Finally, the pellet was suspended with an equal volume of ice-cold 15% glycerol. Electroporation was conducted according to the protocol described in previous reports (Link et al., 1997b; Datsenko & Wanner, 2000). Fifty microliters of CP25e (5′-CCC ATT ATG CTT TGG CAG TTT ATT CTT GAC ATG TAG TGA GGG GGC TGG TAT AAT CAC ATA GTA CTG TTG GGT CTA GAT TAG GGT AAC TTT AAG GAG GTA TTC CTC-3′) and an equal volume of its complementary DNA (200 nM each) were mixed and incubated at 95 °C for 5 min, and then cooled to room temperature. Fifty microliters

of LacUV5 (5′-CTC ACT CAT TAG GCA CCC CAG GCT TTA CAC TTT ATG CTT CCG GCT CGT ATA ATG TGT GGA ATT GTG AGC GGA TAA CAA TTT CAC ACA GGA AAC AGC T-3′) and an equal volume of its complementary DNA (200 nM Proteases inhibitor each) were mixed, and incubated at 70 °C for 10 min, and then cooled to room temperature. These were used as templates for PCR. PCR or cross-over PCR (Link et al., 1997b) was performed with AccuPrime Pfx DNA Polymerase, learn more Platinum Pfx DNA Polymerase (Invitrogen),

or Pyrobest DNA polymerase (Takara Bio Inc.). Primers and templates used in this study are listed in Table S2 (DNA sequences are listed in Table S3). Each PCR product specified by enzyme name in Table S2 was digested with the correspondent enzyme, dephosphorylated with alkaline phosphatase (Takara Bio Inc.), and then introduced into the donor vector with a DNA Ligation Kit Ver.2 (Takara Bio Inc.). The LacI promoter region of pKO3-lacI-35-10 (5′- TGG

CGC AAA ACC TTT CGC GGT ATG GCA TGA TAG CG -3′) was replaced with 5′- TGT TGA CAA ACC TTT CGC GGT ATG GTA TAA TAG CG -3′. Finally, we constructed the plasmids listed in Table S2. The DNA sequences of the constructed plasmids were confirmed by DNA sequencing analysis: the region amplified from genomic DNA was consistent with E. coli genomic DNA (GenBank accession no. NC_000913) or S. cerevisiae genomic DNA (768–995 base of GenBank accession no. X05731 for ubi4, and GenBank accession no. Z74170 for ubp1). Homologous recombination Bay 11-7085 with pKO3 or pKOV was performed according to the procedure reported previously with modifications (Link et al., 1997b). Briefly, derivatives of pKO3 or pKOV were transformed into the target strains. The strains were grown at 43 °C on LB agar plates containing CP (20 μg mL−1). Three colonies were picked and cultivated at 30 °C in 1 mL of LB broth, and 100 μL of cultured bacteria was further cultivated at 37 °C overnight on LB agar plates containing 10% (w/v) sucrose (Wako Pure Chemical Industries, Ltd). Finally, the strains grown only in LB containing sucrose but not in LB containing CP were selected as the strains containing neither pKO3 nor pKOV. Insertion of the target gene fragment into the appropriate sites in the chromosomal DNA was confirmed by PCR amplifying the region spanning both the target gene and the chromosomal site.