Restriction enzymes

DNA manipulations were carried out based on previously published protocols⁶⁷ . Restriction enzymes and T4 DNA ligase were obtained from Roche Diagnostics (Basel, Switzerland), and were used according to the manufacturer’s instructions. PCRs were performed using either Q5® High-Fidelity DNA polymerase (New England Biolabs, Hertfordshire, UK) or Extensor Long Range PCR Enzyme master mix (Thermo Scientific, Glouchester, UK). Synthetic oligonucleotides were synthesized by Eurofins (Ebersberg, Germany) and are listed in supplemental Table S2 and S10. Ird-labelled synthetic oligonucleotides were provided by IDT (Integrated DNA technologies, Dresden, Germany) and are listed in Supplemental Table S2. PCR products were purified with the use of a High-Pure PCR product purification kit (Roche, Basel, Switzerland). Plasmid DNA was introduced into E. coli and B. breve by electroporation, and large-scale preparation of chromosomal DNA from B. breve was performed as described previously^{68 (link)}. Plasmid DNA was obtained from B. breve and E. coli using the Roche High Pure plasmid isolation kit (Roche Diagnostics, Basel, Switzerland). An initial lysis step was performed using 30 mg ml⁻¹ of lysozyme for 30 min at 37 °C as part of the plasmid purification protocol for B. breve.

DNA manipulations were carried out as previously reported (Russell, 2001 ). Restriction enzymes and T4 DNA ligase were obtained from Roche Diagnostics, and were used according to the manufacturer's instructions. PCRs were performed using Extensor Long Range PCR Enzyme master mix (Thermo Scientific). Synthetic oligonucleotides were synthesized by Eurofins (Ebersberg, Germany) and are listed in Table 2. PCR products were purified by the use of a High-Pure PCR product purification kit (Roche). Plasmid DNA was introduced into E. coli and B. breve by electroporation and large-scale preparation of chromosomal DNA from B. breve was performed as described previously (O'Riordan and Fitzgerald, 1999 (link)). Plasmid DNA was obtained from B. breve and E. coli using the Roche High Pure plasmid isolation kit (Roche Diagnostics, Basel, Switzerland). An initial lysis step was performed using 30 mg ml⁻¹ of lysozyme for 30 min at 37°C as part of the plasmid purification protocol for B. breve.

Restriction enzymes (Roche Molecular Biochemicals and New England BioLabs), Antarctic phosphatase (New England BioLabs, Ipswich, MA, USA), and T4 DNA ligase (Roche Molecular Biochemicals, St. Louis, MO, USA) were used according to the manufacturer’s instructions. When necessary, the End-It DNA end repair kit (Epicentre, Middletown, WI, USA) was used to convert 5′ or 3′ protruding ends to blunt-ended DNA. DNA fragments used in cloning procedures were excised from agarose gels and purified with a PureLink Quick Gel Extraction Kit (Invitrogen, Waltham, MA, USA). Bacterial genomic DNA was prepared from overnight LB broth cultures with the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich, St. Louis, MO, USA). Plasmids were purified from overnight LB broth cultures by using the Wizard Plus SV Miniprep DNA Purification System (Promega, Madison, WI, USA).

Cell culture media were from Invitrogen (Life Technologies SAS, Saint Aubin, France). The Escherichia coli Rosetta 2(DE3) cells, used for the recombinant protein expression, were from Novagen-EMD4 Biosciences (Merck Chemicals, Nottingham, United Kingdom). Restriction enzymes, T4 DNA ligase, RPROTKSOL-RO: recombinant PCR grade Proteinase K were from Roche (Basel, Swiss), deoxyribonuclease I from bovine pancreas (DNAse I) was from ThermoFisher Scientific (Lithuania), and calf-intestinal alkaline phosphatase (CIP) was from New England Biolabs France (Evry, France). Snake venom phosphodiesterase 1 from Crotalus adamanteus (SVPDE1) was from Worthington (Biochemical Corporation). The purified human Nudix (nucleoside diphosphate-linked moiety X)-type motif 16 (NUDT16) protein was prepared as described (Palazzo et al., 2015 (link)). Bovine PARG was from Trevigen (Gaithersburg, United States). Bleomycin was from Sanofi-Aventis (France).

The general procedures used for DNA manipulation were essentially those described previously [40] . Restriction enzymes and T4 DNA ligase were obtained from Roche Diagnostics and used according to the manufacturer's instructions. PCRs were performed using Taq PCR master mix (Qiagen GmbH). Synthetic oligonucleotides were synthesized by MWG Biotech AG and are listed in Table S1. PCR products were purified by using a High-Pure PCR product purification kit (Roche). Plasmid DNA was introduced into E. coli and B. breve by electroporation and large-scale preparation of chromosomal DNA from Bifidobacterium spp. was performed as described previously [41] (link). Plasmid DNA was obtained from B. breve and E. coli using a QIAprep spin plasmid miniprep kit (Qiagen GmbH). An initial lysis step was performed using 30 mg/ml of lysozyme for 30 min at 37°C as part of the plasmid purification protocol for B. breve.

DNA manipulations were performed according to standard protocols [39] . Restriction enzymes and the ligase were purchased from Roche. Primers were obtained from STAB VIDA (Caparica, Portugal). Plasmid DNA from E. coli was isolated using a GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich). Sequencing was performed at STAB VIDA (Caparica, Portugal).

All enzymes, as well as plasmid pGEM1, TNT T7 Quick Coupled System and rabbit reticulocyte lysate were from Promega (Madison, WI, USA). ER rough microsomes from dog pancreas were from tRNA Probes (College Station, TX, USA). EasyTag™ EXPRESS³⁵S Protein Labeling Mix, [³⁵S]-L-methionine and ³⁵S-L-cysteine, for in vitro labeling was purchased from Perkin Elmer (Waltham, MA, USA). Restriction enzymes and Endoglycosidase H were from Roche Molecular Biochemicals (Basel, Switerland). Proteinase K was from Sigma-Aldrich (St Louis, MO). The DNA plasmid, RNA clean-up and PCR purification kits were from Thermo Fisher Scientific (Ulm, Germany). All oligonucleotides were purchased from Macrogen (Seoul, South Korea).