Browsing by Subject "Biosensor"

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Application of fluorescent proteins for functional dissection of the drosophila visual system
(2021) Smylla, Thomas; Wagner, Krystina; Huber, Armin
The Drosophila eye has been used extensively to study numerous aspects of biological systems, for example, spatio-temporal regulation of differentiation, visual signal transduction, protein trafficking and neurodegeneration. Right from the advent of fluorescent proteins (FPs) near the end of the millennium, heterologously expressed fusion proteins comprising FPs have been applied in Drosophila vision research not only for subcellular localization of proteins but also for genetic screens and analysis of photoreceptor function. Here, we summarize applications for FPs used in the Drosophila eye as part of genetic screens, to study rhodopsin expression patterns, subcellular protein localization, membrane protein transport or as genetically encoded biosensors for Ca2+ and phospholipids in vivo. We also discuss recently developed FPs that are suitable for super-resolution or correlative light and electron microscopy (CLEM) approaches. Illustrating the possibilities provided by using FPs in Drosophila photoreceptors may aid research in other sensory or neuronal systems that have not yet been studied as well as the Drosophila eye.
Bioprocess exploitation of microaerobic auto-induction using the example of rhamnolipid biosynthesis in Pseudomonas putida KT2440
(2025) Grether, Jakob; Dittmann, Holger; Willems, Leon; Schmiegelt, Tabea; Benatto Perino, Elvio Henrique; Hubel, Philipp; Lilge, Lars; Hausmann, Rudolf; Grether, Jakob; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Dittmann, Holger; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Willems, Leon; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Schmiegelt, Tabea; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Benatto Perino, Elvio Henrique; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Hubel, Philipp; Core Facility Hohenheim, Mass Spectrometry Core Facility, University of Hohenheim, Ottilie-Zeller-Weg 2, 70599, Stuttgart, Germany; Lilge, Lars; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Hausmann, Rudolf; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany
Background: In biomanufacturing of surface-active agents, such as rhamnolipids, excessive foaming is a significant obstacle for the development of high-performing bioprocesses. The exploitation of the inherent tolerance of Pseudomonas putida KT2440, an obligate aerobic bacterium, to microaerobic conditions has received little attention so far. Here low-oxygen inducible promoters were characterized in biosensor strains and exploited for process control under reduction of foam formation by low aeration and stirring rates during biosynthesis of rhamnolipids. Results: In this study, homologous promoters of P. putida inducible under oxygen limitation were identified by non-targeted proteomic analyses and characterized by fluorometric methods. Proteomics indicated a remodeling of the respiratory chain and the regulation of stress-related proteins under oxygen limitation. Of the three promoters tested in fluorescent biosensor assays, the promoter of the oxygen-sensitive cbb3-type cytochrome c oxidase gene showed high oxygen-dependent controllability. It was used to control the gene expression of a heterologous di-rhamnolipid synthesis operon in an auto-inducing microaerobic two-phase bioprocess. By limiting the oxygen supply via low aeration and stirring rates, the bioprocess was clearly divided into a growth and a production phase, and sources of foam formation were reduced. Accordingly, rhamnolipid synthesis did not have to be controlled externally, as the oxygen-sensitive promoter was autonomously activated as soon as the oxygen level reached microaerobic conditions. A critical threshold of about 20% oxygen saturation was determined. Conclusions: Utilizing the inherent tolerance of P. putida to microaerobic conditions in combination with the application of homologous, low-oxygen inducible promoters is a novel and efficient strategy to control bioprocesses. Fermentation under microaerobic conditions enabled the induction of rhamnolipid production by low oxygen levels, while foam formation was limited by low aeration and stirring rates.