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

Abstract

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 cellbased 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 measure large dynamic ranges. Luciferases are good candidates for generation of such signals. They are genetically encoded, versatile, and cost-effective, whose 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 (Vazquez-Vilar, et al., 2020) [*Copy Editor: Please ask Au to confirm that this should be cited here; he removed mention of "GoldenBraid" in this sentence during revision, so just double-checking.] 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

SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome

“…7xE-Box::Renilla was a gift from Koen Venken (Addgene plasmid # 124532 ; http://n2t.net/addgene:124532 ; RRID:Addgene_124532, Sarrion-Perdigones et al, 2020)…”

“…FeatureCounts (Gohr and Irimia, 2019) was used to quantify mapped transcripts from total RNA-seq libraries. DEGs were found using DESeq2 as previously described (Sarrion-Perdigones, Gonzalez and Venken, 2020). DEGs with |L2FC| > 1, p-adjusted value <0.05 were considered significant and used as input in downstream Gene Ontology (GO) analysis (DAVID, v 6.8).…”

Serial Recombineering Cloning to Build Selectable and Tagged Genomic P[acman] BAC Clones for Selection Transgenesis and Functional Gene Analysis using Drosophila melanogaster

“…165879;Matinyan et al, 2021b; Table 1) Standard cloning vector backbone (Figs. 4 and 5) for GB2.0 synthetic assembly cloning (Matinyan et al, 2021b;Sarrion-Perdigones et al, 2013Sarrion-Perdigones et al, 2020;Sarrion-Perdigones et al, 2022 5). Each marker contains a fusion promoter with eukaryotic and prokaryotic activities, a gene encoding the selection/resistance marker, and the minimal terminator of the thymidine kinase gene of the herpes simplex virus.…”

“…5) cassettes using primers that add sequences homologous to the target regions in the P[acman] BAC for the recombineering event. We will then describe cloning and verification of these recombineering templates in a plasmid with a conditional origin of replication that aids recombineering (see Basic Protocol 3) using GoldenBraid 2.0 (GB2.0) synthetic assembly cloning (Matinyan et al, 2021b;Sarrion-Perdigones et al, 2013Sarrion-Perdigones, Gonzalez, & Venken, 2020;Sarrion-Perdigones, Gonzalez, & Venken, 2022). Finally, we will explain how to prepare the recombineering templates (Figs.…”

Synthetic Assembly DNA Cloning to Build Plasmids for Multiplexed Transgenic Selection, Counterselection or Any Other Genetic Strategies Using Drosophila melanogaster

“…7 and 8): 400 ng of plasmid 2.5 μl of rCutSmart buffer 30. On ice, thaw a microcentrifuge tube containing a 50-μl aliquot of home-made chemocompetent DH10B-T1R E. coli cells (Sarrion-Perdigones et al, 2020).…”