Regensburg 2025 – scientific programme
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BP: Fachverband Biologische Physik
BP 14: Poster Session I
BP 14.19: Poster
Tuesday, March 18, 2025, 10:00–12:30, P3
Cell-free protein synthesis measured in flowing nanolitre-droplets — Benno Schedler1, Alexandros Katranidis2, and •Jörg Fitter1,2 — 1AG Biophysik, I. Physikalsiches Institut (IA), RWTH Aachen University, D-52074 Aachen, Germany — 2Forschungszentrum Jülich, ER-C-3, D-52425 Jülich, Germany
Cell-size confinement of biological reactions by utilizing microfluidic water-in-oil droplets has been widely used to revolutionize the field of biomolecular research. This approach capitalizes on the precise control and manipulation of nanoliter-sized droplets within microfluidic channels. The confinement facilitates reduced reagent consumption and improved scalability. Analysing cell-free protein synthesis (CFPS) is an ideal application of this approach and represents a complex multistep process which can be monitored in individual droplets if fluorescent proteins are synthesized [1]. Understanding the physicochemical principles underlying CFPS reactions, including the role of macromolecular crowding, is crucial for optimizing protein synthesis yields and functionality [2]. Here we present the results of our ongoing research using the example of the synthesis of green fluorescent protein (GFP), which we have analysed employing confocal fluorescence microscopy. Focus is set on the high throughput capability of the approach and thus the possibility of analysing several hundred parallel reactions. The latter provide important information about the average synthesis productivity and the distribution widths for reactions under different environmental conditions. [1] Hansen et al., 2016, Nature Nanotechnology, 11, 191; [2] Kempf et al., 2017, Scientific Reports, 7, 46753
Keywords: water-in-oil droplets; cell free protein synthesis; confocal fluorescence microscopy; microfluidics; gene expression noise