Real-time PCR primers are listed in Additional file 1: Table S4. Relative RNA level of a particular gene in mutant strains was normalized to that of wild type using the 2−ΔΔCt method with 16S rRNA or recA as reference gene [55]. Mapping transcriptional start sites The transcriptional start (+1) sites of the promoters were mapped by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) [56]. The RLM-RACE was performed using GeneRacer Kit (Invitrogen) according to manufacturer’s instructions. The B. pseudomallei RNA was isolated as described previously [14]. Sequence motif predication and database
search of motifs in Belinostat research buy the B. pseudomallei KHW genome 150 base pairs of nucleotide sequence upstream of the transcriptional starts of each gene was submitted to the bioinformatics tool – MEME (http://meme.nbcr.net/meme/cgi-bin/meme.cgi) for prediction of DNA motifs [57]. The motif with the highest statistical significance (lowest E-value) was chosen and its data – in Position-Specific Probability Matrix format was submitted to MAST (http://meme.nbcr.net/meme/cgi-bin/mast.cgi) to search for the best matching positions in the upstream sequences of B. pseudomallei KHW genes [58]. Statistical analysis Results were presented as mean ± standard deviation. Student’s t-test was used to find the significant differences between the means, defined as when p < 0.05 (*) and p < 0.01 (**). Acknowledgements
We thank this website M. A. Valvano (University of Western Ontario) for pMLBAD plasmid, S.J. Busby (University of Birmingham) for pRW50 plasmid, S. Korbsrisate (Mahidol University) for BopC antibody, and M.P. Stevens (University of Edinburgh) Prostatic acid phosphatase for BopE antibody. This work is supported by grants T208A3105 from the Ministry of Education to YHG, NMRC/1221/2009 from the National Medical Research Council to YHG, an award from the Pacific Southwest Regional Center of Excellence in Biodefense and Emerging Infectious Diseases (NIH U54 A1065359) to JFM, and grant HDTRA1-11-1-0003 from the Defense Threat Reduction Agency to JFM. We would like to thank Isabelle Chen for her technical assistance. Additional file Additional file 1 Materials and
Methods. Table S1. Summary of Illumina sequencing. Table S2. β-galactosidase activities in E coli DH5α strain containing transcriptional promoter-lacZ fusions and arabinose-inducible bsaN and bicA or empty vector. Table S3. List of additional plasmids used in this study. Table S4. List of Real-Time PCR primers for this study. Figure S1. Secretion of BopC. KHW and ΔbsaM mutant were grown in acidic LB broth for 3 hours. Total protein from the bacterial culture supernatant was precipitated and protein concentration was normalized with respect to the optical density (OD600) of the bacterial cultures. Proteins on membranes were probed with rabbit polyclonal antibodies to BopC and BopE. Figure S2. (A) Intracellular replication of B. pseudomallei KHW and mutants in RAW264.7 cells. Cells were infected at an MOI of 0.
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