Isopropanol → NADPH

Participants

Reaction Steps

This example illustrates a prescreening assay used to identify variant genes encoding ketoreductases capable of reducing isopropanol in the presence of NADP+ to produce acetone and NADPH. An E. coli colony containing a plasmid encoding an engineered ketoreductase was picked using a Q-Bot® robotic colony picker (Genetix USA, Inc., Boston, Mass.) into 96-well shallow well microtiter plates containing 180 μL Terrific Broth (TB), 1% glucose and 30 μg/mL chloramphenicol (CAM). Cells were grown overnight at 30° C. with shaking at 200 rpm. A 10 μL aliquot of this culture was then transferred into 96-deep well plates containing 390 μL Terrific Broth (TB), 1 mM MgSO4 and 30 μg/mL CAM. After incubation of the deep-well plates at 30° C. with shaking at 250 rpm for 2 to 3 hours, recombinant gene expression within the cultured cells was induced by addition of IPTG to a final concentration of 1 mM. The plates were then incubated at 30° C. with shaking at 250 rpm for 18 hours.

Provenance & Evidence