A versatile plasmid architecture for mammalian synthetic biology (VAMSyB)

Data underlying the figures in the publication “A versatile plasmid architecture for mammalian synthetic biology (VAMSyB)”, published in Metabolic Engineering, 2021, 66, 41–50. https://doi.org/10.1016/j.ymben.2021.04.003

Table of contents:

1. Dataset 1; Excel file with the curated data underlying the figures.

Figure 1. (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.

Figure 2. (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

Figure 3. (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.

Figure S1. 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.

Figure S2. 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.