Escherichia coli strain top10 f

The ComC gene of Methanobrevibacter millerae SM9 [21 (link)] was amplified using forward primer 5′-CACCATGAAGATAATGAAGGATAACGAAA and reverse primer 5′-TCATTAATCTTCAAGATAAGAATCTATATC with the reverse primer containing two stop codons. The PCR reaction utilized high-fidelity Hercules II DNA polymerase (1.0 μL; Stratagene, USA) in a 50 μL reaction with 0.2 μM of each primer, 0.3 μM dNTP, approximately 20 ng M. millerae SM9 DNA, and 1 × buffer. The PCR cycling parameters had an initial denaturation of 95°C for 2 min, followed by 35 cycles of 94°C for 30 s, 56°C for 30 s, and 68°C for 40 s. The PCR product was purified using agarose DNA electrophoresis and a Wizard SV Gel and PCR kit (Promega, USA). It was then inserted into pET151D using TOPO cloning in chemically competent Escherichia coli strain TOP 10F according to the manufacturer's instructions (Invitrogen, USA). Colonies were screened by colony PCR using pET151D T7 forward primer and the ComC reverse primer using 2.5 U Taq polymerase (Roche, NZ), and then the recombinant plasmids were isolated using alkaline lysis and purification with a Wizard SV Gel and PCR Clean-up kit (Promega, USA). The plasmid used for expression was sequenced to verify that the gene was in frame and that the sequence was identical to the reference sequence and then transformed into E. coli Rosetta 2 cells (Novagen, USA).

Primers used for isolating the P35S sequences from different transgenic events were designed using Primer Premier 5.0 software (PREMIER Biosoft International, Palo Alto, CA) according to the nucleotide sequences flanking the P35S promoter. Primers were synthesized by Sangon (Shanghai, China); their sequences are given in Supplementary Table S3 online. The PCR samples were prepared using a KOD-Plus kit (Toyobo, Osaka, Japan) in a sample volume of 50 μL containing 20 ng of genomic DNA, 1× KOD-Plus PCR buffer, 200 μM of each dNTP, 1 mM MgSO4, 100 nM of each primer, and 0.5 units of KOD-Plus DNA polymerase. PCRs were performed on a C1000™ Thermal Cycler (Bio-Rad, Hercules, USA) using the following program: 94°C for 2 min (initial denaturation); 35 cycles of 94°C for 15 s (denaturation) and 68°C for 3 min (annealing and extension); and 68°C for 7 min (final extension). PCR products harboring a target band were recovered and subcloned into the pZErO-2 vector (Invitrogen, Carlsbad, CA, USA) via an EcoRV restriction enzyme site. Ligation products were transformed into Escherichia coli strain TOP10F (Invitrogen, Carlsbad, CA, USA), and positive clones screened. Plasmids containing the PCR products were sequenced using M13 forward and reverse primers (Tsingke, Beijing, China).

The fungal strains used in this work and their origin are shown in Table 1. Strain IMI 58289 was derived from Commonwealth Mycological Institute, Kew, United Kingdom. Strains m567 and FSU48 were provided by the Fungal Stock Center at the University Jena, Germany, C1995 by J.F. Leslie, Kansas State University, E282 by S. Tonti, University Bologna, Italy, MRC2276 by W.C.A. Gelderblom, South Africa, and NCIM 1100 was provided by the National Collection of Industrial Microorganisms, India. Strains B14 and B20 were provided by S.-H. Yun, Korea. Strain V64-1 was kindly provided by T. Kyndt from the University Ghent, Belgium.
Escherichia coli strain Top10 F’ (Invitrogen, Groningen, The Netherlands) was used for plasmid propagation. The uracil-auxotrophic Saccharomyces cerevisiae FGSC 9721 (FY 834) was provided by the Fungal Genetics Stock Center (Kansas State University) and used for yeast recombination cloning.