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Nanopores constitute an important class of biotechnologically relevant proteins. Unlike binders and enzymes, their experimental characterization is limited to high-resolution, yet low throughput biophysical methods. Addressing this technological gap, the functional nanopore (FuN) screen now provides a versatile assay to study and engineer nanopores in Escherichia coli combining quantitative resolution with the ease, scalability, and throughput of cellular assays.

Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly developed for different biotechnological applications. Yet, a capacity to study and engineer protein nanopores by combinatorial means has so far been hampered by a lack of suitable assays that combine sufficient experimental resolution with throughput. Addressing this technological gap, the functional nanopore (FuN) screen now provides a quantitative and dynamic readout of nanopore assembly and function in the context of the inner membrane of

Small 17(33), 2101066 (2021). doi:10.1002/smll.202101066, Published by Wiley-VCH, Weinheim

Proteases have emerged as a promising class of enzymes to build post-translationally regulated signalling functions in diverse organisms and cell types ranging from simple prokaryotes to higher eukaryotes and in reconstituted systems in vitro. An expanding repertoire of proteases can now be readily configured to build tailored sensors, switches and transducers, and is increasingly facilitating the construction of complex sensory systems for a variety of biotechnological and biomedical applications. This is complemented by an increasing understanding of the fundamental design principles underlying biological signal processing at both protein-level and circuit-level that is now actively probed through synthesis. This review thus aims to summarize and analyse the most promising conceptual and experimental approaches that can be applied to build artificial signalling functions with proteases while highlighting advances, drawbacks and limitations.