Project summary Multiplexed cellular assays that can efficiently, sensitively, and simultaneously measure multiple signaling pathways in the same cells require orthogonal probes with large dynamic ranges whose measurements can be obtained quickly. Luciferases are genetically encoded, cost-effective, versatile candidates that fulfill these requirements. Commonly used dual luciferase reporter assays currently detect one luciferase coupled to a cellular pathway, and a second luciferase coupled to a control pathway for normalization purposes, effectively resulting in just one activity measurement for a single pathway. To increase the number of cellular signaling pathways that can be simultaneously probed using luciferase reporters, we plan to explore the limits of luciferase multiplexing in this proposal. Preliminary work demonstrates feasibility by expanding multiplexing towards six luciferases that can report on five cellular signaling pathways and one control, effectively increasing the potency of the dual luciferase assay five-fold. To ensure low experimental variation, we adopted a flexible synthetic assembly cloning pipeline that stitches together all six luciferase reporter units into a single vector, resulting in the transfection of equal stoichiometric ratios of each transcriptional unit in each transfected cell. To demonstrate proof of concept, we engineered a luciferase assay tailored to probe pathway fluxes through transcriptional response elements of five known cellular signaling pathways against a constitutive promoter for normalization proposes and assayed the effects of siRNA, ligand, and chemical compound treatments on their target pathways and the four other cellular pathways at the same time. Based on this preliminary knowledge, we propose to explore luciferase multiplexing even further. Four complementary approaches will be pursued: 1. Increase the number of the two already explored substrate-consuming luciferases we can detect in a single experiment. 2. Include a third group of substrate-consuming luciferases, namely vargulin luciferases. 3. Identify quenchers against the coelenterazine and/or vargulin luciferase groups. 4. Develop a streamlined assembly pipeline with as few cloning steps as possible to synthetically stitch together up to twelve luciferase reporter units in a single multiplex luciferase reporter vector, and incorporate a specialized plasmid backbone capable of accommodating large DNA insert cargo encompassing all twelve luciferase reporter units. Furthermore, to demonstrate proof-of-concept for multiplex luciferase assaying using up to twelve luciferase reporter units, we will generate a set of multiplex luciferase reporters that can report on transcriptional readouts for a number of the most commonly known cellular signaling pathways that will be tested for up- and downregulation. Hence, this work promises to expand luciferase multiplexing by growing the available repertoire of substrate-specific, quenchable an...