Avanti jxn 26 centrifuge

A selection plasmid containing an inducible bacterial toxin and target site was transformed into NEB^® Turbo Competent E. coli (High Efficiency) (S7 Fig). We started an 8 mL overnight culture of the cells shaking at 250 rpm at 37°C for 16 hours. In the morning, 200 μL of cells were then inoculated into 50 mL of Terrific Broth (Teknova) with 2.5 μL of 1X10¹¹ pfu/ml M13KO7 Helper Phage (New England Biolabs). After four hours of growing the culture at 37°C at 250 rpm, cells were spun at 4500 g for 10 min in an Avanti JXN-26 centrifuge (Beckman Coulter). The supernatant was collected without disturbing the cell pellet and the centrifugation was repeated. The top 40 mL of supernatant were added to a fresh tube and we mixed with 10 mL of 2.5 M NaCl/20% PEG-8000 (w/v) (VWR). Phages were precipitated overnight at 4°C for 16 hours and then spun at 12,000 g for one hour at 4°C. Pellet was resuspended in 1 mL of TBS and 200 μL of 2.5 M NaCl/20% PEG-8000 (w/v) (VWR) was added. Phages were precipitated for 3 hours on ice and then spun them at 12,000 rpm in a Sorvall^™ Legend^™ Micro 21R centrifuge (Thermo Scientific) for 30 minutes at 4°C. After removing the supernatant, 200 μL of 1X TBS and 200 μL of 50% glycerol were used to resuspend the pellet. The phages were stored at -20°C until the start of the selection.

SA (^NCXCL-SA^C and ^NSA-scFv^C) and Fc (^NFc-CXCL^C) fusion proteins were expressed by transient transfection of suspension-adapted human embryonic kidney (HEK293) cells. Cell lines were not authenticated and were tested for mycoplasma contamination. Protein production was performed either in house using FreeStyle 293 Expression System (Thermo Fisher Scientific) or outsourced to the Protein Expression Core Facility (PECF) of the Life Science Faculty of the EPFL^{64 (link),65 (link)}. At the end of the 7-day phase production, cells were harvested by centrifugation at 15,000 × g for 30 min at 4 °C on an Avanti JXN-26 Centrifuge (Beckman Coulter). Any additional cell debris was removed from the medium by filtration through 0.22-μm PES membrane filters (Thermo Fisher Scientific).

The lab-scale storage stability of the selected strains was tested using a culture fermenter. LAB strains were pre-cultivated for 48 h at 37 °C in MRS broth under anaerobic conditions. Pre-cultured strains in MRS were inoculated into a lab-scale fermenter at a final concentration of 1% (v/v). The fermenter was run at 37 °C, with the pH maintained at 5.5 and stirring at 100 rpm, for 18 h. The pH during fermentation was adjusted using 15% ammonia solution. After fermentation, the culture broth was centrifuged at 4000× g for 20 min at 10 °C (Avanti JXN-26 centrifuge; Beckman Coulter (Brea, CA, USA)). The collected pellet was mixed thoroughly with an equal volume of 10% skim milk as a cryoprotectant. The suspensions were dispensed into Petri dishes and freeze-dried. The lyophilized strains were then stored in aluminum/PE pouches at 25 °C/40% humidity for 4 weeks. The number of CFU was measured four times (once per week over 4 weeks).