Ge et al. BMC Genomics 2010, 11:456 http://www.biomedcentral.com/1471-2164/11/456 Table 3 Primers used in the real-time RT-PCR in this study Genes mgtC rpoE ssaG fliC fumC nuoA rfaH Primers mgtC-F mgtC-R rpoE-K rpoE-R ssaG-F ssaG-R fliC-F fliC-R fumC-F fumC-R nuoA-F unoA-R rfaH-F rfaH-R Sequences 5’-GTCTCTGGTATTGGCTTTCTGG-3’ 5’-TTGGCACAAAGAATAATGATCG-3’ 5’-GACGCGATTGAAGCAGAAA-3’ 5’-CCAGCTCCCGTAAGGTGAT-3’ 5’-TTAGTGGATATGCTCTCCCACA-3’ 5’-TCATTTTGATCAGTGAACTTTCG-3’ 5’-AGCCTGTCGCTGTTGACC-3’ 5’-CGCTGCAGGTTGTGGTTG-3’ 5’-CGGTATGGAACGCAAAGTG-3’ 5’-CTCCTGGCCTAAGGTGAGC-3’ 5’-TGCTGCCTGATGCTGGTA-3’ 5’-CGCTTTCGCGGATAGAAG-3’ 5’-ACTTCAGCGTGCTCAGGAA-3’ 5’-GCGTGGCGTTGATTGTAGT-3’ The protocol for the PCR reaction was an initial dena- turation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 5 s, annealing at 56-64°C for 5 s and extension at 72°C for 10 s. For certain pairs of primers the annealing temperatures might be changed to optimize the yield of products. The amplified specific products were examined by melting temperature curves and agarose gel electrophoresis. Relative gene expression was quantified using a standard curve method plotted from ten-fold serial dilutions of known quantities of cDNA samples. Each standard curve for each gene was then used to transform threshold cycle (Ct) to the rela- tive amount of unknown cDNA of the gene. The analy- sis was based on the Ct values of the specific genes, with rfaH as a normalizer. The amount of the specific gene was divided by the amount of rfaH in the same cDNA preparation to calculate the normalized value. Relative gene expression was presented as ratios of the normalized values of cDNA from bacterial growth in U937 cells to the growth in LB broth. All experiments were performed in triplicate using independent RNA preparations (biological replicates). DNA manipulations and preparation of electrocompetent cells Plasmid DNA from E. coli was purified with a Quantum Prep plasmid miniprep kit (Bio-Rad). Plasmids were transformed into the strain S. Enteritidis PT4 KS8822/ 88 by electroporation. Transformants carrying the Red helper plasmid were made electrocompetent as described previously [72] with some modifications. Cells were grown overnight in LB broth Amp at 30°C and then 1 ml of overnight cultures were used to inoculate 100 ml of LB broth Amp and incubation was continued to an OD600 of 0.15-0.2. L-arabinose (Sigma) was then Page 10 of 13 added to a final concentration of 10 mM and incubation was continued until the OD600 reached 0.7. The suspen- sion was cooled on ice for 20 min, and the cells were made electrocompetent by washing twice with the same volume of ice-cold water and then once with 40 ml of ice-cold 10% glycerol. The cells were finally resuspended in 1 ml of ice-cold 10% glycerol, then divided into 50 μl working aliquots and kept at -70°C for up to two months. Total genomic DNA was isolated according to the manufacturer’s instructions (High Pure PCR tem- plate Preparation kit, Roche). Construction of gene mutants by the l Red mutagenesis method Salmonella genes were disrupted by the method described previously [72,73]. Briefly, purified plasmid pKOBEGA was introduced into Salmonella by electro- poration, and transformants were selected on LB agar Amp after incubation for 24 h at 30°C. The l Red Table 4 Primers used in the gene mutagenesis in this study Genes Primers Sequences ssaN ssaN- 5’- Km-F CTTTGCTATCTCCTTTTACGAGTACAATCGGGCTTCACTGC GGGCAGCAAGTGATGGCCTAAAGCCACGTTGTGTCTCAA- 3’a ssaN- 5’- Km-R CTTCAGACAGTGTAAAATCGATGAATTCGCGGACTTCTCGT CCACGTTCACCAATTAACAGCGCTGAGGTCTGCCTCGTG- 3’a ssaN-F 5’-CGTTGTTAAATGCGTGGTTG-3’ ssaN-R 5’-CCCATTCCCGTACGTTCTAA-3’ ssaR ssaR- 5’- Km-F GCGCTTGTACTTTCCTTATTCATTATGGGGCCGACGCTATT AGCTGTAAAAGAGCGCTGGAAAGCCACGTTGTGTCTCAA- 3’a ssaR- 5’- Km-R ATTAATATGAGCAAAGAATCAGGTTTTATCTTTCTTTTTAT GTTCTTCAGGCCAGGTTCGGCGCTGAGGTCTGCCTCGTG- 3’a ssaR-F 5’-TATCGCACTGTATGGCCTTG-3’ ssaR-R 5’-ACCGCCTGCCAGTAAAAATA-3’ sspH2 sspH2- 5’- Km-F CTTTATGAAGTTTTCCGTCTCACTCAGTCTGTCCAGGAAGA GGCTGAATGCGTCGGCGTTAAAGCCACGTTGTGTCTCAA- 3’a sspH2- 5’- Km-R ACACTGGTTATTCCTGATAATAATCTGACCAGCCTGCCGGC GCTGCCGCCAGAACTGCGGGCGCTGAGGTTGTGTCTCAA- 3’a sspH2-F 5’-TCATCTTCAGCCAGTTGTGC-3’ sspH2-R 5’-GTAATCGCCGCATTTATCGT-3’ a: 80-nucleotide (nt)-long primers including 60 nt homology extensions complementary to the targeted regions of the genes and 20 nt priming sequences (underlined) for the synthesis of the kanamycin resistance cassette gene from E. coli MC4100 ybeW::Km.