Engineering

The initial objective was to engineer a cascaded detection system capable of combining antigen sensing with threshold activation inside a single cell. This was conceptualised as a Dual SynNotch circuit: the first receptor (SynNotch_1), upon engagement with Ligand A, would release an intracellular transcription factor (TF1) that activates a promoter driving expression of a second SynNotch receptor (SynNotch_2) on the plasma membrane. SynNotch_2, once expressed and subsequently engaged by Ligand B, would release a second transcription factor (TF2) to activate the final reporter gene. The intended logic was a true sequential AND gate, ensuring that only cells exposed to Ligand A followed by Ligand B would express the reporter. The architecture was designed for modularity, allowing the extracellular recognition domains and internal promoters to be interchanged without re-engineering the cascade. This concept rested on idealised assumptions: that SynNotch activation would behave as a digital switch; that the downstream promoters would fire only when their cognate transcription factors crossed a sharp activation threshold; that SynNotch_2 would traffic uniformly and rapidly to the membrane; and that basal activity in both receptors and promoters would be negligible.