Team:UiOslo Norway/Experiments/In vivo Assay Methanol Dehydrogense

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In vivo Methanol Dehydrogenase Assay

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• Inoculate a preculture M9 minimal media containing the appropriate antibiotic and incubate at 37 °C overnight shaking at 220 rpm.

• Inoculate a mainculture (50 ml) with an OD600 of at least 0.3, add IPTG to a final concentration of 0.1 mM and grow the culture until the stationary phase is reached.

• Measure OD600 of the culture and harvest a cell pellet of an OD600 of 1 in a volumne of 50 ml by centrifugation at 8000 x g for 10 minutes at roomtemperature

• Resuspend the cell pellet in 50 ml M9 minimal media and measure the actual OD600

• Start the experiment by adding 2 ml of 10 % (v/v) methanol

• Directly after adding the methanol take a 600 µl sample

• Take a 600 µl sample every 5 minutes over a 1 hour

• Pellet the harvested cells by centrifugation at 11000 x g for 5 minutes at 4 °C

• Use 500 µl of the supernatant with 500 µl of Nash reagent and measure absorption at 412 nm and determine the concentration of formaldehyde