Escherichia coli HS996/pSC101-BAD-gbaA (Wang et al., 2006) was provided by Youming Zhang, Gene Bridges, Germany. Escherichia coli DH10B was used for the functional recombineering elements’ http://www.selleckchem.com/products/PD-98059.html integration. Escherichia coli strains were routinely grown in Luria–Bertani (LB) media. Antibiotics were added at the following concentrations for plasmid selection (μg mL−1): gentamicin (25), tetracycline (12.5), ampicillin (100), kanamycin (30) and chloramphenicol (12.5). Strains containing pSC101-BAD-gbaA were incubated at 30 °C unless otherwise mentioned. Escherichia coli strain culture, competent cell preparation, DNA transformation,

plasmid extraction, restriction enzyme digestion and agarose gel electrophoresis were performed as per standard protocols (Sambrook & Russel, 2001). Amplification of the homology arm (in recombineering research, the short homologous DNA sequence used for the recombination is often called the ‘homology arm’) flanked neo was performed in a 50-μL reaction with 100 ng of pKD4, 0.2 mM dNTP each, 0.25 μM of each sense and antisense primer

and 2.5 U of Pfu (NEB). The PCR cycling conditions consisted of an initial denaturation step at 95 °C for 5 min, followed by 30 cycles of 95 °C for 45 s, 60 °C for 60 s and 72 °C for 2 min and a final extension step at 72 °C for 10 min. The PCR product was analyzed by agarose gel electrophoresis, followed by ethanol precipitation and dissolved in a suitable volume of 10 mM Tris-Cl (pH 8.0); the DNA concentration was adjusted to 100 ng μL−1. find more Short primers (≤60-mer) were purchased from Sangon Co. Ltd (China) and long primers (>60-mer) were purchased from Integrated DNA Technologies Inc. The primers used in this study are

listed in Table 1. The vector pGR harboring the functional recombineering elements for E. coli DH10B genome integration was constructed as follows: first, 0.8 kb aacC1 was amplified from pBAD322G with primers GRK1 and GRK2, 1.1 kb araC was amplified with primers GRK3 and GRK4 from pKD46, then the XhoI- and SacI-digested aacC1 and the SacI- and BamHI-digested araC were ligated and cloned into the XhoI- and BamHI-treated pBluescript KS(−), creating pKAC. With E. coli DH10B genomic DNA as a template, 420 bp endA1 upstream sequences were amplified with the primers EA1 and EA2 and digested with EcoRI and XhoI, and 370 bp endA1 Methane monooxygenase downstream sequences were amplified with primers EA3 and EA4 and digested with XhoI and KpnI. The two fragments were then ligated and cloned into EcoRI- and KpnI-treated pBluescript KS(−) to obtain pENLR. Finally, 3.2 kb λ Red genes and the recA containing XhoI–BamHI fragment excised from pSC101-BAD-gbaA and the 2.0 kb aacC1 and the araC containing BamHI–XhoI fragment excised from pKAC were ligated and cloned into the XhoI site of pENLR, generating pGR. Recombineering experiments with pKD46 (Datsenko & Wanner, 2000) and pSC101-BAD-gbaA (Wang et al.