Bromothymol Blue

Kinase experiment:

For all three enzymes, an expression in the 96-deep well scale is performed. Therefore, electrocompetent E. coli ArcticExpress(DE3) cells are transformed with the corresponding plasmid. Single clones are picked using the CP7200 Colony Picker and transferred to 96-deep well plates filled with 1.2 mL autoinduction media by a MicroFlo Select dispenser. After incubation (36 h, 37°C at 1,000 rpm), further processing is done manually. First, 100 μL of cell culture is transferred into a 96-well plate (U-shaped bottom) and harvested by centrifugation (4,570 rpm, 10 min at RT) while the supernatant is discarded. The frozen pellets (1 h at -20°C) are thawed at room temperature for one hour to improve cell lysis. Lysis is continued by the addition of 30 μL lysis buffer (3 h, 1,000 rpm, 37°C) containing 2 mM KPi, pH 7.0, 2 mM MgCl2, 10 μg/mL DNaseI, 100 μg/mL lysozyme. Next, 120 μL buffer (2 mM KPi, pH 7.0) is added followed by centrifugation (3,000 rpm, 15 min at RT). For the photometric measurement, 20 μL of the crude extract is transferred to a 96-well plate (F-shaped bottom) and the reaction is started by adding 180 μL master mix 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). The measurements are carried out for 60 min at 2-min intervals. Depending on the enzyme, different time windows are used for the activity calculation[1].

References:

[1]. Pick A, et al. Identification and characterization of two new 5-keto-4-deoxy-D-Glucarate Dehydratases/Decarboxylases. BMC Biotechnol. 2016 Nov 17;16(1):80.
[2]. Yang W, et al. A plate method for rapid screening of Ketogulonicigenium vulgare mutants for enhanced 2-keto-l-gulonic acid production. Braz J Microbiol. 2017 Jul - Sep;48(3):397-402.