A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells

João Pedro Fonseca; Alain R. Bonny; G. Renuka Kumar; Andrew H. Ng; Jason Town; Qiu Chang Wu; Elham Aslankoohi; Susan Y. Chen; Patrick Harrigan; Lindsey C. Osimiri; Amy L. Kistler; Hana El-Samad

. CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/506188 doi: bioRxiv preprint Cell lysates were prepared in RIPA lysis and extraction buffer (Thermo #89900) containing protease inhibitors (Sigma #P8340) for Western blotting. Lysates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane, and subjected to Western blotting using primary antibodies: rabbit The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/506188 doi: bioRxiv preprint transfected cells were seeded into 8 wells of 96-well plates for 2 days in duplicates. Nano luciferase assays for minigenome function and cell titer glo assays for cell viability were performed as per manufacturer's instructions (Promega). For minigenome assays in ZEBOV-4cis stable cells, 500 ng each of p2.0-T7-3E5E-nLuc or p2.0-T7-3E5E-eGFP and 500ng of T7-expression plasmid were co-transfected. Nano luciferase levels were assayed as above and eGFP signal was captured via microscopy (Leica, 4X magnification). The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/506188 doi: bioRxiv preprint Gedunin (5uM), or 6-Azauridine (5uM). After 2 days cells were processed for nano luciferase assay for functional minigenome activity and cell titer glo assays for cell viability. Levels of signal relative to DMSO control is plotted. Bar plots represent mean of biological replicates (n=2).