Supplementary Figure 3: AND-logic gate module optimization using hammerhead ribozymes.
From: Programmable full-adder computations in communicating three-dimensional cell cultures
a) Logic description of trigger-inducible AND-logic gate modules responsive to ID, IP and IV with corresponding truth tables. The modules were optimized using relevant inducer combinations as shown using grey boxes (only in the presence of the trigger-inducible transcription factor). b) Performance of three unmodified input-programmable AND-logic gate modules (rtTA/PD, VanR-KRAB/PV-ON, TtgR-KRAB/PP-ON). Two versions of PV-ON-inducible promoters were designed using different constitutive promoters (PSV40 and PhCMV); however, for simplicity, both were named PV-ON. HEK293T cells were engineered with respective AND-logic gate components and induced with corresponding inducer molecules. After 48 h, the activity of the secreted reporter protein SEAP was quantified. OFF-state reporter activity levels are displayed. c) Performance and schematic symbol of ribozyme-optimized AND-logic gate modules, in which hammerhead ribozymes (HHRs) env140 or sTRSV were installed into the 3’-UTR of indicated reporter plasmids. As illustrated, the SEAP levels in the OFF-states are reduced in the presence of HHRs compared to unmodified modules. Partial data are reproduced from Supplementary Fig. 2a. c) Logic description, performance and optimization of the dimerization-dependent AND-logic gate module based on Gal4-Coh2 and VP16-DocS. As illustrated, the introduction of the sTRSV hammerhead ribozyme into the 3’-UTR of the reporter plasmid reduced SEAP levels in the OFF-state. Partial data are reproduced from Supplementary Fig. 2b. Supplementary Table 2 describes the transfection details. Error bars represent the means ± s.d. of n=3 independent experiments performed in triplicate.
