Plusone

The selected anticandidal bacteriocins were analyzed using glycine or tricine sodium dodecyl sulfate-polyacrylamide gel (Mini-Protein™ IV, BIO-RAD laboratory, Hercule, CA, USA) with a multicolor low-range protein ladder (Spectra™, Fermentus, Vilnius, Lithuania). They were visualized using the PlusOne Silver Staining Kit (PlusOne™, GE HEALTHCARE (AB)”, Pollards Wood, Nightingales Lane, Chalfont St. Giles, Buckinghamshire, HP8 4SP, UK). The molecular weights of the protein bands were calculated.

All chemical reagents purchased were 2D-DIGE grade (i.e., PlusOne, GEHealthcare, Little Chalfont, United Kingdom). Whole protein extracts were obtained from 75 cm² plates using cell lysis buffer composed of 40 mM Tris-HCl pH 7.8, 8 M urea, 2 M thiourea, 4% (w/v) 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), 50 mM DL-Dithiothreitol DTT, and protease inhibitor (Complete Mini, Roche Diagnostics). Cells were disrupted by passing them through a 23 gauge syringe 10 times. The homogenates were centrifuged at 20,000 g for 1 h at 4 °C. The supernatant was collected and conserved at −20 °C. An aliquot of crude protein extract was precipitated and washed according to the 2D clean-up kit procedure (GEHealthcare). Pellets were resuspended in 50 µL of Urea, Thiourea, CHAPS (UTC) buffer (8 M urea, 2 M thiourea, 4% CHAPS). Protein concentrations were measured using the Detergent compatible (DC)kit protein assay kit (Biorad) in microplate assays (BMG Labtech GmbH, Ortenberg, Germany). To verify the integrity of protein extracts and correct quantification of protein concentrations, 5 µg of each sample was separated in Bis-Tris NuPAGE Novex Midi Gels (Life Technologies, Carlsbad, CA, United States of America, under 3-(N-Morpholino)propanesulfonic acid, 4-Morpholinepropanesulfonic acid (MOPS) conditions. The gel was then stained using Coomassie staining solution.

The device was designed using Inventor 2016 (Autodesk) and printed on a Makergear M2 Rev E using PLA. An automated version of the stretcher included a high torque motor, an L293D motor driver chip (Texas Instruments) and a Raspberry Pi. Functionalized membranes of around 750 µm thick were manufactured by spincoating PDMS (1 : 20) on silicon wafers, then baking for 2.5 h at 75°C. With the edges covered, the membrane was exposed to oxygen plasma (2 min exposure at 100 W, Diener). They were then treated for 3 h with a solution consisting of 94% ethanol, 0.5% acetic acid and 0.1% Bind Silane (PlusOne, GE), followed by 1 mM BS³ (Thermo Fisher) and an overnight incubation with 100 µg ml⁻¹ fibronectin (Corning) or laminin (Sigma).