DOI: 10.5281/zenodo.4980395
title: A versatile plasmid architecture for mammalian synthetic biology (VAMSyB)

description: Data underlying the figures in the publication “A versatile plasmid architecture for mammalian synthetic biology (VAMSyB)”, published in <em>Metabolic Engineering</em>, <strong>2021</strong>, 66, 41–50. https://doi.org/10.1016/j.ymben.2021.04.003 Table of contents: <strong>1. Dataset 1</strong>; Excel file with the curated data underlying the figures. <strong>Figure 1.</strong> (F) Comparison of promoters cloned in the tier-1 scaffold, including minimal promoters (PTKmin, Pmin, PCMVmin-2, PMLP, and PCMVmin-1) and constitutive promoters (PTK, PSV40, PmPGK1, PhCMV, PhEF1a, and PRPBSA) was assessed in terms of SEAP production. (G-H) Modulation of the rtTA-based tet-ON system with different (G) minimal promoters and (H) suppressor (TS) 5’UTR sequences or destabilizing ribozymes (sTRSV and env140) in the 3’UTR was assessed in terms of SEAP production. <strong>Figure 2.</strong> (C-D) Expression characterization of each cassette of the tier-2 construct is tested by inserting expression cassettes controlled by the moderately strong phospho-glycerate kinase (PmPGK1) or strong human cytomegalovirus-derived (PhCMV) promoter. (C) Expression comparison of each cassette of the tier-2 construct tested individually was assessed in terms of Nluc production. (D) Characterization of interference between promoters of different strengths (PhCMV and PmPGK1) encoded in individual constructs or cloned into a single tier-2 construct. PhCMV expression was assessed in terms of SEAP production and PmPGK1 expression was assessed in terms of Nluc production. The reporters were transfected individually, cotransfected (+), or expressed from the same plasmid (|). (E) Characterization of interference between a constitutive promoter (PmPGK1) and an inducible promoter (PCRE). PmPGK1 driving firefly luciferase (Fluc) and a synthetic inducible cAMP-responsive (PCRE) promoter driving Nluc were cloned into different expression cassettes of a single tier-2 construct. Levels of Nluc activity are normalized to Fluc activity. (G-J) Generation of a single tier-2 construct encoding four different well-established synthetic gene switches: (G) the doxycycline-regulated rtTA system, (H) the doxycycline-regulated tTA system, (I) the vanillic acid-regulated VanA system, and (J) the phloretin-regulated TtgA system were all assessed in terms of SEAP production <strong>Figure 3. </strong>(C) Characterization of polyclonal stable cell lines generated with the tier-3 PB and SB constructs encoding Tet-responsive inducible SEAP-p2A-iRFP670 in the first cassette (A1), constitutively expressed rtTA in the second cassette (A2), and constitutively expressed YPet-p2A-PuroR in the third cassette (A3) demonstrated dose-dependent induction of SEAP expression in response to doxycycline. (F) Characterization of polyclonal stable cell lines generated with the tier-3 lentiviral constructs encoding Tet-responsive inducible SEAP-p2A-iRFP670 in the first cassette (A1), constitutively expressed rtTA in the second cassette (A2), and constitutively expressed YPet-p2A-PuroR in the third cassette (A3) show dose-dependent induction of SEAP expression in response to doxycycline. (J) Characterization monoclonal lines generated with the CRISPR tier-3 constructs encoding Tet-responsive inducible SEAP-p2A-iRFP670 in the first cassette (A1), constitutively expressed rtTA in the second cassette (A2) show dose-dependent induction of SEAP expression in response to doxycycline. <strong>Figure S1.</strong> Characterization of functional genetic elements that can reduce interference between promoters of different strengths (PhCMV and PmPGK1) encoded in a single tier-2 construct. PhCMV-driven expression was assessed in terms of SEAP production and PmPGK1-driven expression was assessed in terms of Nluc production. The reporters were transfected individually, cotransfected (+), or expressed from the same plasmid (|). Functional genetic elements, including a plasmid backbone spacer sequence [spacer], a synthetic poly(A) signal/transcriptional pause site derived from a commercial vector [pGL3], a co-transcriptional cleavage element [CoTC], a transcriptional termination sequence derived from the human β-globin gene [Tactb], or an insulator sequence consisting of two repeats of chicken hypersensitive site 4 [2xcHS4] were introduced directly 5’ of the PmPGK1-NLuc expression cassette encoded in either the (A) A2 or (B) A3 insertion site. <strong>Figure S2. </strong>Selection of high-performing sub-populations derived from polyclonal stable cell lines generated with the tier-3 PiggyBac (PB; pcTS50), Sleeping Beauty (SB; pcTS51), or lentiviral (Lenti; pcVH38) construct as presented in Figure 3. The polyclonal stable cell lines were induced with doxycycline for 48 h and then sorted based on iRFP670 expression to isolated high-expressing clones. (A-B) The sorted PB generated sub-populations were assayed for (A) dose-dependent induction of SEAP, as well as (B) iRFP670 reporter mean fluorescence intensity (MFI) output in response to doxycycline. (D-E) The sorted SB generated sub-populations were assayed for (D) dose-dependent induction of SEAP expression, as well as (E) iRFP670 reporter MFI output in response to doxycycline. (G-H) The sorted lentiviral generated sub-population was assayed for (G) dose-dependent induction of SEAP expression, as well as (H) iRFP670 reporter MFI output in response to doxycycline.