evaluate their effects on damage accumulation, physiological changes, and cellular decline. In Aim 2, we will genetically rewire the core Sir2-HAP circuit under the guidance of computational modeling and examine how these engineered circuits govern cell- fate decisions and cell deterioration dynamics. In Aim 3, we will use high-throughput microfluidics to identify the gene expression programs associated with Mode 1, Mode 2, and Mode 3 and examine how perturbations of these programs affect multi-generational deterioration dynamics. These analyses will uncover the genes and processes that underlie the missing connections between the Sir2-HAP core circuit and downstream modules that underlie cellular decline leading to cell death. They will enable us to expand our computational model and improve its predictive power. Throughout the study, we will construct deterministic and stochastic models, which will produce testable predictions and guide engineering of s...