Construction of soil metagenome cosmid library

A soil sample of ∼1 kg was collected from Half Moon Bay, Kaikōura New Zealand. A sample of 250 g was weighed into a 1 L centrifuge bottle, with 270 mL heated (70 °C) lysis buffer (100 mM Tris–HCl, 100 mM Na EDTA, 1.5 M NaCl, 1% (w/v) cetyl trimethyl ammonium bromide, pH 8.0) added. This was mixed to ensure the soil was thoroughly wetted, and returned to the water bath to reach 70 °C. A volume of 30 mL heated (70 °C) 20% SDS (w/v) was added to the centrifuge tube, inverted to mix, and returned to the water bath. The sample was incubated for 2 h, with gentle inversion every 30 min. The sample was then rapidly cooled in an ice/water bucket for 30 min, with inversion once in this time. The soil and chilled precipitate were then separated from the clarified lysate by centrifugation at 4500 rcf for 35 min at 4 °C. The supernatant was recovered, volume measured, and returned to room temperature by brief incubation in the warm water bath. Isopropanol was added to 0.7 × the supernatant volume, and mixed by gentle inversion to precipitate the eDNA. Following a 30 min room temperature incubation, the precipitated eDNA was collected by centrifugation at 4500 rcf for 35 min at 4 °C. The supernatant was discarded, and the eDNA pellet and centrifuge bottle was washed with 100 mL of ice cold 70% (v/v) ethanol. The sample was again centrifuged at 4500 rcf for 10 min at 4 °C. The supernatant was discarded, and the bottle briefly centrifuged again to collect the remaining ethanol, which was removed by pipette. The eDNA pellet was briefly air dried (no more than 15 min). A minimum volume of TE buffer was added to cover the eDNA pellet, and this was left to slowly resuspend overnight at room temperature.

The eDNA substrate for cosmid library construction was then size selected by agarose gel extraction in 0.8% agarose 1× TAE gel run at 80 V for 2 h alongside λ HindIII marker, then overnight in fresh TAE at 18 V. DNA above the 23 kb marker was excised from the gel and electroeluted, then concentrated using a 30 000 kDa molecular weight cut-off column centrifugal concentrator. The DNA sample was quantified by nanodrop, then end-repaired using End-It DNA End-Repair Kit (Epicentre).

The cosmid vector pWEB::tnc was prepared by digested with SmaI restriction enzyme (NEB), and dephosphorylation with Antarctic phosphatase (NEB) according to the manufacturer's instructions. The prepared cosmid vector (250 ng) and eDNA substrate (125 ng) were then ligated in a final reaction volume of 5 μL using Fast-link DNA ligase (Epicentre) according to the manufacturer's instructions. Phage packaging extract was prepared in house according to Winn and Norris (2010), and used to package the ligated DNA. The resulting diluted phage heads were added in a 1 : 10 ratio to an ice cold, day culture of E. coli EC100 (or EC100 ΔentD) grown to OD₆₀₀ of 1.0 in LB 10 mM MgSO₄. The phage head/cell mixture was incubated at room temperature for 20 min, then aliquoted across 96 wells and incubated at 37 °C 200 rpm for 75 min. The recovered, transfected E. coli cells were then diluted in LB Amp Chl for cosmid selection, and titre samples of select wells plates plated on LB Amp Chl agar, for colony counting and library size estimations. The remaining cultures were incubated overnight at 37 °C 200 rpm to replicate the library clones in liquid culture. The following morning, samples were taken from each of the 96 aliquots to make glycerol stocks for long term storage, alongside minipreps of each library “well”.