For LS-GR, a single fresh colony of LS-GR carrying pACYC184 or pECBAC1 was inoculated into LB with chloramphenicol; after overnight growth at 37 °C, 1 : 100 of the culture was transferred to 30 mL LB with chloramphenicol. Once the A600 nm reached about 0.2, l-arabinose was added at a final concentration of 0.2% w/v to induce the expression of λ Red genes. After 60 min, the

culture was pelleted at 4 °C, washed three times with ice-cold Nutlin-3a manufacturer 10% glycerol and finally resuspended in 200 μL ice-cold 10% glycerol. For each DNA transformation, 100 ng of PCR products were added to a 50-μL aliquot of the competent cells, gently mixed and then transferred to a 0.1-cm cuvette for electroporation using a Bio-Rad Gene-Pulser II (1.8 kV, 25 μF, 200 Ω). After pulsing, 1 mL of LB was added to the cells and the suspension was incubated overnight at 37 °C, after which 0.5 mL was used for plasmid extraction learn more and E. coli DH10B DNA retransformation. Single kanamycin-resistant colonies were cultured for plasmid extraction and restriction enzyme digestion analysis. Another 0.5 mL was diluted to count the number of viable cells by plating on LB plates with chloramphenicol (for pACYC184 modification) or on the LB plates without an antibiotic

(for pECBAC1 modification). The recombineering strategy using LS-GR is illustrated in Fig. 1. Prophage-based and integrative form λ Red recombineering strains now in use are either grown at 30 °C, which slows the experimental process, or lack gam, which protects the incoming DNA Bupivacaine from degradation. To obtain an integrative form recombineering strain that circumvents the shortcomings, we constructed a new integrative form λ Red recombineering strain by integrating the functional recombineering elements, including λred genes, araC, recA and aacC1, into the E. coli DH10B genome. Escherichia coli DH10B serves as an ideal starting strain because of its well-characterized genetic background as well as its ability to

hold a large construct (Durfee et al., 2008). endA1, the nonsense mutation of nonessential gene endA encoding the DNA-specific endonuclease, was selected as the integration region. Many E. coli genome integration methods are available; here, the λ Red recombineering strategy was chosen and longer homologous DNA fragments (420 and 370 bp for the left and the right side, respectively) were used for the recombination. After pSC101-BAD-gbaA transformation, l-arabinose-induced electrocompetent cells of DH10B harboring pSC101-BAD-gbaA were prepared and the 5.8-kb PvuII fragment of pGR containing the functional recombineering elements was electroporated. Transformants were selected on an LB plate with gentamicin at 37 °C. To eliminate pSC101-BAD-gbaA that may still exist in the strain after transformation, four colonies obtained above were each inoculated into 3 mL of LB without an antibiotic to grow to the stationary phase. After three rounds of serial culture with 100-fold dilution, the cells were plated on LB plates with gentamicin.