Molecular biology methods

2.5.1 DNA isolation

Cell pellets (M. ruminantium M1 or protozoa-associated methanogen community) were frozen in liquid nitrogen and ground into a fine powder with a sterile mortar and pestle. For the pellet containing protozoa-associated methanogens, the pellet was heated to 70 °C for 10 min to de-activate any remaining deoxyribonucleases before freeze- grinding. DNA was then extracted using QIAGEN genomic tip 500/G according to the manufacturer’s recommendations (Qiagen, Germany).

2.5.2 Plasmid DNA isolation

Cell pellets obtained from E. coli cultures (5 mL) were obtained from centrifugation at 12,000 × g for 5 min at room temperature. Plasmid DNA was extracted using QIAGEN plasmid DNA isolation kit according to the manufacturer’s recommendations. For larger

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scale plasmid preparations (from 20-50 mL of E. coli culture), PureLink® HiPure Plasmid Midiprep Kit (Life Technologies, CA, USA) was used according to the manufacturer’s instructions.

2.5.3 Colony PCR

A PCR master mix was prepared for the appropriate primer pair, and 25 μL of the master mix was dispensed into 0.2 mL PCR tubes. For amplification of 18S rRNA gene sequences, the reaction mix contained 1× PCR buffer, primer pair RP841F/Reg1302R (0.5 μM for each primer), 1.5 mM MgCl2, 0.02% bovine serum albumin (BSA) (w/v), and 0.15 U/μL of Taq polymerase. PCR cycling parameters used for this reaction are described in Table 2.2. For amplification of phage display library inserts, the reaction mix contained 1× PCR buffer, 2.5 mM MgCl2, primer pair pspF03/pspR03 (0.2 μM for each primer), and 0.02 U/μL of Taq polymerase. PCR cycling parameters used for this reaction are described in Table 2.3. For each PCR reaction, a random colony was picked up using a sterile pipette tip, spot inoculated onto a Petri plate containing the appropriate medium and antibiotics, and then the remaining bacterial cells were transferred into the PCR reaction mix by pipetting up and down. PCR amplicons were generated using a Mastercycle Pro thermocycler (Eppendorf, Germany).

Table 2.2. Thermal profile for PCR amplification of partial 18S rRNA gene sequences.

Initial denaturation: 94 °C for 3 min Amplification (35 cycles): 94 °C for 30 s

54 °C for 45 s 72 °C for 1 min Final extension: 72 °C for 7 min

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Table 2.3. Thermal profile for PCR amplification of phage display library inserts. Initial denaturation: 95 °C for 2 min

Amplification (30 cycles): 95 °C for 15 s 59 °C for 30 s 72 °C for 1 min Final extension: 72 °C for 7 min

2.5.4 Preparation of PCR amplicons for sequencing

2.5.4.1 Sanger sequencing of the 16S archaeal gene from M. ruminantium M1

Archaeal 16S rRNA gene sequences were amplified by PCR using the primer pair Ar915F/Ar1386R (1 μM for each primer) in a reaction mix containing 1.5 mM MgCl2, 0.02% BSA (w/v), and 0.05 U/μL of Taq polymerase. PCR cycling parameters used for this reaction is described in Table 2.4. Sequence analysis was performed by Massey Genome Service (Palmerston North, New Zealand).

Table 2.4. Thermal profile for PCR amplification of archaeal 16S rRNA gene sequences.

Initial denaturation: 95 °C for 2 min Amplification (30 cycles): 95 °C for 15 s

59 °C for 30 s 72 °C for 1 min Final extension: 72 °C for 7 min

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2.5.4.2 Sanger sequencing of the partial 18S rRNA gene sequence from rumen

protozoa

Protozoal 18S rRNA gene sequences were amplified by PCR using the primer pair RP841F/Reg1302R (0.5 μM for each primer) in a reaction mix containing 1.5 mM MgCl2, 0.02% BSA (w/v), and 0.15 U/μL of Taq polymerase. PCR cycling parameters used for this reaction is described in Table 2.2. Sequence analysis was performed by Macrogen Inc. (Seoul, Korea).

2.5.4.3 Sanger sequencing of phage display inserts

Insert sequences present in the phage display vector pYW01 were amplified by PCR using the primer pair pspF03/pspR03 (0.2 μM for each primer) in a reaction mix containing 2.5 mM MgCl2 and 0.02 U/μL of Taq polymerase. PCR cycling parameters used for this reaction are described in Table 2.3. Sequence analysis was performed either by Massey Genome Service (Palmerston North, NZ) or Macrogen (Seoul, Korea).

2.5.4.4 16S and 18S rRNA gene sequencing by pyrosequencing

Archaeal 16S rRNA gene sequences were amplified by PCR using the primer pair Ar915F/Ar1386R (1 μM for each primer) in a reaction mix containing 1.5 mM MgCl2, 0.02% BSA (w/v), and 0.05 U/μL of Taq polymerase. Bacterial 16S rRNA gene sequences were amplified by PCR using primer pair Ba9F/Ba515Rmod1 (0.1 μM for each primer) in a reaction mix containing 2.5 mM MgCl2 and 0.02 U/μL of Taq polymerase. Protozoal 18S rRNA gene sequences were amplified by PCR using the primer pair RP841F/Reg1302R (0.5 μM for each primer; final concentration) in a reaction mix containing 1.5 mM MgCl2, 0.02% BSA (w/v), 50 pg/μL of DNA, and 0.15 U/μL of Taq polymerase. Barcoded PCR reactions were performed as described previously (Kittelmann et al., 2013).

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2.5.4.5 PacBio sequencing of phage display inserts

Insert sequences present in the phage display vector pYW01 were amplified by PCR using the primer pair PelBF1/pspR03 (0.5 μM for each primer; final concentration) in a reaction mix containing 2.5 mM MgCl2 and 0.02 U/μL of Taq polymerase. PCR cycling parameters used for this reaction is described in Table 2.3. For the libraries prior to biopanning against protozoal bait, PAM1 and PAM2, the PCR amplicons were electrophoresed on 1% (w/v) agarose gels. Fragments between 0.4-1.5 kb were excised and purified using the Wizard® SV Gel and PCR Clean-Up System (Promega; Wisconsin, USA). For the PAM_FINAL library (after two rounds of biopanning against protozoal cells as bait), PCR amplicons were purified and size selected by using NucleoMag NGS Clean-up and Size Select magnetic beads (medi’Ray; Auckland, NZ). DNA samples were submitted to the Leibniz-Institut DSMZ (Germany) for sequencing.

2.5.5 Denaturing gradient gel electrophoresis (DGGE)

Protozoal 18S rRNA gene sequences were amplified by PCR using the primer pair Syl316F and Syl539-GC (1 μM for each primer) in a reaction mix containing 2 mM MgCl2, 0.05% BSA (w/v), and 1 U/μL of Taq polymerase, as described previously (Sylvester et al., 2005). PCR cycling parameters used for the reaction are detailed in Table 2.5. Denaturing gradient gel electrophoresis (DGGE) was performed on the amplicons in an 8% agarose gel with a gradient from 25% to 45% denaturants (where 100% denaturants represents 7 M urea and 40% formamide) at 60 V for 16 h at 60 °C in 0.5× TAE buffer (Sylvester et al., 2005). The gel was stained with SYBRgold (Life Technologies) according to manufacturer’s instructions.

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Table 2.5. Thermal profile for PCR amplification of protozoal 18S rRNA gene sequences in preparation for DGGE.

First cycle: 94 °C for 4 min

56 °C for 30 s 72 °C for 1 min

35 cycles: 94 °C for 1 min

56 °C for 30 s 72 °C for 1 min

Final cycle: 94 °C for 4 min

56 °C for 30 s 72 °C for 30 min