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Condensation of RNA and proteins is central to cellular functions, and the ability to program it would be valuable in synthetic biology and synthetic cell science. Here we introduce a modular platform for engineering synthetic RNA condensates from tailor-made, branched RNA nanostructures that fold and assemble co-transcriptionally. Up to three orthogonal condensates can form simultaneously and selectively accumulate fluorophores through embedded fluorescent light-up aptamers. The RNA condensates can be expressed within synthetic cells to produce membrane-less organelles with a controlled number and relative size, and showing the ability to capture proteins using selective protein-binding aptamers. The affinity between otherwise orthogonal nanostructures can be modulated by introducing dedicated linker constructs, enabling the production of bi-phasic RNA condensates with a prescribed degree of interphase mixing and diverse morphologies. The in situ expression of programmable RNA condensates could underpin the spatial organization of functionalities in both biological and synthetic cells. 

As membrane-mediated antibiotic resistance continues to evolve in Gram-positive bacteria, the development of new approaches to elucidate the membrane properties involved in antibiotic resistance has become critical. Membrane vesicles (MVs) secreted by the cytoplasmic membrane of Gram-positive bacteria contain native components, preserving lipid and protein diversity, nucleic acids, and sometimes virulence factors. Thus, MV-derived membrane platforms present a great model for Gram-positive bacterial membranes. In this work, we report the development of a planar bacterial cytoplasmic membrane-based biosensor using MVs isolated from the Bacillus subtilis WT strain that can be coated on multiple surface types such as glass, quartz crystals, and polymeric electrodes, fostering the multimodal assessment of drug–membrane interactions. Retention of native membrane components such as lipoteichoic acids, lipids, and proteins is verified. This biosensor replicates known interaction patterns of the antimicrobial compound, daptomycin, with the Gram-positive bacterial membrane, establishing the applicability of this platform for carrying out biophysical characterization of the interactions of membrane-acting antibiotic compounds with the bacterial cytoplasmic membrane. We report changes in membrane viscoelasticity and permeability that correspond to partial membrane disruption when calcium ions are present with daptomycin but not when these ions are absent. This biomembrane-based biosensing platform enables an assessment of membrane biophysical characteristics during exposure to antibiotic drug candidates to aid in identifying compounds that target membrane disruption as a mechanism of action.