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

TUs with varied promoter parts driving mAzamiGreen expression, flanked by the bovine growth hormone (Bgh) pA 3' UTR. In an adjacent TU, a CAG promoter expresses mScarlet with the rabbit beta-globin (Rgl) pA 3' UTR ( Fig. 2a) . We transiently transfected each multi-TU plasmid into HEK293T cells and normalized the mAzamiGreen expression by mScarlet to control for plasmid copy number. This suite of promoters span a range of 2 orders of magnitude over background, with the strongest promoter, CMV, more than 300-fold greater than a promoter-less mAzamiGreen. The ranked expression strength of the promoters revealed a smooth continuum of expression between 10-fold and 300-fold (Fig. 2a) . Thus this suite of promoters can be used to tailor TU expression levels and leverage overlapping promoter strengths to avoid repeated elements in multi-TU plasmids. The ranked expression of the promoters across human and mouse demonstrates that their relative strengths are largely maintained (Fig. Supplementary Fig. 1a, b) , suggesting that these promoters are portable across different systems. Additionally, the rank of promoter effect on gene expression is consistent between transient and stably integrated expression, leaving this suite of promoters as a reliable metric for TU design (Fig. Supplementary Fig. 1c, d) .