Rapid and Efficient Synthetic Assembly of Multiplex Luciferase Reporter Plasmids for the Simultaneous Monitoring of Up to Six Cellular Signaling Pathways.

High-throughput cell-based screening assays are valuable tools in the discovery of chemical probes and therapeutic agents. Such assays are designed to examine the effects of small compounds on targets, pathways, or phenotypes participating in normal and disease processes. While most cell-based assays measure single quantities, multiplexed assays seek to address these limitations by obtaining multiple simultaneous measurements. The signals from such measurements should be independently detectable and cover large dynamic ranges. Luciferases are good candidates for generation of such signals. They are genetically encoded, versatile, and cost-effective, and their output signals can be sensitively detected. We recently developed a multiplex luciferase assay that allows monitoring the activity of five experimental pathways against one control simultaneously. We used synthetic assembly cloning to assemble all six luciferase reporter units into a single vector over eight stitching rounds. Because all six reporters are on a single piece of DNA, a single vector ensures stoichiometric ratios of each transcriptional unit in each transfected cell, resulting in lower experimental variation. Our proof-of-concept multiplex hextuple luciferase assay was designed to simultaneously monitor the p53, TGF-β, NF-κβ, c-Myc, and MAPK/JNK signaling pathways. The same synthetic assembly cloning pipeline allows the stitching of numerous other cellular pathway luciferase reporters. Here we present an improved three-step synthetic assembly protocol to quickly and efficiently generate multiplex hextuple luciferase reporter plasmids for other signaling pathways of interest. This improved assembly protocol provides the opportunity to analyze any five desired pathways at once much more quickly. Protocols are provided on how to prepare DNA components and destination vector plasmids, design synthetic DNA, perform assembly cloning of new transcriptional reporter elements, implement multipartite synthetic assembly cloning of single-pathway luciferase reporters, and carry out one-step assembly of final multiplex hextuple luciferase vectors. We present protocols on how to perform multiplex hextuple luciferase in an accompanying Current Protocols in Molecular Biology article. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of DNA parts and destination vectors for synthetic assembly cloning Basic Protocol 2: DNA synthesis and assembly cloning of a typical transcriptional reporter element Alternate Protocol: DNA synthesis and assembly cloning of a challenging transcriptional reporter element Basic Protocol 3: Multipartite synthetic assembly cloning of individual pathway luciferase reporters Basic Protocol 4: One step assembly into final multiplex hextuple luciferase vectors Support Protocol: Generation of home-made chemocompetent E. coli DH10B-T1R cells.