Journal: BMC Microbiology

Article Title: Biochemical and physiological characterization of the GTP-binding protein Obg of Mycobacterium tuberculosis

doi: 10.1186/1471-2180-11-43

Figure Lengend Snippet: Immunoblot analysis of Obg of M. tuberculosis . A . Immunoblot analysis of Obg from M. tuberculosis strains harboring plasmids. M . tuberculosis strains were grown in 7H9-OADC-TW broth at 37°C to early log phase and lysates prepared using a bead beater and separated (100 μg protein for each lane) on SDS-PAGE. The immunoblots were probed with anti-Obg antiserum (1:500 dilution) followed by alkaline phosphatase labeled anti-rabbit IgG (1:1000 dilution, Zymed). The antibody-incubated blots were then developed with NBT/BCIP substrates. Lane 1, M. tuberculosis carrying the plasmid pMV261(empty vector control); Lane 2, M. tuberculosis carrying the plasmid pMVOBG (plasmid overexpressing Obg). B . Immunoblot analysis of Obg at different growth points in M . tuberculosis culture. Wild type M. tuberculosis was grown in 7H9-OADC-TW broth at 37°C. Lysates were prepared from wild-type M. tuberculosis grown to different ODs at 600 nm, separated (200 μg protein for each lane) on SDS-PAGE, and probed with anti-Obg antiserum (1:500 dilution) followed by peroxidase-labeled anti-rabbit IgG (1:10,000 dilution, Sigma). The blots were developed with an ECL kit (Amersham) and autoradiographed. "Obg" indicates the Obg protein reacting with anti-Obg antiserum. Values below each band indicate the OD value at 600 nm at the time of harvest. The graph above the bands gives the levels of Obg, based on density of the bands using Image J software. C . Immunoblots of Obg in separated soluble vs membrane fractions of M. tuberculosis lysates. The bacteria were grown in 7H9-OADC-TW broth at 37°C to mid-log phase. Lysates were prepared using a bead beater, and the soluble and pellet fractions separated by centrifugation. The protein fractions (200 μg protein for each lane) were separated by SDS-PAGE, blotted and probed with anti-Obg antiserum (1:500 dilution) (marked as Obg) or anti-SigH antiserum (1:1000 dilution) (marked as SigH), followed by peroxidase-labeled anti-rabbit IgG (1:10,000 dilution, Sigma). The blots were developed with an ECL kit (Amersham) and autoradiographed. In the figure, lanes labeled Whole, Supernatant and Pellet represent extracts of whole M. tuberculosis , of the 49,000 g supernatant, and of the 49,000 g pellet, respectively.

Article Snippet: M. tuberculosis H37Rv was grown either in Middlebrook 7H9 broth medium containing Tween (0.05%) and OADC (10%) (7H9-TW-OADC) broth, or in Middlebrook 7H10 agar medium containing Tween (0.05%) and OADC (10%) (7H10-TW-OADC).

Techniques: Western Blot, SDS Page, Labeling, Incubation, Plasmid Preparation, Software, Centrifugation

Journal: BMC Microbiology

Article Title: Biochemical and physiological characterization of the GTP-binding protein Obg of Mycobacterium tuberculosis

doi: 10.1186/1471-2180-11-43

Figure Lengend Snippet: Obg cofractionation with ribosomal subunits . M. tuberculosis was grown in 7H9-OADC-TW broth at 37°C, and lysates prepared using a bead beater. About 500 g protein was separated in 10-40% sucrose gradient. A . The ODs of the separated fractions were measured (manually) at 260 nm. B . The proteins in the fractions were then precipitated with ethanol and separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-Obg antiserum (1:500 dilution), followed by peroxidase-labeled anti-rabbit IgG (1:10,000 dilution, Sigma). The blots were developed with an ECL kit (Amersham) and autoradiographed. Lane C is a whole-cell extract from M. tuberculosis . Lanes 1-15 represent fractions from the top (10% sucrose) to the bottom (40% sucrose) of the sucrose gradient. Fraction 16 was not analyzed in immunoblot.

Article Snippet: M. tuberculosis H37Rv was grown either in Middlebrook 7H9 broth medium containing Tween (0.05%) and OADC (10%) (7H9-TW-OADC) broth, or in Middlebrook 7H10 agar medium containing Tween (0.05%) and OADC (10%) (7H10-TW-OADC).

Techniques: SDS Page, Labeling, Western Blot

Journal: BMC Microbiology

Article Title: Biochemical and physiological characterization of the GTP-binding protein Obg of Mycobacterium tuberculosis

doi: 10.1186/1471-2180-11-43

Figure Lengend Snippet: Growth of M. tuberculosis strains at different time points . M. tuberculosis was grown in 7H9-OADC-TW broth at 37°C. Growth was followed by measuring the OD at 600 nm using 1 ml aliquots. Closed circles: M. tuberculosis carrying the plasmid pMV261 (empty vector control); squares: M. tuberculosis carrying the plasmid pMVOBG (plasmid overexpressing Obg). The data shown are representative findings from three different. experiments.

Article Snippet: M. tuberculosis H37Rv was grown either in Middlebrook 7H9 broth medium containing Tween (0.05%) and OADC (10%) (7H9-TW-OADC) broth, or in Middlebrook 7H10 agar medium containing Tween (0.05%) and OADC (10%) (7H10-TW-OADC).

Techniques: Plasmid Preparation

Journal: Tuberculosis (Edinburgh, Scotland)

Article Title: The Mycobacterium tuberculosis β-oxidation genes echA5 and fadB3 are dispensable for growth in vitro and in vivo

doi: 10.1016/j.tube.2011.06.006

Figure Lengend Snippet: Growth kinetics of M. tuberculosis (H37Rv), echA5 mutant and fadB3 mutant in (a) Middlebrook 7H9-Tw-Glycerol-OADC medium and (b) Sauton’s minimal medium plus 0.2% glycerol. Results shown are the average of three independent experiments. Error bars represent SD.

Article Snippet: M. tuberculosis H 37 Rv was grown in Middlebrook 7H9-Tw-Glycerol-OADC liquid broth (supplemented with 0.2% glycerol, 0.05% Tween-80 and 10% OADC) at 37 °C shaken at 150 r.p.m. in an orbital shaker, on Middlebrook 7H11 solid media supplemented with 0.5% glycerol and 10% OADC (Becton Dickinson) or in Sauton’s Minimal Medium supplemented with 0.2% glycerol, 0.0001% ZnSO 4 and 0.025% Tyloxapol as described previously.

Techniques: Mutagenesis

Journal: Tuberculosis (Edinburgh, Scotland)

Article Title: The Mycobacterium tuberculosis β-oxidation genes echA5 and fadB3 are dispensable for growth in vitro and in vivo

doi: 10.1016/j.tube.2011.06.006

Figure Lengend Snippet: Growth kinetics of M. tuberculosis (H37Rv), echA5 mutant and fadB3 mutant in (a) Middlebrook 7H9-Tw-Glycerol-OADC medium and (b) Sauton’s minimal medium plus 0.2% glycerol. Results shown are the average of three independent experiments. Error bars represent SD.

Article Snippet: For example, the Middlebrook 7H9-Tw-Glycerol-OADC medium used for propagation of these strains contains glycolytic carbon sources in the form of glycerol and glucose as well as the β-oxidation substrate oleic acid, including the oleic acid released during hydrolysis of Tween-80.

Techniques: Mutagenesis