![]() Golden Gate ( Marillonnet & Grützner, 2020), GoldenBraid ( Vazquez-Vilar, et al., 2020) and their successors use type IIs restriction enzymes for DNA assembly ( Sarrion-Perdigones et al., 2011 Engler et al., 2008 Sarrion-Perdigones et al., 2013 Vazquez-Vilar et al., 2017 Weber et al., 2011 Werner et al., 2012). GB2.0 is a second version of the initial GoldenBraid system ( Sarrion-Perdigones et al., 2011), which itself was founded on the multipartite Golden Gate cloning pipeline ( Engler et al., 2008). In the previous work, we utilized the GoldenBraid2.0 (GB2.0) cloning system ( Sarrion-Perdigones et al., 2013, 2014) to carry out the serial assembly of the multiplex hextuple luciferase reporter vector over eight rounds of serial assembly cloning ( Sarrion-Perdigones et al., 2019). We present this simplified method in this unit. Here, however, we improved that method by developing a convenient three-step synthetic assembly protocol to quickly and efficiently construct multiplex hextuple luciferase reporter plasmids for other signaling pathways of interest. Using the same synthetic assembly cloning pipeline would allow us and others to construct, other multiplex reporters tailored to any five cellular signaling pathways. It also carried a control constitutive hCMV-IE1 promoter ( Yew et al., 1997) serving as a normalization standard. The vector carried five different transcriptional response elements purposed to report on p53 ( Osada et al., 2005), TGF-β ( Zawel et al., 1998), NF-κβ ( Oeckinghaus and Ghosh, 2009), c-Myc ( Walhout et al., 1997 Allevato et al., 2017), and MAPK/JNK ( Carter et al., 2001) pathway signaling events. In our prior work, we implemented a previously described synthetic assembly cloning pipeline to build a proof-of-concept multiplex hextuple luciferase reporter vector using an eight-step synthetic assembly protocol. Multiplex monitoring allows us to measure the effects on a pathway of interest, as well as collateral “off-target” modulations on other signaling pathways. Using this method, we were able to monitor the effects of genetic, ligand and small molecule chemical compound treatments of five cellular pathways of interest. Recently, we developed a multiplex hextuple luciferase assay that allows for the simultaneous recording of the activity of six luciferases within the same sample ( Sarrion-Perdigones et al., 2019). We present protocols on how to perform multiplex hextuple luciferase in the accompanying Current Protocols in Molecular Biology article (see Sarrion-Perdigones, et al., in press). Protocols are provided on how to prepare DNA components and destination vector plasmids, design synthetic DNA, and 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. This improved assembly protocol provides opportunities to analyze any five desired pathways at once much quicker. 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. The same synthetic assembly cloning pipeline allows the stitching of numerous other cellular pathway luciferase reporters. 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. 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. We used synthetic assembly cloning ( Vazquez-Vilar, et al., 2020) to assemble all six luciferase reporter units into a single vector, over eight stitching rounds. We recently developed a multiplex luciferase assay that allows monitoring the activity of five experimental pathways against one control simultaneously. ![]() They are genetically encoded, versatile, and cost-effective, whose output signals can be sensitively detected. Luciferases are good candidates for generation of such signals. ![]() The signals from such measurements should be independently detectable and measure large dynamic ranges. While most cell-based assays measure single quantities, multiplexed assays seek to address these limitations by obtaining multiple simultaneous measurements. Such assays are designed to examine the effects of small compounds on targets, pathways, or phenotypes participating in normal and disease processes. High-throughput cell-based screening assays are valuable tools in the discovery of chemical probes and therapeutic agents.
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