All cells require energy and chemical building blocks to grow, proliferate, and respond to environmental cues. As a consequence, pathologies including diabetes, cancer, developmental disorders and infectious diseases are etiologically linked to central metabolism. Interpreting these pathologies and developing novel therapeutic interventions requires understanding the systems-level regulation of organismal physiology that has its basis in enzymatic activities and steady-state levels of metabolites. Unfortunately, measuring these properties in living cells is generally beyond the capability of current technology. Advances in metabolomics have enabled measuring the concentration and fluxes for hundreds of metabolites while spectroscopic techniques allow for the low resolution imaging of isotopically labeled compounds. However, all such techniques are inherently limited by a combination of invasiveness, generality, imaging resolution, and throughput. To this end, the goal of this proposal is to enable the rapid development of genetically-encoded fluorescent biosensors with a large dynamic range capable of measuring the metabolic state of living cells. Recent work has shown that highly engineered variants of the green fluorescent protein can act as reporters of intracellular metabolite concentration. Constructing these sensors has historically been a slow, error-prone exercise in rational protein engineering, and thus there are only a handful of known useful biosensors. The goal of our work is to accelerate this process by several orders of magnitude. My laboratory is developing a multiplex directed evolution approach for the rapid assay of large biosensor libraries, enabling us to simultaneously optimize for superior spectral properties while also constructing many different sensors in parallel. As a proof of principle, we are applying this strategy for the construction of biosensors to measure metabolites associated with cellular proliferation, including glucose, glutamine, and lactate. In related work, we propose to use these sensors as genetic screening tools for the in vitro study of pathway regulation and as in vivo imaging probes in a C. elegans cancer model. If successful, these studies will enable the high-throughput measurement and microscopic imaging of metabolic function in living cells and open up new avenues of physiologic research at the cellular, tissue, and whole-animal level.
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