Fosmid library construction and screening

Genomic DNA was further purified by cesium chloride gradient ultracentrifugation prior to library creation [70]. A large insert library was constructed as described previously [71] using the CopyControl™ Fosmid Library Production Kit with pCC1FOS™ Vector Kit (EpiCentre). This resulted in a library containing 4608 clones. Functional screening was performed according to procedures by Mewis et al. [42] with modifications. Screening was carried out in phosphate buffer (25 mM sodium phosphate, pH 6.0), containing 100 µM each of the three fluorogenic substrates (6-chloro-4-methylumbelliferyl β-cellobioside, 6-chloro-4-methylumbelliferyl β-xylobioside, and 6-chloro-4-methylumbelliferyl β-D-xylopyranoside). Screening was performed at a temperature of 37 °C, which is the body temperature of Castor canadensis [72]. Fosmids chosen for sequencing (z-score > 3 for each substrate) were rearrayed using an automated colony-picking robot (Qpix2, Molecular Devices), into a 96 well plate (Costar 3370) containing 200 µL of LB chloramphenicol (12.5 µg/mL) and 10% glycerol. This master plate was incubated overnight at 37 °C and then stored at −80 °C.

The frozen master plate was then used to assay the clones against a panel of eleven different fluorogenic substrates, see supplemental methods for detailed description.