Screening Fosmid Libraries for Lipase/Esterase Activity

Fosmid clones obtained by plating the constructed libraries on LB agar plates were arrayed in 384-microtiter plates (1 clone/well) or alternatively in 96-microtiter plates (pools of approximately 40 clones/well) containing LB medium and chloramphenicol (12.5 μg/mL). The plates were incubated at 37°C overnight, and the day after replication, the plates were produced and used in the screening assay. Glycerol (20% [vol/vol], final concentration) was added to the original plates, which were stored at −80°C. Gel diffusion and colorimetric assays were adapted for the screening of the desired activities. The detection of lipase/esterase activity was carried out on LB agar supplemented with chloramphenicol (12.5 μg/mL), fosmid autoinduction solution (2 mL/L) (Epicentre), and 0.3% (vol/vol) tributyrin emulsified with gum arabic (2:1, vol/vol) by sonication. The previously prepared microtiter plates were printed on the surface of large (22.5 cm by 22.5 cm) LB agar plates using 384-pin polypropylene replicators and incubated for 18 to 48 h at 37°C. Lipolytic activity was identified as a clear zone around the colonies where tributyrin was hydrolyzed (12 (link)).