Fig. 6
Wild-type landscapes for all three enzymes. Graphical representation of 96-well wild-type landscapes for all three enzymes. a KdgDAb (Z’ = 0.75), b KdgDCt (Z’ = 0.67), c KdgDPn (Z’ = 0.48); controls are empty vector and only media. Each 96-well plate contained 15 controls and 81 wells with the wild type enzyme. 100 μl of cell culture was centrifuged and the supernatant discarded. Lysis of the cells was done in 30 μl lysis buffer (2 mM KPi, pH 7.0, 2 mM MgCl2, 10 μg/ml DNaseI, 100 μg/ml lysozyme). Afterwards, 120 μl buffer (2 mM KPi, pH 7.0) was added and the lysed cells centrifuged. 20 μl of the crude extract was transferred into a new plate and 180 μl master mix was added to give a final volume of 200 μl (2.5 mM KPi, pH 7.0, 2 mM MgCl2, 25 μg/ml BTB and 5 mM keto-deoxy-d-glucarate)