E coli jm109 competent cells

Microbial genomic DNA was extracted from sludge samples using the FAST DNA Spin Kit for soil (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer’s instructions. The PCR amplification of 16S rRNA gene fragments for clone library analyses was conducted as described previously with a primer set of 27F and U533R (18 (link), 19 (link)). PCR products were purified with a QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany), ligated into the pGEM-T Easy Vector (Promega, Madison, WI, USA), and cloned into E. coli JM109 competent cells (Promega). The sequences of the cloned PCR products were elucidated at the Biomedical Center of TAKARA Bio.
The sequences obtained were assigned to phylotypes using BLASTClust (2 (link)) with a cut-off value of 97% sequence identity. Phylotypes were phylogenetically classified with the Classifier program in the Ribosomal Database Project (47 (link)) and were compared to sequences in the GenBank nucleotide sequence database using the BLAST program (2 (link)). Phylogenetic trees were constructed using the neighbor-joining method (37 (link)) with the program MEGA7 (24 (link)). Bootstrap resampling was conducted with 1,000 replicates to validate the robustness of the phylogenetic trees. The nucleotide sequence data obtained in this study have been submitted to the GenBank database under Accession Nos. LC145217–LC145287.

The pUCP26-estA plasmid was constructed with the insertion of a full-length estA gene from P. aeruginosa PAO1 (GenBank® accession number AF005091) at the EcoRI and BamHI restriction sites of the shuttle vector pUCP26 [14 ], kindly provided by Professor Herbert Schweizer from Colorado State University. Thus, a synthetic DNA fragment including the signal peptide and its own ribosomal binding site (Shine-Dalgarno) was inserted under the control of the lac promoter. The plasmid was designed in silico and later synthesized by Epoch BioLabs®. and transformed into E.coli JM109 competent cells (Promega) according to the manufacturer's instructions, generating the strain E.coli JM109-estA. For the construction of the P. aeruginosa-estA strain, the shuttle vector pUCP26estA was transformed into P. aeruginosa PAO1 by heat shock, as previously described [15 (link)]. All strains were stored at -80°C in sterile solutions of 30 to 50% glycerol (Table 1). With the exception of the RML-producing medium, all liquid cultures were grown in Luria-Bertani (LB) broth (Sigma Aldrich). For the solid medium, 2% bacteriological agar (Vetec®) was added. Bacterial growth for the pre-inoculum was carried out in a liquid medium, in 50 mL conical bottom tubes, with 5 to 10 mL medium, rotation at 170 rpm, and 30°C.

A 350 base pair (bp) fragment from the Nc-5 region was amplified from N. caninum Bahía strain total DNA using high fidelity KAPA HiFi HotStart DNA polymerase (KAPA Biosystems, Woburn, MA, USA) and a previously reported set of primers: Np21+ (5′-CCC AGT GCG TCC AAT CCT GTA AC-3′) / Np6+ (5′-CTC GCC AGT CAA CCT ACG TCT TCT-3′) [28 (link)]. The amplified product was ligated into the pGEM-T easy vector (Promega, Madison, WI, USA) multiple cloning site and then used for transforming E. coli JM109 competent cells (Promega), following the manufacturer’s recommendations. Extracted plasmid DNA was confirmed by sequencing and used as amplification template in all LAMP standardization assays.

Microbial cells for clone library analysis were collected from early stationary phases of the 4th enrichment cultures. Genomic DNA was extracted using the FAST DNA Spin Kit for soil (MP Biomedicals) according to the manufacturer’s instructions. PCR amplification of 16S rRNA gene fragments was performed using a primer pair of 27F (5′- AGA GTT TGA TYM TGG CTC AG -3′) and 533R (5′- TTA CCG CGG CKG CTG RCA C -3′) as described previously27 (link). PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN), ligated into pGEM-T Easy Vector (Promega), and cloned into E. coli JM109 competent cells (Promega). Sequences of the cloned PCR products were determined at the Dragon Genomics Center (TAKARA Bio).

Reactions progress was monitored by analytical thin-layer chromatography (TLC) on precoated aluminum foil (Silica Gel 60 F254-plate, Sigma–Aldrich, St. Louis, MO, USA), and the products were visualized by UV light. Purity of all compounds (>98%) was verified by thin layer chromatography and NMR measurements [46 (link),47 (link)]. The chemicals required for validation, all of analytical grade, and all reagents required for bacterial cell culture and the kit for plasmid extraction were purchased from Sigma–Aldrich (St. Louis, MO). E. coli JM109 competent cells were from Promega, (Fitchburg, WI, USA). Ni-NTA agarose and 3–12 mL 10kDa Slide-A-Lyzer™ Dialysis Cassette used for protein purification were purchased from Thermo Fisher Scientific (Waltham, MA, USA). SDS-PAGE experiments were performed using Mini-PROTEAN^® II handcast systems (Bio-Rad, Hercules, CA, USA) kits for both equipment and reagents, unless specified. Monoacylglycerol lipase (human recombinant, 50 μg, hMAGL) was purchased from Cayman Chemical (Ann Arbor, MI, USA). For bioluminescence experiments, LH₂ potassium salt was purchased from Promega Italia Srl, while ATP magnesium salt was from Sigma–Aldrich.

After five successive subcultures, enriched microorganisms were collected by centrifugation. DNA was extracted using the FAST DNA Spin Kit for soil (MP Biomedicals, Irvine, US) according to the manufacturer's instructions. Partial 16S rRNA gene fragments were amplified by PCR with a primer pair of 27F and 533R for bacteria and A25F and A958R for archaea, as described previously (Kato et al., 2010, 2015). PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany), ligated into pGEM‐T Easy Vector (Promega, Madison, US) and cloned into E. coli JM109 competent cells (Promega). Sequences of the cloned PCR products were determined at the Biomedical Center, TAKARA Bio (Kusatsu, Japan). The nucleotide sequence data reported here have been submitted to GenBank under Accession Nos. LC111538–LC111564.