Rapid flow sterile

Isolation of the rOMVs carrying GFPmut2 was achieved following a previously described protocol [18 (link)] with minor modifications. The supernatant fraction from the previous centrifugation step was filtered through a Rapid-Flow sterile disposable filter unit made of polyethersulfone (PES) with 0.45 μm pore size (Thermo Scientific, USA). The culture filtrate was then concentrated by ultrafiltration (centrifugation at 5,000 ×g, 4°C) using a Macrosep Advance centrifugal device with a 100K molecular weight cutoff PES membrane (Pall Corporation, USA). The rOMVs were pelleted from the concentrated culture filtrate via ultracentrifugation at 150,000 ×g at 4°C for 3 h in an Optima XE-100 ultracentrifuge (Beckman Coulter, USA). The pelleted OMVs were resuspended in 900 μl of phosphate-buffered saline (PBS), pH 7.4, and stored at -20°C.

Water and FFT cells were retrieved by filtering ~1.5 L water through 0.22 μm Rapid-Flow sterile disposable filters (Thermo Fisher Scientific (Waltham, MA, USA)) and from ca. 50 g wet FFT, respectively, and stored at −20 °C until DNA was extracted. DNA was extracted and purified from water column samples using the DNeasy PowerWater DNA Isolation Kit (Qiagen, Hilden, Germany), while ca. 0.5 g of each FFT sample was extracted using the FastDNA Spin Kit for Soil (MP Biomedical, Irvine, CA, USA). DNA concentration and quality were confirmed with a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) using a Qubit High-Sensitivity dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and DNA quality was assessed using an Epoch Microplate Spectrophotometer with Take3 plate (BioTek, Winooski, VT, USA).