Fig. 1

Workflow for the pulse-chase SILAC experiment. For biofilm generation, E. coli PHL644 cells in stationary phase were spun down onto poly-l-lysine coated slides placed in 9-deep-well plates. Triplicate sets of biofilms were initially matured in minimal M63 media supplemented with either [2,6-²H₂]-l-phenylalanine (rows 1-3, filled blue circles) or [4,4,5,5-²H₄]-l-lysine (rows 4–6, filled orange circles). After 3 days of maturation, biofilms from rows 1, 4 were harvested (as denoted by the crosses) to prepare protein samples. Labelled media were replaced with fresh unlabelled media (empty blue circles for l-phenylalanine and empty orange circles for l-lysine) for the chase phase. Following a further 3 days of incubation, the biofilms in rows 2, 5 that had matured for 6 days were harvested to extract proteins. The media of the remaining biofilms was again exchanged; fresh media containing [2,6-²H₂]-l-phenylalanine were provided to the biofilms that had been previously supplemented with lysine, and conversely fresh labelled media supplemented with [4,4,5,5-²H₄]-l-lysine were provided to the biofilms that had previously been supplemented with phenylalanine. At day 9, biofilms from rows 3 and 6 were harvested