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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.

The functional analysis of protein nanopores is typically conducted in planar lipid bilayers or liposomes exploiting high-resolution but low-throughput electrical and optical read-outs. Yet, the reconstitution of protein nanoporesin vitrostill constitutes an empiric and low-throughput process. Addressing these limitations, nanopores can now be analysed using the functional nanopore (FuN) screen exploiting genetically-encoded fluorescent protein sensors that resolve distinct nanopore-dependent Ca2+in- and efflux patterns across the inner membrane ofEscherichia coli. With a primary proof-of-concept established for the S2168 holin, and thereof based recombinant nanopore assemblies, the question arises to what extent alternative nanopores can be analysed with the FuN screen. Focussing on self-assembling membrane peptides, three sets of 13 different nanopores are assessed for their capacity to form nanopores in the context of the FuN screen. Nanopores tested comprise both natural and computationally-designed nanopores while a comparison with high-resolution biophysical studies provides an experimental benchmark for future studies.

A broad variety of biomolecules is industrially produced in bacteria and yeasts. These microbial expression hosts can be optimized through genomic engineering using CRISPR tools. Here, we designed and characterized such a modular genome editing system based on the Cas12a-like RNA guided nuclease MAD7 in E. coli. Our system enables the efficient generation of single nucleotide polymorphisms (SNPs) or gene deletions and can directly be used with donor DNA from benchtop DNA assembly to increase throughput. We combined multiple edits to engineer an E. coli strain with reduced overflow metabolism and increased plasmid yield, highlighting the versatility and industrial applicability of our approach.

Planar lipid bilayers constitute a versatile method for measuring the activity of protein channels and pores on a single molecule level. Ongoing efforts attempt to tailor this method for detecting biomedically relevant target analytes or for high-throughput screening of drugs. To improve the mechanical stability of bilayer recordings, we use a thin-film epoxy resist ADEX as septum in free-standing vertical bilayers. Defined apertures with diameters between 30 µm and 100 µm were micro-fabricated by photolithography. The performance of these septa was tested by functional reconstitution of the K+ channel KcvNTS in lipid bilayers spanned over apertures in ADEX or Teflon films; the latter is conventionally used in bilayer recordings and serves as reference. We observe that the functional properties of the K+ channel are identical in both materials while ADEX provides no advantage in terms of capacitance and signal-to-noise ratio. In contrast to Teflon, however, ADEX enables long-term experimental recordings while the stability of the lipid bilayer is not compromised by pipetting solutions in and out of the recording chamber. Combined with the fact that the ADEX films can be cleaned with acetone, our results suggest that ADEX carries great potential for multiplexing bilayer chambers in robust and reusable sensing devices. Graphical Abstract

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 read-out of nanopore assembly and function in E. coli. The assay is based on genetically-encoded fluorescent protein sensors that resolve the nanopore-dependent influx of Ca2+ across the inner membrane of E. coli. Illustrating its versatile capacity, the FuN screen is first applied to dissect the molecular features that underlie the assembly and stability of nanopores formed by the S2168 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S2168 with different ring-shaped oligomeric protein structures that feature defined hexa-, hepta- and octameric geometries. Library screening highlights substantial plasticity in the ability of the S2168 transmembrane module to oligomerize in alternative geometries while the functional properties of the resultant nanopores can be fine-tuned through the identity of the connecting linkers. Overall, the FuN screen is anticipated to facilitate both fundamental studies and complex nanopore engineering endeavors with many potential applications in biomedicine, biotechnology and synthetic biology.

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

A nanopore‐based CuII‐sensing system is reported that allows for an ultrasensitive and selective detection of CuII with the possibility for a broad range of applications, for example in medical diagnostics. A fluorescent ATCUN‐like peptide 5/6‐FAM‐Dap‐β‐Ala‐His is employed to selectively bind CuII ions in the presence of NiII and ZnII and was crafted into ion track‐etched nanopores. Upon CuII binding the fluorescence of the peptide sensor is quenched, permitting the detection of CuII in solution. The ion transport characteristics of peptide‐modified nanopore are shown to be extremely sensitive and selective towards CuII allowing to sense femtomolar CuII concentrations in human urine mimics. Washing with EDTA fully restores the CuII‐binding properties of the sensor, enabling multiple repetitive measurements. The robustness of the system clearly has the potential to be further developed into an easy‐to‐use, lab‐on‐chip CuII‐sensing device, which will be of great importance for bedside diagnosis and monitor of CuII levels in patients with copper‐dysfunctional homeostasis., Capturing copper: The Dap‐β‐Ala‐His sequence, which is generated from the amino‐terminal‐CuII and NiII‐binding motif, is used for a selective binding of CuII in presence of NiII and ZnII and was crafted to an ion track‐etched PET membrane. Cu detection was achieved through fluorescence quenching and I–V measurements, enabling femtomolar CuII concentrations to be sensed.

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.

Cells of multicellular organisms are surrounded by and attached to a matrix of fibrous polysaccharides and proteins known as the extracellular matrix. This fibrous network not only serves as a structural support to cells and tissues but also plays an integral part in the process as important as proliferation, differentiation, or defense. While at first sight, the extracellular matrices of plant and animals do not have much in common, a closer look reveals remarkable similarities. In particular, the proteins involved in the adhesion of the cell to the extracellular matrix share many functional properties. At the sequence level, however, a surprising lack of homology is found between adhesion-related proteins of plants and animals. Both protein machineries only reveal similarities between small subdomains and motifs, which further underlines their functional relationship. In this review, we provide an overview on the similarities between motifs in proteins known to be located at the plant cell wall-plasma membrane-cytoskeleton interface to proteins of the animal adhesome. We also show that by comparing the proteome of both adhesion machineries at the level of motifs, we are also able to identify potentially new candidate proteins that functionally contribute to the adhesion of the plant plasma membrane to the cell wall.