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

Given the urgent need that outbreaks of emerging viral agents pose to human health, we sought to test if the MTK could be harnessed to streamline and accelerate the generation of BSL2 reagents like the stable EBOV RNP cell lines. We synthesized the EBOV RNP genes and used the MTK multicistronic workflow with Left connector parts that encode P2A ribosomal skipping site elements to rapidly assemble a 4-cistronic construct of Zaire ebolavirus (ZEBOV-4cis) (Fig. 7a ) directly and simultaneously into five different Part 0 destination vectors ( Supplementary Fig. 5a ). These vectors provide distinct types and numbers of genome integration events ( via transposase (PiggyBac) or integrases (PhiC31, BxBI)) . Digestion followed by gel electrophoresis confirmed high cloning efficiency and correct assembly of ZEBOV-4cis in all 5 destination vectors ( Supplementary Fig. 5a ). In transient transfections, all ZEBOV-4cis constructs showed no impact on cell viability and displayed levels of minigenome activity similar to the 6-plasmid system and ~40-60-fold higher activity than the cells lacking Lpol (Fig. 7b, Supplementary Fig. 5b ).