Growth assay for cobalamin-deficient E. coli

Growth assays were performed as previously described^{31 (link)}. Briefly, the cobalamin-deficient E. coli ΔFEC strain was transformed with various expression vectors to express BacA_Mt, ECF-CbrT or ECF-FolT2. Single colonies were then innoculated into M9 medium supplemented with 0.00001% (w/v) arabinose (Sigma-Aldrich) and 50 µg ml⁻¹ methionine (Sigma-Aldrich). Cells were grown at 37 °C for 24 h and were then diluted with a ratio of 1:250 into 200 µl fresh M9 medium supplemented with 0.00001% (w/w) arabinose and either 1 nM cyano-cobalamin or 50 µg ml⁻¹ methionine (Sigma-Aldrich) in 96-well plates (Greiner). Plates were sealed with sterile and gas-permeable foil (Breathe-Easy, Diversified Biotech) and incubated at 37 °C for 24 h. The OD₆₀₀ was measured every 10 min on a SpectraMax ABS plus Microplate reader (Molecular Devices). The measurements were performed as biological triplicates each containing technical triplicates and collected using the instruments’ SoftMax Pro version 7.1.2 (Molecular Devices).