Construction of Metagenomic Fosmid Libraries

High-molecular-weight DNA extracted from all enrichment cultures was combined in equimolar amounts and used to prepare two metagenomic fosmid libraries, IS_Lib1 (Cavascura enrichments) and IS_Lib2 (Maronti enrichments) using the CopyControl fosmid library pCC2FOS production kit (Epicentre Technologies, Madison, WI, USA). DNA was end-repaired to generate blunt-ended 5′-phosphorylated fragments according to the manufacturer’s instructions. Subsequently, DNA fragments in the range of 30 to 40 kbp were resolved by gel electrophoresis (2 V cm⁻¹ overnight at 4°C) and recovered from 1% low-melting-point agarose gel using GELase 50× buffer and GELase enzyme (Epicentre). Nucleic acid fragments were then ligated to the linearized CopyControl pCC2FOS vector following the manufacturer’s instructions. After the in vitro packaging into the phage lambda (MaxPlax lambda packaging extract, Epicentre), the transfected phage T1-resistant EPI300-T1^RE. coli cells were spread on Luria-Bertani (LB) agar medium containing 12.5 μg mL⁻¹ chloramphenicol and incubated at 37°C overnight to determine the titer of the phage particles. The resulting libraries had estimated titers of 14 × 10⁴ and 1 × 10⁴ nonredundant fosmid clones in the IS_Lib1 and IS_Lib2 libraries, respectively. For long-term storage, E. coli colonies were washed from the agar surface using liquid LB medium containing 20% (vol/vol) sterile glycerol, and the aliquots were stored at −80°C.