Phusion high fidelity dna polymerase

All PCR reactions to clone or sequence IDF_13, IDF_15, ef2833, ef2847, ef2850, ef2169, and ef2170 genes were performed in a Mastercycler Eppendorf with Phusion high-fidelity DNA polymerase (NEB) and according to manufacturer’s instructions. PCR products and DNA restriction fragments were purified with QIAquick kits (QIAGEN, Hilden, Germany) when necessary. Electro-transformation of E. coli, L. lactis, and E. faecalis were carried out as previously described [64 (link),65 (link)] using a Gene Pulser apparatus (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Transformations of chemically competent E. coli ER2566 were carried out by a heat shock procedure.

Genomic DNA was extracted from lactococci with Wizard Genomic DNA Purification Kit (Promega). Before extraction, bacteria were incubated in 10 mM Tris-HCl, pH 8.0 supplemented with 25% sucrose and 30 mg mL⁻¹ lysozyme at 37 °C for 1 h. Plasmid DNA was extracted from E. coli with QIAgen Spin Miniprep Kit, and from L. cremoris with the same kit, with a 1 h-pre-incubation with lysozyme as described above. PCR products were amplified with Phusion High-Fidelity DNA Polymerase (NEB) in a Mastercycler (Eppendorf), digested with restriction enzymes (NEB) and purified with a NucleoSpin Gel and PCR CleanUp kit (Macherey-Nagel). Ligations were performed with Lucigen Fast-Link DNA Ligation Kit. Alternatively, fragments were assembled by the strand overlap extension method using Gibson Assembly Master Mix (NEB). All plasmid constructs were verified by sequencing (Eurofins) using the primers pairs, pET21-f/pET21-r, msp3545-f/msp3545-r and pMalE/pMal-rev for pET21, pNZ8048 and pMal-c4X vectors, respectively (Table S2).

Isolation of total RNA from mycelium of P. sapidus at culture day six and cDNA synthesis were performed as described previously [13 (link)] using the primer 5′-AAGCAGTGGTATCAACGCAGAGT ACGCTTTTTTTTTTTTTTTTTTT-3′ for reverse transcription. Specific primers for gene amplification were deduced from the ORF-start (P1: 5′-ATGACTACACCTGCACCACCCCTCGACCTC-3′) and -stop (P2: 5′-TCAAGCAGAGATTGGAGCTTGGGTSWGAGGA-3′) region of the homologous peroxidase of Pleurotus ostreatus PC15 (GenBank accession no. KDQ22873.1). PCRs were performed with Phusion High-Fidelity DNA Polymerase and the Master Cycler gradient (Eppendorf, Hamburg, Germany) as described elsewhere [45 (link)]. The cycler program was as follows: denaturation for 2 min at 98 °C, 35 cycles at 98 °C for 1 min, 62 °C for 30 s and 72 °C for 90 s, and a final elongation at 72 °C for 10 min. Analysis of PCR products, ligation, transformation in Escherichia coli, colony PCR, and sequencing were performed as described by Behrens et al. [13 (link)]. Translation of DNA sequences was performed using SnapGene^® (GSL Biotech LLC, Chicago, IL, USA). Sequence homology was examined using BLAST [46 (link)]. Alignments were produced by ClustalOmega [47 (link)].