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Subject: bacterial growth ×

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    Real-time image processing for label-free enrichment of Actinobacteria cultivated in picolitre droplets
    (London [u.a.] : Royal Society of Chemistry, 2013) Zang, E.; Brandes, S.; Tovar, M.; Martin, K.; Mech, F.; Horbert, P.; Henkel, T.; Figge, M.T.; Roth, M.
    The majority of today's antimicrobial therapeutics is derived from secondary metabolites produced by Actinobacteria. While it is generally assumed that less than 1% of Actinobacteria species from soil habitats have been cultivated so far, classic screening approaches fail to supply new substances, often due to limited throughput and frequent rediscovery of already known strains. To overcome these restrictions, we implement high-throughput cultivation of soil-derived Actinobacteria in microfluidic pL-droplets by generating more than 600000 pure cultures per hour from a spore suspension that can subsequently be incubated for days to weeks. Moreover, we introduce triggered imaging with real-time image-based droplet classification as a novel universal method for pL-droplet sorting. Growth-dependent droplet sorting at frequencies above 100 Hz is performed for label-free enrichment and extraction of microcultures. The combination of both cultivation of Actinobacteria in pL-droplets and real-time detection of growing Actinobacteria has great potential in screening for yet unknown species as well as their undiscovered natural products.
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    NERNST: a genetically-encoded ratiometric non-destructive sensing tool to estimate NADP(H) redox status in bacterial, plant and animal systems
    ([London] : Springer Nature, 2023) Molinari, Pamela E.; Krapp, Adriana R.; Weiner, Andrea; Beyer, Hannes M.; Kondadi, Arun Kumar; Blomeier, Tim; López, Melina; Bustos-Sanmamed, Pilar; Tevere, Evelyn; Weber, Wilfried; Reichert, Andreas S.; Calcaterra, Nora B.; Beller, Mathias; Carrillo, Nestor; Zurbriggen, Matias D.
    NADP(H) is a central metabolic hub providing reducing equivalents to multiple biosynthetic, regulatory and antioxidative pathways in all living organisms. While biosensors are available to determine NADP+ or NADPH levels in vivo, no probe exists to estimate the NADP(H) redox status, a determinant of the cell energy availability. We describe herein the design and characterization of a genetically-encoded ratiometric biosensor, termed NERNST, able to interact with NADP(H) and estimate E NADP(H). NERNST consists of a redox-sensitive green fluorescent protein (roGFP2) fused to an NADPH-thioredoxin reductase C module which selectively monitors NADP(H) redox states via oxido-reduction of the roGFP2 moiety. NERNST is functional in bacterial, plant and animal cells, and organelles such as chloroplasts and mitochondria. Using NERNST, we monitor NADP(H) dynamics during bacterial growth, environmental stresses in plants, metabolic challenges to mammalian cells, and wounding in zebrafish. NERNST estimates the NADP(H) redox poise in living organisms, with various potential applications in biochemical, biotechnological and biomedical research.