Fosmid autoinduction solution

The metagenomic libraries were screened for carboxylesterase and lipase activity as follows. The fosmid library was grown on LB agar plates containing 12.5 μg mL⁻¹ chloramphenicol at 37°C overnight to yield single colonies. Then, 3,456 clones were arrayed in 9- by 384-well microtiter plates and cultivated at 37°C in LB medium supplemented with 12.5 μg mL⁻¹ chloramphenicol. Those original microtiter plates were stored at –80°C after the addition of glycerol, at a final concentration of 20% (vol/vol). For screening clone libraries, 384-pin replicators were used to print clones onto the surface of large LB agar square plates (245 mm by 245 mm) containing 12.5 μg mL⁻¹ chloramphenicol, and 2 mL liter⁻¹ fosmid autoinduction solution (Epicentre), and each plate contained 0.3% (vol/vol) tributyrin (Sigma-Aldrich, Gillingham, United Kingdom) as described previously (27 (link)). After an initial overnight growth at 37°C, the LB agar plates were incubated for 48 h at 37, 50, or 70°C. Positive hits were confirmed by retesting the corresponding fosmid clones taken from the original microtiter plate.

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)).