Generating Fluorescent Mycobacterium Mutants

Fluorescent M. fortuitum was generated using the pVV16-eGFP replicative vector (Vilchèze et al., 2014 (link)). Electro-competent M. fortuitum were generated as previously described (Viljoen et al., 2018 (link)). M. fortuitum was placed on ice for 2 h prior to pelleting by centrifugation (3,000 × g at 4°C for 15 min). Following centrifugation, bacteria were resuspended in decreasing volumes of wash buffer (10% glycerol (v/v) and 0.025% Tyloxapol in distilled water) and pelleted by centrifugation for a total of 4 washes. For electroporation transformation, 1 μg of plasmid DNA was added to 200 μL of electrocompetent bacteria and transferred to a chilled 0.2 cm electrode gap GenePulser electroporation cuvette (Bio-rad) and transformed using a GenePulser Cxell electroporator (Bio-rad) (2.5 kV, 1, 000 Ω and 25 μF). Bacteria were recovered in 800 μL of 7H9^OADC/T and placed at 37°C overnight. For selection of green fluorescent colonies, M. fortuitum was plated on 7H10^OADC supplemented 50 μg/mL kanamycin (Euromedex). Positive colonies were selected based on fluorescence and maintained in 7H9^OADC/T supplemented with 50 μg/mL kanamycin. Fluorescent M. abscessus has been previously described (Bernut et al., 2014a (link)).