Cytexpert software v 2

For expression quantification assays, fluorescence was measured with a Spark microplate reader (Tecan). Green fluorescence from GFP was analysed using excitation at 485±20 nm and emission at 530±20 nm and blue fluorescence from mTagBFP2 using excitation at 399±20 nm and emission at 454±20 nm. Cultures were performed directly within the microplate reader at 37 °C with orbital shaking (175 rpm) using black 96-well plates with clear bottoms in which wells were filled with 200 µl of LB supplemented with vitamin B12 (150 nM) and/or EA at indicated concentrations. OD600nm and fluorescence signals were recorded every 20 minutes.
Fluorescence quantification was also performed by flow cytometry (Cytoflex, Beckman Coulter). FSC (forward scatter), SSC (side scatter), FITC (488 nm exciter, 525±20 nm emitter) and PB450 (405 nm exciter, 450±22 nm emitter) axes were set to log display. For sample analysis, the following gating strategy was applied: bacterial cells were gated based on a FSC and SSC plot, and then analysed for BFP signal. A new gate covering BFP + cells, which correspond to bacterial cells of our reporter strain was selected and then analysed for GFP signal. At least 10,000 events of BFP + cells were recorded for each sample and data were analysed with CytExpert software v2.2 (Beckman Coulter) or with Floreada tool (https://floreada.io).