Sn glycerol 3 phosphate

Cells were grown in M63 glucose minimal medium to OD600 ~1.0. Cells were pelleted and washed with M63 glycerol medium, pH 7.0 or pH 5.5. Cells were then resuspended in same volume of the same medium and grown at 37˚C for 3 h. Cells (500 µl) were pelleted and resuspended in 500 µl of lysis buffer (25 mM Tris-HCl , 10 mM NaCl and 0.4% Triton X100).
Cells were lysed by adding 0.6 g of glass beads and vortexing 30 s followed by 30 s incubation on ice, repeated 5X. The cells were then centrifuged at 20,000 × g for 2 min at 4˚C, and the lysate was used to measure the dehydrogenase activity. A method monitoring MTT reduction to quantitate the dehydrogenase activity of GlpD (Yeh et al., 2008) was modified as follows. Each 225 µl microcuvette contained the following: 25 mM Tris/HCl pH 7.4, 100 mM NaCl, 1 mM MTT (Sigma Aldrich), 3 mM phenazine methosulfate (PMS, Sigma Aldrich) and 100 µl of lysate. This was used as the blank, and the reaction was initiated by the addition of 3.7 mM snglycerol-3-phosphate (Sigma Aldrich). The reduction of MTT at 570 nm was continuously monitored on a BMG LABTECH plate reader for 118 min at room temperature.