pyogenes. For the preparation of competent cells, Ku-0059436 chemical structure strain GT01 was harvested at early- to mid-log phase
(OD660 = 0.4 to 0.5) and washed twice with 0.5 M sucrose buffer. The constructed suicide vector nga::aad9/pFW12 was transformed into strain GT01 by electroporation. The conditions of electroporation were 1.25 kV/mm, 25 μF capacitance and 200 Ω resistance, using Gene Pulser II (Bio-Rad, Hercules, CA, USA). After incubation at 37°C for 3 h, competent cells were spread onto BHI agar plates containing 0.3% yeast extract and spectinomycin (final concentration 100 μg/ml). Selected colonies on the plates were cultured. Cultured bacteria were washed once with saline, resuspended in 10 mM Tris, 1 mM EDTA and boiled for 10 min. Genomic DNA was obtained from the supernatant of boiled bacteria. The double-crossover replacement was analyzed using genomic DNA by PCR and successful double-crossover replacement was further confirmed by DNA sequencing. Cloning of nga gene All PCR reactions for plasmid construction were undertaken as previously Selleckchem Fedratinib described . The nga GT01 of
S. pyogenes strain GT01 was amplified by PCR with Extaq DNA polymerase using primers nga-n4Eco (5′-GGAATTCATGAGAAACAAAAAAGTAAC-3′) and sloC2 (5′-ATCATCCGTTTTCTGACCTG-3′) and cloned into pGEM-T easy (Promega, Madison, WI, USA) to yield pNGIe1, whose insert was sequenced. Oligonucleotide nga-n4Eco contained a restriction site for EcoRI endonuclease (shown in bold in the primer sequence). The nga GT01 gene is oriented in the opposite direction as the lacUV5 promoter. An EcoRI fragment
containing the nga GT01 gene of pNGIe1 was sub-cloned into pLZ12-Km2  to yield pLZN2, whose insert was sequenced for verification. To construct pLZN-RBS, inverse PCR with Pyrobest DNA polymerase (Takara) using the primers LZ-R0 (5′-CCGTCGACCTCGAGGGGGGGC-3′) and nga-RBS1 (5′-CCGCTCGAG ATATAAGGTGGTTTAC A TGAGAAACAAAAAAGTAAC-3′) was performed to add a MAPK Inhibitor Library cell line potential ribosome-binding site (16 bp) to nga encoded on pLZN2. Oligonucleotides nga-RBS1 and LZ-R0 contained a restriction site for XhoI endonuclease, C1GALT1 the potential ribosome binding site and/or start codon for the nga gene, respectively (shown in bold, underline and italic in the primer sequence, respectively). The amplification product was digested with XhoI and self-ligated. The insert was sequenced for verification. To construct pLZN-RBSII2, inverse PCR with PrimeSTAR™ HS DNA polymerase (Takara) using the primers nga-RBS2 (5′-CCGGGGCCCTTAAAAATAATATAAGGTGGTTTAC A TGAG-3′) and LZ-R3 (5′-CTCGAGGGGGGGCCCATCAGTC-3′) was performed to add the further upstream DNA sequence (10 bp) to the potential ribosome-binding site encoded on pLZN-RBS. A oligonucleotide nga-RBS2 contained the upstream DNA sequence, the potential ribosome binding site and start codon for the nga gene (shown in dotted underline, underline and italic in the primer sequence, respectively).