Mem amino acid solution

LACL-derived ligamentocytes were harvested from six adult males, healthy 12-month-old ACLs of New Zealand Rabbits, obtained from the regional slaughterhouse. The explanted LACLs were split into 2-mm² pieces and were cultivated in T25 culture flasks in growth medium (Dulbecco's modified Eagle’s medium [DMEM]/Ham’s F12 medium [1:1, Merck KGaA, Darmstadt, Germany] supplemented with 10% fetal bovine serum [FBS, Merck KGaA], 1% penicillin/streptomycin solution [Merck KGaA], 25 μg/ml ascorbic acid [Sigma-Aldrich, Munich, Germany], 2.5 μg/ml amphotericin B [Merck KGaA], MEM amino acid solution [Sigma-Aldrich]). After 7–10 days, ligamentocytes emigrated and were expanded using 0.05% trypsin/0.02% ethylenediaminetetraacetic acid (EDTA) for the subsequent experiments.

A sequence containing the E. coli EDL933 mgrB gene (Genbank Z2872) fused to a C-terminal His-6 tag (Supplementary Fig. 1d) was designed, synthesised (Eurofins), and subcloned into the XbaI and XhoI restriction sites of pBluescript KS(-) to obtain plasmid pmgrB-H6. This is the mgrB sequence of the first EHEC isolate sequenced (strain EDL933)^{81 (link)} which is identical to the ones found in BL21 or K12. The plasmid was then transformed into E. coli TOP10 cells to isolate sufficient amounts of plasmid DNA and verify the sequence. For overexpression, pmgrB-H6 was transfected into E. coli BL21 (DE3) pLysS (NEB) and plated on ampicillin-containing agar plates (100 µg/ml). In all, 16–24 h later, freshly grown colonies were transferred to 30 ml 1x M9 Salt Solution (Sigma-Aldrich) supplemented with 1x MEM Amino Acid Solution (Sigma-Aldrich). After 24–48 h, this bacterial culture was harvested by centrifugation and resuspended in M9 medium supplemented with the amino acid mix and 0.1 mM IPTG (Sigma-Aldrich). After different time points, 10 ml samples were collected, the bacteria were harvested by centrifugation and the supernatant was sterile filtrated using 0.2 µM filters (Filtropur S, Sarsted). All samples were immediately frozen and stored at −20 °C until use.

LACL-derived fibroblasts were isolated from 7 female, healthy ACLs of New Zealand Rabbits (approximate 12 months) derived from the abattoir. Explanted LACLs were sliced into 2 mm² pieces and placed into a T25 culture flask with growth medium for several weeks (Dulbecco’s Modified Eagle´s Medium (DMEM)/Ham’s F12 medium (1:1, Merck KGaA, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS, Merck KGaA), 1% penicillin/streptomycin solution (Merck KGaA), 25 μg/mL ascorbic acid (Sigma-Aldrich, Munich, Germany), 2.5 μg/mL amphotericin B (Merck KGaA), MEM amino acid solution (Sigma-Aldrich). After 7–10 days, emigrating cells could be harvested using 0.05% trypsin/0.02% EDTA treatment and be further expanded.