• cell engineering
• co-culture assay
• enzymatic signaling cascade
• fold-induction increase
• leakiness reduction
• ligand-induced cleavage
• signal transduction engineering
• SynNotch
• synthetic biology
• synthetic signal amplification

In this work, we addressed the limitations of the synthetic receptor platform SynNotch in mammalian cells, namely, the low fold-induction and the noticeable ligand-independent basal activity. To evaluate the new receptors that we engineered, we set up a co-culture assay composed of receiver and sender-cells. The receiver-cells expressed the engineered receptor in addition to a luciferase (NanoLuc) reporter cassette. On the other hand, the sender-cells expressed the membrane ligand for the receptor activation through cell-cell interaction. The different approaches tested included the engineering of a new reporter construct, the introduction of a multi-component trans-activation domain based on protein dimerization, and the replacement of the intracellular part with an enzymatic signaling cascade triggered by the proteolytic cleavage of the receptor. Eventually, the platform was improved, particularly in terms of reduced leakiness, and the impact of the different approaches was evaluated.