The droplet interface bilayer (DIB) method offers simple control over initial leaflet compositions in model membranes, enabling an experimental path to filling gaps in our knowledge about the interplay between compositional lipid asymmetry, membrane properties, and the behaviors of membrane-active species. Yet, the stability of lipid leaflet asymmetry in DIBs has received very little attention, particularly in the presence of peptides and ion channels that are often studied in DIBs. Herein, we demonstrate for the first time parallel, capacitance-based measurements of intramembrane potential with arrays of asymmetric DIBs assembled in a microfluidic device to characterize the stability of leaflet asymmetry over many hours in the presence and absence of membrane-active peptides. DIBs assembled from opposing monolayers of the ester (DPhPC) and ether (DOPhPC) forms of diphytanoyl-phosphatidylcholine yielded asymmetric bilayers with leaflet compositions that were stable for at least 18 h as indicated by a stable |137 mV| intramembrane potential. In contrast, the addition of surface-bound alamethicin peptides caused a gradual, concentration-dependent decrease in the magnitude of the dipole potential difference. Intermittent current-voltage measurements revealed that alamethicin in asymmetric DIBs also shifts the threshold voltage required to drive peptide insertion and ion channel formation. These outcomes take place over the course of 1 to 5 h after membrane formation, and suggest that alamethicin peptides promote lipid flip-flop, even in the un-inserted, surface-bound state, by disordering lipids in the monolayer to which they bind. Moreover, this methodology establishes the use of parallel electrophysiology for efficiently studying membrane asymmetry in arrays of DIBs.
A primary focus of the rapidly growing field of plant synthetic biology is to develop technologies to precisely regulate gene expression and engineer complex genetic circuits into plant chassis. At present, there are few orthogonal tools available for effectively controlling gene expression in plants, with most researchers instead using a limited set of viral elements or truncated native promoters. A powerful repressible-and engineerable-binary system that has been repurposed in a variety of eukaryotic systems is the Q-system from Neurospora crassa. Here, we demonstrate the functionality of the Q-system in plants through transient expression in soybean (Glycine max) protoplasts and agroinfiltration in Nicotiana benthamiana leaves. Further, using functional variants of the QF transcriptional activator, it was possible to modulate the expression of reporter genes and to fully suppress the system through expression of the QS repressor. As a potential application for plant-based biosensors (phytosensors), we demonstrated the ability of the Q-system to amplify the signal from a weak promoter, enabling remote detection of a fluorescent reporter that was previously undetectable. In addition, we demonstrated that it was possible to coordinate the expression of multiple genes through the expression of a single QF activator. Based on the results from this study, the Q-system represents a powerful orthogonal tool for precise control of gene expression in plants, with envisioned applications in metabolic engineering, phytosensors, and biotic and abiotic stress tolerance.
Microbial genetic manipulation requires access to engineerable plasmids that can be programmed to perturb genes, pathways and genomes. The extensive repertoire of plasmids available for model microbes, such as Escherichia coli, has facilitated fundamental biology studies and synthetic biology applications. However, the scarcity of plasmids for non-model microbes hinders efforts to broaden our biological knowledge and constrains the development of biotechnological solutions. In this study, we introduce a molecular toolkit and multiplexed screen to evaluate functional plasmids in non-model microbes. We constructed a collection of genetic parts consisting of 22 origins of replication (ORIs), 20 antibiotic selectable markers, and 30 molecular barcodes, which can be assembled combinatorially to create a library of plasmids trackable by next-generation DNA sequencing. We demonstrate our approach by delivering a pooled library of 22 ORIs to 12 bacterial species including extremophiles, electroactive bacteria and bioproduction strains. We report, for the first time, DNA delivery by conjugation and functional ORIs for Halomonas alkaliphila, Halomonas neptunia, and Shewanella electrodiphila. Furthermore, we expand the list of functional ORIs for Duganella zoogloeoides, Pseudomonas alcaliphila, Shewanella oneidensis and Shewanella putrefaciens. This screen provides a scalable high-throughput system to rapidly build and identify functional plasmids to establish genetic tractability in non-model microbes.
While the installation of complex genetic circuits in microorganisms is relatively routine, the synthetic biology toolbox is severely limited in plants. Of particular concern is the absence of combinatorial analysis of regulatory elements, the long design-build-test cycles associated with transgenic plant analysis, and a lack of naming standardization for cloning parts. Here, we use previously described plant regulatory elements to design, build, and test 91 transgene cassettes for relative expression strength. Constructs were transiently transfected into Nicotiana benthamiana leaves and expression of a fluorescent reporter was measured from plant canopies, leaves, and protoplasts isolated from transfected plants. As anticipated, a dynamic level of expression was achieved from the library, ranging from near undetectable for the weakest cassette to a ∼200-fold increase for the strongest. Analysis of expression levels in plant canopies, individual leaves, and protoplasts were correlated, indicating that any of the methods could be used to evaluate regulatory elements in plants. Through this effort, a well-curated 37-member part library of plant regulatory elements was characterized, providing the necessary data to standardize construct design for precision metabolic engineering in plants.
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