4b). Deletion constructs made by SOE PCR retained the start and stop codons of mglA (fusion of 1st four and last two codons) and sspA (fusion of 1st four and last 4 codons) in frame with 0.8 kb
of flanking sequence. The constructs were cloned into pMP590 (Table 1) and sequenced to confirm the integrity of the flanking DNA sequence. Allelic exchange was achieved learn more by transformation, selection for plasmid co-integrates, counter selection on sucrose containing media and confirmed via PCR analysis for replacement of the wild type with the deletion mutant allele as described . Each mutation was confirmed by DNA sequence analysis. Extracellular β-galactosidase assay Overnight cultures of lacZ reporter strains were diluted 1:10 in Chamberlains defined media and cultured until mid exponential phase (0.2-0.8 OD600). β-galactosidase activity was measured as OD420using the substrate ONPG (Sigma) as described
elsewhere . Relative promoter activity was normalized using OD600 of culture, time of development, and cell to buffer ratio (CBR). Statistical analysis was performed to determine the mean Miller units and standard deviation from Captisol supplier three independent cultures and significance calculated using an unpaired two tailed t test with unequal variance. SDS-PAGE and FlAsH™ labelling Proteins were separated by SDS-PAGE. Total protein loaded in each sample was equivalent as determined by a BCA assay (Pierce). FlAsH™ labeling was accomplished using the manufactor’s protocols (Invitrogen). In gel fluorescence of the arsenical fluoriscein and total protein stain was conducted on a Typhoon 9200 laser scanner (488 nm laser/520 nm BP 40 filter and 633 nm laser/670 nm BP 30 filter). Densitometry was conducted using ImageQuant XL software and sample comparisons made using the same gel and scan. Mean intensity and standard deviation of four
samples from independent cultures was calculated and significance determined using an unpaired two tailed t test with unequal variance. Acknowledgements We thank Allen Honeyman for sending us the lacZ containing plasmids pALH109 and pALH122. This work was supported by a Southeast Regional Center of Excellence in Biodefense and Emerging Infections grant (NIH/NIAID U54-AI057157) and by the National Institutes of Health (AI069339). References 1. Markowitz LE, Hynes NA, de la Cruz Metalloexopeptidase P, Campos E, Barbaree JM, Plikaytis BD, Mosier D, Kaufmann AF: Tick-borne tularemia. An outbreak of lymphadenopathy in children. Jama 1985,254(20):2922–2925.CrossRefPubMed 2. Centers for Disease Control and Prevention (CDC): Doramapimod in vitro tularemia transmitted by insect bites–Wyoming, 2001–2003. MMWR Morb Mortal Wkly Rep 2005,54(7):170–173. 3. Reintjes R, Dedushaj I, Gjini A, Jorgensen TR, Cotter B, Lieftucht A, D’Ancona F, Dennis DT, Kosoy MA, Mulliqi-Osmani G, Grunow R, Kalaveshi A, Gashi L, Humolli I: Tularemia outbreak investigation in Kosovo: case control and environmental studies.