N glycolylneuraminic acid neu5gc

E. coli strains were grown at 37 °C aerobically in lysogeny broth (LB) (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl, pH 7.5) or in minimal medium (MM). MM is M9 mineral medium (33.7 mM Na₂HPO₄, 22.0 mM KH₂PO₄, 8.55 mM NaCl, 9.35 mM NH₄Cl) supplemented with 2 mM MgSO₄ and 0.1 mM CaCl₂. As carbon and energy source, either 27.8 mM [0.5% w/v] glucose, 6.47 mM [0.2% w/v] 5-N-acetylneuraminic acid (Neu5Ac) or 6.15 mM [0.1% w/v] N-glycolylneuraminic acid (Neu5Gc) (all purchased from Sigma-Aldrich, Taufkirchen, Germany) was added. If appropriate, the following antibiotics were used: ampicillin sodium salt (150 μg/ml) or kanamycin (50 μg/ml). For solid media, 1.5% agar (w/v) was added. For all growth experiments, bacterial strains were grown in LB medium overnight at 37 °C aerobically, washed twice in PBS and then adjusted to an optical density at 600 nm (OD₆₀₀) of 0.005 in the desired liquid growth medium, or streaked on agar plates. Growth curves were obtained from bacterial cultures incubated at 37 °C with gentle agitation in 96-well microtitre plates containing 200 μl medium. The OD₆₀₀ was measured by an automatic reader (Epoch2T; BioTek, Bad Friedrichshall, Germany) at appropriate time intervals as indicated.