Structured Review

Difco 7h9 broth
Biofilm and pellicle formation under low and high nitrogen condition. A . M. bovis, wild type M. smegmatis and MSFP were grown <t>7H9</t> medium to form biofilm in low and high nitrogen medium. B . Biofilm formation assayed using the 1% crystal violet (CV) staining assay. Cells in low nitrogen (black bars), High nitrogen (crossed bars) and control (grey bars) in 7H9 media were grown in low and high nitrogen on polystyrene plates. The experiments were repeated three times with similar result. Control, medium only. C . Pellicle formation at the air-liquid interface of the standing 7H9 culture by strains M. bovis (i) , M. smegmatis (ii) and MSFP (iii) in low and high nitrogen condition. Results are representative of at least three independent experiments. LN, low nitrogen; HN, high nitrogen.
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Images

1) Product Images from "Poly-L-glutamate/glutamine synthesis in the cell wall of Mycobacterium bovis is regulated in response to nitrogen availability"

Article Title: Poly-L-glutamate/glutamine synthesis in the cell wall of Mycobacterium bovis is regulated in response to nitrogen availability

Journal: BMC Microbiology

doi: 10.1186/1471-2180-13-226

Biofilm and pellicle formation under low and high nitrogen condition. A . M. bovis, wild type M. smegmatis and MSFP were grown 7H9 medium to form biofilm in low and high nitrogen medium. B . Biofilm formation assayed using the 1% crystal violet (CV) staining assay. Cells in low nitrogen (black bars), High nitrogen (crossed bars) and control (grey bars) in 7H9 media were grown in low and high nitrogen on polystyrene plates. The experiments were repeated three times with similar result. Control, medium only. C . Pellicle formation at the air-liquid interface of the standing 7H9 culture by strains M. bovis (i) , M. smegmatis (ii) and MSFP (iii) in low and high nitrogen condition. Results are representative of at least three independent experiments. LN, low nitrogen; HN, high nitrogen.
Figure Legend Snippet: Biofilm and pellicle formation under low and high nitrogen condition. A . M. bovis, wild type M. smegmatis and MSFP were grown 7H9 medium to form biofilm in low and high nitrogen medium. B . Biofilm formation assayed using the 1% crystal violet (CV) staining assay. Cells in low nitrogen (black bars), High nitrogen (crossed bars) and control (grey bars) in 7H9 media were grown in low and high nitrogen on polystyrene plates. The experiments were repeated three times with similar result. Control, medium only. C . Pellicle formation at the air-liquid interface of the standing 7H9 culture by strains M. bovis (i) , M. smegmatis (ii) and MSFP (iii) in low and high nitrogen condition. Results are representative of at least three independent experiments. LN, low nitrogen; HN, high nitrogen.

Techniques Used: Staining

Growth of the mycobacterial strains in low and high nitrogen broth culture. A . OD 600 of wild type M. bovis was inoculated to an initial optical density of 0.006 - 0.008 in 7H9 medium containing (●) low nitrogen (3.8 mM ammonium sulphate) and (▲) high nitrogen (60 mM ammonium sulphate). B . OD 600 of wild type M. smegmatis and MSFP in low and high nitrogen broth culture. Wild type M. smegmatis, low nitrogen (■), high nitrogen (□); MSFP, low nitrogen (●), high nitrogen (○). Data is mean ± SD of values obtained from three independent cultures. LN, low nitrogen; HN, high nitrogen.
Figure Legend Snippet: Growth of the mycobacterial strains in low and high nitrogen broth culture. A . OD 600 of wild type M. bovis was inoculated to an initial optical density of 0.006 - 0.008 in 7H9 medium containing (●) low nitrogen (3.8 mM ammonium sulphate) and (▲) high nitrogen (60 mM ammonium sulphate). B . OD 600 of wild type M. smegmatis and MSFP in low and high nitrogen broth culture. Wild type M. smegmatis, low nitrogen (■), high nitrogen (□); MSFP, low nitrogen (●), high nitrogen (○). Data is mean ± SD of values obtained from three independent cultures. LN, low nitrogen; HN, high nitrogen.

Techniques Used:

2) Product Images from "Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis"

Article Title: Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0200145

Rv1460 truncation mutants are impaired for growth in vitro under standard culture conditions. (A–C) Growth of H37Rv (wild-type), three truncation (Δ Rv1460 stop) mutants and their complemented strains under standard conditions in 7H9 OADC. The results shown are the mean and standard deviation of three experiments.
Figure Legend Snippet: Rv1460 truncation mutants are impaired for growth in vitro under standard culture conditions. (A–C) Growth of H37Rv (wild-type), three truncation (Δ Rv1460 stop) mutants and their complemented strains under standard conditions in 7H9 OADC. The results shown are the mean and standard deviation of three experiments.

Techniques Used: In Vitro, Standard Deviation

Succinate dehydrogenase and aconitase activity is not impaired in Rv1460 truncation mutants. (A) Succinate dehydrogenase activity and (B) aconitase activity in the H37Rv (wild-type), Δ Rv1460 stop_5.20 and complemented strains cultured in 7H9 OADC. Activity was standardised relative to total protein. The results shown are the mean and standard deviation of five and three experiments respectively.
Figure Legend Snippet: Succinate dehydrogenase and aconitase activity is not impaired in Rv1460 truncation mutants. (A) Succinate dehydrogenase activity and (B) aconitase activity in the H37Rv (wild-type), Δ Rv1460 stop_5.20 and complemented strains cultured in 7H9 OADC. Activity was standardised relative to total protein. The results shown are the mean and standard deviation of five and three experiments respectively.

Techniques Used: Activity Assay, Cell Culture, Standard Deviation

3) Product Images from "Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis"

Article Title: Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0200145

Rv1460 truncation mutants are impaired for growth in vitro under standard culture conditions. (A–C) Growth of H37Rv (wild-type), three truncation (Δ Rv1460 stop) mutants and their complemented strains under standard conditions in 7H9 OADC. The results shown are the mean and standard deviation of three experiments.
Figure Legend Snippet: Rv1460 truncation mutants are impaired for growth in vitro under standard culture conditions. (A–C) Growth of H37Rv (wild-type), three truncation (Δ Rv1460 stop) mutants and their complemented strains under standard conditions in 7H9 OADC. The results shown are the mean and standard deviation of three experiments.

Techniques Used: In Vitro, Standard Deviation

Succinate dehydrogenase and aconitase activity is not impaired in Rv1460 truncation mutants. (A) Succinate dehydrogenase activity and (B) aconitase activity in the H37Rv (wild-type), Δ Rv1460 stop_5.20 and complemented strains cultured in 7H9 OADC. Activity was standardised relative to total protein. The results shown are the mean and standard deviation of five and three experiments respectively.
Figure Legend Snippet: Succinate dehydrogenase and aconitase activity is not impaired in Rv1460 truncation mutants. (A) Succinate dehydrogenase activity and (B) aconitase activity in the H37Rv (wild-type), Δ Rv1460 stop_5.20 and complemented strains cultured in 7H9 OADC. Activity was standardised relative to total protein. The results shown are the mean and standard deviation of five and three experiments respectively.

Techniques Used: Activity Assay, Cell Culture, Standard Deviation

4) Product Images from "Lansoprazole is an antituberculous prodrug targeting cytochrome bc1"

Article Title: Lansoprazole is an antituberculous prodrug targeting cytochrome bc1

Journal: Nature Communications

doi: 10.1038/ncomms8659

LPZS is a highly selective antituberculous drug with in vivo activity. ( a ) Intracellular ratio of LPZ ( m/z 370.0834, g mol −1 ) and its metabolite ( m/z 354.0884, g mol −1 ) determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) over a 48-h period in MRC-5 cells. Representative example of three individual experiments; the complete data set can be found in Supplementary Table 2 . ( b ) ESI–MS mass spectra in the range m/z 350–375 measured for experiments performed on the cell lysate of MRC-5 fibroblasts exposed to LPZ (extracted ion chromatograms can be found in Supplementary Fig. 4a,b ). ( c ) ESI–MS spectrum at m/z 354.0884 corresponding to the LPZS standard in methanol. ( d ) Structures of LPZ and LPZS. LPZS is missing the sulfoxide (red), which is essential for LPZ activity on the human proton pump. ( e ) LPZ/LPZS ratio determined by ESI-Q-TOF-MS over a 48-h period in 7H9 broth. Representative example of three individual experiments; the complete data set can be found in Supplementary Table 2 . ( f ) Dose–response curve of LPZS for Mtb grown in 7H9 broth (mean±s.d. of three individual experiments). ( g ) Survival of Mtb -infected MRC-5 fibroblasts was quantified at different concentrations of LPZS (mean±s.d. of three individual experiments). ( h ) Efficacy of LPZS in the mouse model of acute tuberculosis. Bacterial burden (c.f.u.) was determined in the lungs of four mice treated with the vehicle control (TPGS) or four mice treated with LPZS at 300 mg kg −1 b.i.d. given by oral gavage (mean±s.d., Student's t -test was used to compare groups).
Figure Legend Snippet: LPZS is a highly selective antituberculous drug with in vivo activity. ( a ) Intracellular ratio of LPZ ( m/z 370.0834, g mol −1 ) and its metabolite ( m/z 354.0884, g mol −1 ) determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) over a 48-h period in MRC-5 cells. Representative example of three individual experiments; the complete data set can be found in Supplementary Table 2 . ( b ) ESI–MS mass spectra in the range m/z 350–375 measured for experiments performed on the cell lysate of MRC-5 fibroblasts exposed to LPZ (extracted ion chromatograms can be found in Supplementary Fig. 4a,b ). ( c ) ESI–MS spectrum at m/z 354.0884 corresponding to the LPZS standard in methanol. ( d ) Structures of LPZ and LPZS. LPZS is missing the sulfoxide (red), which is essential for LPZ activity on the human proton pump. ( e ) LPZ/LPZS ratio determined by ESI-Q-TOF-MS over a 48-h period in 7H9 broth. Representative example of three individual experiments; the complete data set can be found in Supplementary Table 2 . ( f ) Dose–response curve of LPZS for Mtb grown in 7H9 broth (mean±s.d. of three individual experiments). ( g ) Survival of Mtb -infected MRC-5 fibroblasts was quantified at different concentrations of LPZS (mean±s.d. of three individual experiments). ( h ) Efficacy of LPZS in the mouse model of acute tuberculosis. Bacterial burden (c.f.u.) was determined in the lungs of four mice treated with the vehicle control (TPGS) or four mice treated with LPZS at 300 mg kg −1 b.i.d. given by oral gavage (mean±s.d., Student's t -test was used to compare groups).

Techniques Used: In Vivo, Activity Assay, Mass Spectrometry, Infection, Mouse Assay

5) Product Images from "Lansoprazole is an antituberculous prodrug targeting cytochrome bc1"

Article Title: Lansoprazole is an antituberculous prodrug targeting cytochrome bc1

Journal: Nature Communications

doi: 10.1038/ncomms8659

LPZS is a highly selective antituberculous drug with in vivo activity. ( a ) Intracellular ratio of LPZ ( m/z 370.0834, g mol −1 ) and its metabolite ( m/z 354.0884, g mol −1 ) determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) over a 48-h period in MRC-5 cells. Representative example of three individual experiments; the complete data set can be found in Supplementary Table 2 . ( b ) ESI–MS mass spectra in the range m/z 350–375 measured for experiments performed on the cell lysate of MRC-5 fibroblasts exposed to LPZ (extracted ion chromatograms can be found in Supplementary Fig. 4a,b ). ( c ) ESI–MS spectrum at m/z 354.0884 corresponding to the LPZS standard in methanol. ( d ) Structures of LPZ and LPZS. LPZS is missing the sulfoxide (red), which is essential for LPZ activity on the human proton pump. ( e ) LPZ/LPZS ratio determined by ESI-Q-TOF-MS over a 48-h period in 7H9 broth. Representative example of three individual experiments; the complete data set can be found in Supplementary Table 2 . ( f ) Dose–response curve of LPZS for Mtb grown in 7H9 broth (mean±s.d. of three individual experiments). ( g ) Survival of Mtb -infected MRC-5 fibroblasts was quantified at different concentrations of LPZS (mean±s.d. of three individual experiments). ( h ) Efficacy of LPZS in the mouse model of acute tuberculosis. Bacterial burden (c.f.u.) was determined in the lungs of four mice treated with the vehicle control (TPGS) or four mice treated with LPZS at 300 mg kg −1 b.i.d. given by oral gavage (mean±s.d., Student's t -test was used to compare groups).
Figure Legend Snippet: LPZS is a highly selective antituberculous drug with in vivo activity. ( a ) Intracellular ratio of LPZ ( m/z 370.0834, g mol −1 ) and its metabolite ( m/z 354.0884, g mol −1 ) determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) over a 48-h period in MRC-5 cells. Representative example of three individual experiments; the complete data set can be found in Supplementary Table 2 . ( b ) ESI–MS mass spectra in the range m/z 350–375 measured for experiments performed on the cell lysate of MRC-5 fibroblasts exposed to LPZ (extracted ion chromatograms can be found in Supplementary Fig. 4a,b ). ( c ) ESI–MS spectrum at m/z 354.0884 corresponding to the LPZS standard in methanol. ( d ) Structures of LPZ and LPZS. LPZS is missing the sulfoxide (red), which is essential for LPZ activity on the human proton pump. ( e ) LPZ/LPZS ratio determined by ESI-Q-TOF-MS over a 48-h period in 7H9 broth. Representative example of three individual experiments; the complete data set can be found in Supplementary Table 2 . ( f ) Dose–response curve of LPZS for Mtb grown in 7H9 broth (mean±s.d. of three individual experiments). ( g ) Survival of Mtb -infected MRC-5 fibroblasts was quantified at different concentrations of LPZS (mean±s.d. of three individual experiments). ( h ) Efficacy of LPZS in the mouse model of acute tuberculosis. Bacterial burden (c.f.u.) was determined in the lungs of four mice treated with the vehicle control (TPGS) or four mice treated with LPZS at 300 mg kg −1 b.i.d. given by oral gavage (mean±s.d., Student's t -test was used to compare groups).

Techniques Used: In Vivo, Activity Assay, Mass Spectrometry, Infection, Mouse Assay

6) Product Images from "Lansoprazole is an antituberculous prodrug targeting cytochrome bc1"

Article Title: Lansoprazole is an antituberculous prodrug targeting cytochrome bc1

Journal: Nature Communications

doi: 10.1038/ncomms8659

LPZS is a highly selective antituberculous drug with in vivo activity. ( a ) Intracellular ratio of LPZ ( m/z 370.0834, g mol −1 ) and its metabolite ( m/z 354.0884, g mol −1 ) determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) over a 48-h period in MRC-5 cells. Representative example of three individual experiments; the complete data set can be found in Supplementary Table 2 . ( b ) ESI–MS mass spectra in the range m/z 350–375 measured for experiments performed on the cell lysate of MRC-5 fibroblasts exposed to LPZ (extracted ion chromatograms can be found in Supplementary Fig. 4a,b ). ( c ) ESI–MS spectrum at m/z 354.0884 corresponding to the LPZS standard in methanol. ( d ) Structures of LPZ and LPZS. LPZS is missing the sulfoxide (red), which is essential for LPZ activity on the human proton pump. ( e ) LPZ/LPZS ratio determined by ESI-Q-TOF-MS over a 48-h period in 7H9 broth. Representative example of three individual experiments; the complete data set can be found in Supplementary Table 2 . ( f ) Dose–response curve of LPZS for Mtb grown in 7H9 broth (mean±s.d. of three individual experiments). ( g ) Survival of Mtb -infected MRC-5 fibroblasts was quantified at different concentrations of LPZS (mean±s.d. of three individual experiments). ( h ) Efficacy of LPZS in the mouse model of acute tuberculosis. Bacterial burden (c.f.u.) was determined in the lungs of four mice treated with the vehicle control (TPGS) or four mice treated with LPZS at 300 mg kg −1 b.i.d. given by oral gavage (mean±s.d., Student's t -test was used to compare groups).
Figure Legend Snippet: LPZS is a highly selective antituberculous drug with in vivo activity. ( a ) Intracellular ratio of LPZ ( m/z 370.0834, g mol −1 ) and its metabolite ( m/z 354.0884, g mol −1 ) determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) over a 48-h period in MRC-5 cells. Representative example of three individual experiments; the complete data set can be found in Supplementary Table 2 . ( b ) ESI–MS mass spectra in the range m/z 350–375 measured for experiments performed on the cell lysate of MRC-5 fibroblasts exposed to LPZ (extracted ion chromatograms can be found in Supplementary Fig. 4a,b ). ( c ) ESI–MS spectrum at m/z 354.0884 corresponding to the LPZS standard in methanol. ( d ) Structures of LPZ and LPZS. LPZS is missing the sulfoxide (red), which is essential for LPZ activity on the human proton pump. ( e ) LPZ/LPZS ratio determined by ESI-Q-TOF-MS over a 48-h period in 7H9 broth. Representative example of three individual experiments; the complete data set can be found in Supplementary Table 2 . ( f ) Dose–response curve of LPZS for Mtb grown in 7H9 broth (mean±s.d. of three individual experiments). ( g ) Survival of Mtb -infected MRC-5 fibroblasts was quantified at different concentrations of LPZS (mean±s.d. of three individual experiments). ( h ) Efficacy of LPZS in the mouse model of acute tuberculosis. Bacterial burden (c.f.u.) was determined in the lungs of four mice treated with the vehicle control (TPGS) or four mice treated with LPZS at 300 mg kg −1 b.i.d. given by oral gavage (mean±s.d., Student's t -test was used to compare groups).

Techniques Used: In Vivo, Activity Assay, Mass Spectrometry, Infection, Mouse Assay

7) Product Images from "The Rip1 Protease of Mycobacterium tuberculosis Controls the SigD Regulon"

Article Title: The Rip1 Protease of Mycobacterium tuberculosis Controls the SigD Regulon

Journal: Journal of Bacteriology

doi: 10.1128/JB.01537-14

Activation of SigD-dependent transcription requires Rip1 cleavage of RsdA. Quantitative real-time PCR was used to measure the mRNA levels of rpfC (A) or rv1815c (B) in the strains as indicated. Strains were grown in 7H9 medium to log phase for RNA collection.
Figure Legend Snippet: Activation of SigD-dependent transcription requires Rip1 cleavage of RsdA. Quantitative real-time PCR was used to measure the mRNA levels of rpfC (A) or rv1815c (B) in the strains as indicated. Strains were grown in 7H9 medium to log phase for RNA collection.

Techniques Used: Activation Assay, Real-time Polymerase Chain Reaction

8) Product Images from "Application of Fluorescent Protein Expressing Strains to Evaluation of Anti-Tuberculosis Therapeutic Efficacy In Vitro and In Vivo"

Article Title: Application of Fluorescent Protein Expressing Strains to Evaluation of Anti-Tuberculosis Therapeutic Efficacy In Vitro and In Vivo

Journal: PLoS ONE

doi: 10.1371/journal.pone.0149972

Evaluation of plasmid stability and the absence of effects on bacterial growth. A. Growth curves for M . bovis BCG strains carrying L5-tdTomato, Hsp60-tdTomato or the vector plasmids alone (no fluorescent protein expressed). B. Optical density (OD) of cultures at 600 nm in 7H9 media with (kan 25) or without (no kan) 25 μg/ml kanamycin over 18 days of culture. C. Fluorescence changes of the strain in media with or without kanamycin over 18 days of culture. D. Correlation between fluorescence and OD at 600 nm for the strain grown in medium without kanamycin. E. Correlation between fluorescence and OD at 600 nm for the strain grown in medium with kanamycin. For correlation analyses, samples for OD 600 within the range of 0.05–1 were selected. Data represent one of at least three independent replicate experiments. Values in the Y-axis represent mean of fluorescence or OD values. The error bars are standard deviations.
Figure Legend Snippet: Evaluation of plasmid stability and the absence of effects on bacterial growth. A. Growth curves for M . bovis BCG strains carrying L5-tdTomato, Hsp60-tdTomato or the vector plasmids alone (no fluorescent protein expressed). B. Optical density (OD) of cultures at 600 nm in 7H9 media with (kan 25) or without (no kan) 25 μg/ml kanamycin over 18 days of culture. C. Fluorescence changes of the strain in media with or without kanamycin over 18 days of culture. D. Correlation between fluorescence and OD at 600 nm for the strain grown in medium without kanamycin. E. Correlation between fluorescence and OD at 600 nm for the strain grown in medium with kanamycin. For correlation analyses, samples for OD 600 within the range of 0.05–1 were selected. Data represent one of at least three independent replicate experiments. Values in the Y-axis represent mean of fluorescence or OD values. The error bars are standard deviations.

Techniques Used: Plasmid Preparation, Fluorescence

9) Product Images from "Rifampin Stability in 7H9 Broth and L?wenstein-Jensen Medium ▿"

Article Title: Rifampin Stability in 7H9 Broth and L?wenstein-Jensen Medium ▿

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.01951-10

Serial monitoring outcomes of RIF stability in L-J medium and 7H9 broth when kept at 37°C and 4°C. (a) Stability of RIF in 7H9 and L-J media when kept at 37°C. (b) Stability of RIF in 7H9 and L-J media when kept at 4°C.
Figure Legend Snippet: Serial monitoring outcomes of RIF stability in L-J medium and 7H9 broth when kept at 37°C and 4°C. (a) Stability of RIF in 7H9 and L-J media when kept at 37°C. (b) Stability of RIF in 7H9 and L-J media when kept at 4°C.

Techniques Used:

10) Product Images from "Mode of Action of Clofazimine and Combination Therapy with Benzothiazinones against Mycobacterium tuberculosis"

Article Title: Mode of Action of Clofazimine and Combination Therapy with Benzothiazinones against Mycobacterium tuberculosis

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.00395-15

Effect of menaquinone supplementation on clofazimine (CZM), BDQ, and isoniazid (INH) against M. tuberculosis H37Rv by REMA. Cells were grown in a normal 7H9 medium (no MK-4) or in medium supplemented with increasing concentrations of menaquinone (10, 100, and 1,000 μM MK-4). REMA results are presented as mean ± SD values of triplicates. Drug concentrations are in micrograms per milliliter.
Figure Legend Snippet: Effect of menaquinone supplementation on clofazimine (CZM), BDQ, and isoniazid (INH) against M. tuberculosis H37Rv by REMA. Cells were grown in a normal 7H9 medium (no MK-4) or in medium supplemented with increasing concentrations of menaquinone (10, 100, and 1,000 μM MK-4). REMA results are presented as mean ± SD values of triplicates. Drug concentrations are in micrograms per milliliter.

Techniques Used:

11) Product Images from "The Chromosomal parDE2 Toxin–Antitoxin System of Mycobacterium tuberculosis H37Rv: Genetic and Functional Characterization"

Article Title: The Chromosomal parDE2 Toxin–Antitoxin System of Mycobacterium tuberculosis H37Rv: Genetic and Functional Characterization

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.00886

MParDE2 Promoter identification and regulation. (A) M. smegmatis recombinants viz., MS1, harboring a promoter-less lacZ construct; MS2, containing P 363parD2 -lacZ fusion construct and MS12, vector control strains were grown in 7H9 medium at 37°C and β-galactosidase activity was measured spectrophotometrically, after regular intervals. (B) The transcriptional start site (+1), putative -35 and -10 promoter elements along with parD2 and parE2 translational start sites are indicated in rectangular boxes determined by 5′-RACE PCR analysis. MParD2 amino acid residues substituted with alanine are underlined. (C) Regulation of P parDE2 in M. smegmatis strains. β-galactosidase activity from different M. smegmatis strains harboring 363 bp parDE2 promoter sequence fused with lacZ gene, co-expressing MParD2 or MParDE2 protein complex under the control of groEL promoter. Empty pMV261 vector, parD2 and ParDE2 sequences without promoter were used as negative controls. Error bars indicate mean ± SD from three independent experiments.
Figure Legend Snippet: MParDE2 Promoter identification and regulation. (A) M. smegmatis recombinants viz., MS1, harboring a promoter-less lacZ construct; MS2, containing P 363parD2 -lacZ fusion construct and MS12, vector control strains were grown in 7H9 medium at 37°C and β-galactosidase activity was measured spectrophotometrically, after regular intervals. (B) The transcriptional start site (+1), putative -35 and -10 promoter elements along with parD2 and parE2 translational start sites are indicated in rectangular boxes determined by 5′-RACE PCR analysis. MParD2 amino acid residues substituted with alanine are underlined. (C) Regulation of P parDE2 in M. smegmatis strains. β-galactosidase activity from different M. smegmatis strains harboring 363 bp parDE2 promoter sequence fused with lacZ gene, co-expressing MParD2 or MParDE2 protein complex under the control of groEL promoter. Empty pMV261 vector, parD2 and ParDE2 sequences without promoter were used as negative controls. Error bars indicate mean ± SD from three independent experiments.

Techniques Used: Construct, Plasmid Preparation, Activity Assay, Polymerase Chain Reaction, Sequencing, Expressing

12) Product Images from "Bioluminescence for Assessing Drug Potency against Nonreplicating Mycobacterium tuberculosis"

Article Title: Bioluminescence for Assessing Drug Potency against Nonreplicating Mycobacterium tuberculosis

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.00528-15

Growth curves for M. tuberculosis 18b-Lux and evaluation of luciferase expression. (A) 18b-Lux was grown at 37°C, with shaking, in glucose- or acetate-containing 7H9 medium with 50 μg/ml STR. (B) SS18b-Lux was incubated under the same
Figure Legend Snippet: Growth curves for M. tuberculosis 18b-Lux and evaluation of luciferase expression. (A) 18b-Lux was grown at 37°C, with shaking, in glucose- or acetate-containing 7H9 medium with 50 μg/ml STR. (B) SS18b-Lux was incubated under the same

Techniques Used: Luciferase, Expressing, Incubation

Related Articles

Clone Assay:

Article Title: Mycobacterium tuberculosis Proteasome Accessory Factor A (PafA) Can Transfer Prokaryotic Ubiquitin-Like Protein (Pup) between Substrates
Article Snippet: For M. smegmatis , we grew bacteria in 7H9 broth (Difco) supplemented with 0.2% glycerol and 0.05% Tween 80 supplemented with 50 µg ml−1 hygromycin. .. To make strain EHD1491, M. smegmatis dop was cloned into the expression plasmid pETDUET (Novagen, Inc.) at MCS2.

Article Title: Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis
Article Snippet: M . tuberculosis H37Rv and Mycobacterium smegmatis mc2 155 were cultured in 7H9 broth (Difco) with 0.05% Tween-80 supplemented with 0.2% glycerol and ADC (Bovine Albumin fraction V (50 g/L), dextrose (20 g/L) and catalase (0.0375 g/L)) or Middlebrook OADC (oleic acid ADC), or on Middlebrook 7H10 (Difco) supplemented with ADC or OADC. .. E . coli XL1 Blue strain was used for cloning, while recombinant protein expression was performed using the E . coli Arctic express (DE3) (Agilent Technologies) and the BL21(DE3)pLysS (Novagen) strains.

Article Title: The Chromosomal parDE2 Toxin–Antitoxin System of Mycobacterium tuberculosis H37Rv: Genetic and Functional Characterization
Article Snippet: E. coli strains used for cloning were grown under standard conditions in Luria-Bertani (LB) medium (Difco). .. M. tuberculosis and M. smegmatis strains were grown in Middlebrook 7H10 agar (Difco) containing 10% (v/v) oleic acid, albumin, dextrose and catalase (OADC; Becton Dickinson) enrichment and 0.5% (v/v) glycerol, or in 7H9 broth (Difco) supplemented with 10% (v/v) albumin, dextrose and catalase (ADC; Becton Dickinson), 0.2% (v/v) glycerol and 0.05% (v/v) Tween 80 ( ).

Article Title: Poly-L-glutamate/glutamine synthesis in the cell wall of Mycobacterium bovis is regulated in response to nitrogen availability
Article Snippet: M. bovis and M. smegmatis strains were routinely cultured in 7H9 broth (Difco) supplemented with 10% (v/v) albumin, dextrose and catalase (ADC), 0.2% (v/v) glycerol and 0.05% (v/v) Tween 80, at 37°C with shaking at 150 rpm. .. Escherichia coli DH5α (Novagen) was used for cloning experiments.

Centrifugation:

Article Title: Downregulation of vimentin in macrophages infected with live Mycobacterium tuberculosis is mediated by Reactive Oxygen Species
Article Snippet: Prior to experiments bacteria were inoculated into 7H9 broth (271310, Difco) from LJ slants. .. On the day of infection log phase culture of bacteria was collected and passed through a syringe for 20 times and kept for 10 min to remove the clumps and the required volume of the culture was pelleted by centrifugation at 3000 g for 10 min. Then the pellet was resuspended in RPMI and added to the macrophage monolayer.

Selection:

Article Title: Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis
Article Snippet: M . tuberculosis H37Rv and Mycobacterium smegmatis mc2 155 were cultured in 7H9 broth (Difco) with 0.05% Tween-80 supplemented with 0.2% glycerol and ADC (Bovine Albumin fraction V (50 g/L), dextrose (20 g/L) and catalase (0.0375 g/L)) or Middlebrook OADC (oleic acid ADC), or on Middlebrook 7H10 (Difco) supplemented with ADC or OADC. .. Kanamycin (50 μg/ml), hygromycin (50 μg/ml), 5-Bromo-4 Chloro-3 Indolyl β-D-galactosidase (X-gal) (40 μg/ml) and sucrose (5%) were used for selection purposes when applicable.

Article Title: Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis
Article Snippet: Bacterial strains and culture conditions M . tuberculosis H37Rv and Mycobacterium smegmatis mc2 155 were cultured in 7H9 broth (Difco) with 0.05% Tween-80 supplemented with 0.2% glycerol and ADC (Bovine Albumin fraction V (50 g/L), dextrose (20 g/L) and catalase (0.0375 g/L)) or Middlebrook OADC (oleic acid ADC), or on Middlebrook 7H10 (Difco) supplemented with ADC or OADC. .. Kanamycin (50 μg/ml), hygromycin (50 μg/ml), 5-Bromo-4 Chloro-3 Indolyl β-D-galactosidase (X-gal) (40 μg/ml) and sucrose (5%) were used for selection purposes when applicable.

Expressing:

Article Title: Mycobacterium tuberculosis Proteasome Accessory Factor A (PafA) Can Transfer Prokaryotic Ubiquitin-Like Protein (Pup) between Substrates
Article Snippet: For M. smegmatis , we grew bacteria in 7H9 broth (Difco) supplemented with 0.2% glycerol and 0.05% Tween 80 supplemented with 50 µg ml−1 hygromycin. .. To make strain EHD1491, M. smegmatis dop was cloned into the expression plasmid pETDUET (Novagen, Inc.) at MCS2.

Article Title: Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis
Article Snippet: M . tuberculosis H37Rv and Mycobacterium smegmatis mc2 155 were cultured in 7H9 broth (Difco) with 0.05% Tween-80 supplemented with 0.2% glycerol and ADC (Bovine Albumin fraction V (50 g/L), dextrose (20 g/L) and catalase (0.0375 g/L)) or Middlebrook OADC (oleic acid ADC), or on Middlebrook 7H10 (Difco) supplemented with ADC or OADC. .. E . coli XL1 Blue strain was used for cloning, while recombinant protein expression was performed using the E . coli Arctic express (DE3) (Agilent Technologies) and the BL21(DE3)pLysS (Novagen) strains.

Article Title: Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis
Article Snippet: Bacterial strains and culture conditions M . tuberculosis H37Rv and Mycobacterium smegmatis mc2 155 were cultured in 7H9 broth (Difco) with 0.05% Tween-80 supplemented with 0.2% glycerol and ADC (Bovine Albumin fraction V (50 g/L), dextrose (20 g/L) and catalase (0.0375 g/L)) or Middlebrook OADC (oleic acid ADC), or on Middlebrook 7H10 (Difco) supplemented with ADC or OADC. .. E . coli XL1 Blue strain was used for cloning, while recombinant protein expression was performed using the E . coli Arctic express (DE3) (Agilent Technologies) and the BL21(DE3)pLysS (Novagen) strains.

Mutagenesis:

Article Title: Lansoprazole is an antituberculous prodrug targeting cytochrome bc1
Article Snippet: .. Recombineering method for target confirmation Mtb H37Rv carrying plasmid pJV53 was grown to mid-log phase in 7H9 broth containing 25 μg ml−1 kanamycin and exposed to 0.2% acetamide for 16 h. Competent cells were co-transformed with 100 ng of single-stranded oligos (lagging and leading strand) (5′-CTCACTGCCTGACGACCTGCTGTCGGGACTCGGTCCGCGCGCGGCACTCTCGTCGATCACGCTGGGTATGC-3′—for the L176P mutation) or (5′-GGCGGGCTCGCAGCCAGACTTCTACATGATGTGGGCCGAGGGTCTGGCCCGGATCTGGCCGCCGTGGGAG-3′—for the T313A mutation) and pYUB412 (50 ng) followed by plating on 7H10 agar plates containing 50 μg ml−1 hygromycin. .. SNPs in hygromycin-resistant clones were confirmed by PCR and sequencing using oligos 5′-TGCTGATCACCGGCGTGTAT-3′ and 5′-AAGATGATCCCCGGCAACAG-3′ for L176P or 5′-TTCAAGTCCGGCGCATTTTT-3′ and 5′-TAGACGAACGGCGGCAGAAT-3′ for T313A.

Generated:

Article Title: Mode of Action of Clofazimine and Combination Therapy with Benzothiazinones against Mycobacterium tuberculosis
Article Snippet: M. tuberculosis strains H37Rv and 18b were grown at 37°C with shaking in 7H9 broth (Difco) supplemented with Middlebrook albumin-dextrose-catalase enrichment, 0.2% glycerol, 0.05% Tween 80, and, in the case of 18b, 50 μg/ml streptomycin (STR) or on solid Middlebrook 7H10 medium (Difco) supplemented with 0.5% glycerol, Middlebrook oleic acid-albumin-dextrose-catalase (OADC), and, in the case of 18b, 50 μg/ml STR. .. Nonreplicating streptomycin-starved 18b cultures (SS18b) were generated as previously described ( ).

Article Title: Bioluminescence for Assessing Drug Potency against Nonreplicating Mycobacterium tuberculosis
Article Snippet: Mycobacterium tuberculosis strains 18b and 18b-Lux were grown at 37°C, with shaking, in 7H9 broth (Difco) supplemented with 10% albumin-dextrose-catalase (ADC), 0.2% glycerol, 0.05% Tween 80, and 50 μg/ml STR or on solid Middlebrook 7H10 agar (Difco) supplemented with 0.5% glycerol, 10% oleic acid-albumin-dextrose-catalase (OADC), and 50 μg/ml STR. .. Nonreplicating STR-starved 18b-Lux (SS18b-Lux) was generated as follows.

Cell Culture:

Article Title: Downregulation of vimentin in macrophages infected with live Mycobacterium tuberculosis is mediated by Reactive Oxygen Species
Article Snippet: Paragraph title: Cell culture, bacteria and infection ... Prior to experiments bacteria were inoculated into 7H9 broth (271310, Difco) from LJ slants.

Article Title: Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis
Article Snippet: The sansanmycin analogues were diluted in fresh RPMI-1640 cell culture medium and added to corresponding wells. .. Positive controls were dissolved in 100% dimethyl sulfoxide (DMSO) and diluted in 7H9 broth (Difco Becton Dickinson) with 10% ADC, 0.05% glycerol and 0.05% Tween 80 before adding to the wells.

Article Title: Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis
Article Snippet: .. M . tuberculosis H37Rv and Mycobacterium smegmatis mc2 155 were cultured in 7H9 broth (Difco) with 0.05% Tween-80 supplemented with 0.2% glycerol and ADC (Bovine Albumin fraction V (50 g/L), dextrose (20 g/L) and catalase (0.0375 g/L)) or Middlebrook OADC (oleic acid ADC), or on Middlebrook 7H10 (Difco) supplemented with ADC or OADC. .. Kanamycin (50 μg/ml), hygromycin (50 μg/ml), 5-Bromo-4 Chloro-3 Indolyl β-D-galactosidase (X-gal) (40 μg/ml) and sucrose (5%) were used for selection purposes when applicable.

Article Title: Poly-L-glutamate/glutamine synthesis in the cell wall of Mycobacterium bovis is regulated in response to nitrogen availability
Article Snippet: .. M. bovis and M. smegmatis strains were routinely cultured in 7H9 broth (Difco) supplemented with 10% (v/v) albumin, dextrose and catalase (ADC), 0.2% (v/v) glycerol and 0.05% (v/v) Tween 80, at 37°C with shaking at 150 rpm. .. Escherichia coli DH5α (Novagen) was used for cloning experiments.

Article Title: The Rip1 Protease of Mycobacterium tuberculosis Controls the SigD Regulon
Article Snippet: M. tuberculosis strains (all based on the wild-type strain Erdman EG1, which is animal passaged) were grown aerobically at 37°C in 7H9 (broth) or 7H10 (agar) (Difco) medium with oleic acid-albumin-dextrose-catalase (OADC) enrichment, 0.5% glycerol, and 0.05% Tween 80 (broth medium only). .. Mycobacterium smegmatis strains were cultured at 37°C on Luria-Bertani (LB) medium containing 0.5% dextrose, 0.5% glycerol, and 0.05% Tween 80 (broth).

Article Title: Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis
Article Snippet: .. Bacterial strains and culture conditions M . tuberculosis H37Rv and Mycobacterium smegmatis mc2 155 were cultured in 7H9 broth (Difco) with 0.05% Tween-80 supplemented with 0.2% glycerol and ADC (Bovine Albumin fraction V (50 g/L), dextrose (20 g/L) and catalase (0.0375 g/L)) or Middlebrook OADC (oleic acid ADC), or on Middlebrook 7H10 (Difco) supplemented with ADC or OADC. .. Kanamycin (50 μg/ml), hygromycin (50 μg/ml), 5-Bromo-4 Chloro-3 Indolyl β-D-galactosidase (X-gal) (40 μg/ml) and sucrose (5%) were used for selection purposes when applicable.

Concentration Assay:

Article Title: Rifampin Stability in 7H9 Broth and L?wenstein-Jensen Medium ▿
Article Snippet: 7H9 broth (Difco) was prepared according to the manufacturer's instruction. .. For the RIF (Sigma, St. Louis, MO)-containing broth preparation, a freshly made RIF– N , N -dimethylformamide (DMF) solution was added to achieve a final concentration of 40 mg/liter.

Article Title: Bioluminescence for Assessing Drug Potency against Nonreplicating Mycobacterium tuberculosis
Article Snippet: Mycobacterium tuberculosis strains 18b and 18b-Lux were grown at 37°C, with shaking, in 7H9 broth (Difco) supplemented with 10% albumin-dextrose-catalase (ADC), 0.2% glycerol, 0.05% Tween 80, and 50 μg/ml STR or on solid Middlebrook 7H10 agar (Difco) supplemented with 0.5% glycerol, 10% oleic acid-albumin-dextrose-catalase (OADC), and 50 μg/ml STR. .. Alternatively, 18b-Lux was grown in 7H9 medium with acetate (0.1% final concentration) supplemented with 10% albumin-NaCl, 0.05% Tween 80, and 50 μg/ml STR.

Incubation:

Article Title: Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis
Article Snippet: Supernatant was then removed from all wells, THP-1 cells were washed with 200 μl phosphate buffered saline (PBS) three times and were subsequently replenished with fresh RPMI-1640 cell culture medium and incubated for a further 24 h at 37 °C and 5% CO2 . .. Positive controls were dissolved in 100% dimethyl sulfoxide (DMSO) and diluted in 7H9 broth (Difco Becton Dickinson) with 10% ADC, 0.05% glycerol and 0.05% Tween 80 before adding to the wells.

Article Title: Lansoprazole is an antituberculous prodrug targeting cytochrome bc1
Article Snippet: Culture conditions and REMA assay of other microorganisms Mycobacterium strains were grown in 7H9 broth (Difco) supplemented with Middlebrook ADC enrichment, 0.2% glycerol, 0.05% Tween-80. .. Twofold serial dilutions of each test compound were prepared in 96-well plates containing bacteria in a total volume of 100 μl and then incubated at 37 or 30 °C (as required) before addition of 10 μl of 0.025% resazurin.

Inhibition:

Article Title: Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis
Article Snippet: Paragraph title: Mtb inhibition using infected macrophage culture ... Positive controls were dissolved in 100% dimethyl sulfoxide (DMSO) and diluted in 7H9 broth (Difco Becton Dickinson) with 10% ADC, 0.05% glycerol and 0.05% Tween 80 before adding to the wells.

Infection:

Article Title: Downregulation of vimentin in macrophages infected with live Mycobacterium tuberculosis is mediated by Reactive Oxygen Species
Article Snippet: Paragraph title: Cell culture, bacteria and infection ... Prior to experiments bacteria were inoculated into 7H9 broth (271310, Difco) from LJ slants.

Article Title: Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis
Article Snippet: Paragraph title: Mtb inhibition using infected macrophage culture ... Positive controls were dissolved in 100% dimethyl sulfoxide (DMSO) and diluted in 7H9 broth (Difco Becton Dickinson) with 10% ADC, 0.05% glycerol and 0.05% Tween 80 before adding to the wells.

Mass Spectrometry:

Article Title: Lansoprazole is an antituberculous prodrug targeting cytochrome bc1
Article Snippet: .. After spinning at 15,000g at 4 °C, samples were shock-frozen in liquid nitrogen and stored at −80 °C for MS. Quantification in 7H9 broth was performed with 500 nM of LPZ, acetonitrile was added in a 1:1 ratio at given time points followed by spinning and shock freezing. .. The UPLC separation was done on an Agilent 1290 Infinity LC system including the 1290 Infinity LC system binary pump with an integrated degasser, the high-performance autosampler and a thermostatted column compartment.

Sonication:

Article Title: Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis
Article Snippet: A cell suspension of sonicated Mtb H37Ra in RPMI-1640 cell culture medium was used to infect differentiated THP-1 cells at a multiplicity of infection of 5 for 4 h at 37 °C at 5% CO2 . .. Positive controls were dissolved in 100% dimethyl sulfoxide (DMSO) and diluted in 7H9 broth (Difco Becton Dickinson) with 10% ADC, 0.05% glycerol and 0.05% Tween 80 before adding to the wells.

Transformation Assay:

Article Title: Application of Fluorescent Protein Expressing Strains to Evaluation of Anti-Tuberculosis Therapeutic Efficacy In Vitro and In Vivo
Article Snippet: Strains and Growth Conditions Plasmids were transformed into M . smegmatis and M . bovis BCG. .. Bacteria were grown in 7H9 broth (Difco, Detroit, MI) supplemented with 0.5% glycerol, 10% OAD (oleic acid dextrose complex without catalase) and 0.05% Tween 80 (M-OADTW broth), or Middlebrook 7H9 supplemented with 10% OAD and 15 g/l Bacto agar (M-OAD agar, Difco) or on 7H11 selective agar (Difco).

Recombinant:

Article Title: Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis
Article Snippet: M . tuberculosis H37Rv and Mycobacterium smegmatis mc2 155 were cultured in 7H9 broth (Difco) with 0.05% Tween-80 supplemented with 0.2% glycerol and ADC (Bovine Albumin fraction V (50 g/L), dextrose (20 g/L) and catalase (0.0375 g/L)) or Middlebrook OADC (oleic acid ADC), or on Middlebrook 7H10 (Difco) supplemented with ADC or OADC. .. E . coli XL1 Blue strain was used for cloning, while recombinant protein expression was performed using the E . coli Arctic express (DE3) (Agilent Technologies) and the BL21(DE3)pLysS (Novagen) strains.

Article Title: The Rip1 Protease of Mycobacterium tuberculosis Controls the SigD Regulon
Article Snippet: Escherichia coli DH5α was used for all recombinant DNA manipulations and grown in Luria-Bertani broth at 37°C. .. M. tuberculosis strains (all based on the wild-type strain Erdman EG1, which is animal passaged) were grown aerobically at 37°C in 7H9 (broth) or 7H10 (agar) (Difco) medium with oleic acid-albumin-dextrose-catalase (OADC) enrichment, 0.5% glycerol, and 0.05% Tween 80 (broth medium only).

Article Title: Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis
Article Snippet: Bacterial strains and culture conditions M . tuberculosis H37Rv and Mycobacterium smegmatis mc2 155 were cultured in 7H9 broth (Difco) with 0.05% Tween-80 supplemented with 0.2% glycerol and ADC (Bovine Albumin fraction V (50 g/L), dextrose (20 g/L) and catalase (0.0375 g/L)) or Middlebrook OADC (oleic acid ADC), or on Middlebrook 7H10 (Difco) supplemented with ADC or OADC. .. E . coli XL1 Blue strain was used for cloning, while recombinant protein expression was performed using the E . coli Arctic express (DE3) (Agilent Technologies) and the BL21(DE3)pLysS (Novagen) strains.

Plasmid Preparation:

Article Title: Mycobacterium tuberculosis Proteasome Accessory Factor A (PafA) Can Transfer Prokaryotic Ubiquitin-Like Protein (Pup) between Substrates
Article Snippet: For M. smegmatis , we grew bacteria in 7H9 broth (Difco) supplemented with 0.2% glycerol and 0.05% Tween 80 supplemented with 50 µg ml−1 hygromycin. .. To make strain EHD1491, M. smegmatis dop was cloned into the expression plasmid pETDUET (Novagen, Inc.) at MCS2.

Article Title: Lansoprazole is an antituberculous prodrug targeting cytochrome bc1
Article Snippet: .. Recombineering method for target confirmation Mtb H37Rv carrying plasmid pJV53 was grown to mid-log phase in 7H9 broth containing 25 μg ml−1 kanamycin and exposed to 0.2% acetamide for 16 h. Competent cells were co-transformed with 100 ng of single-stranded oligos (lagging and leading strand) (5′-CTCACTGCCTGACGACCTGCTGTCGGGACTCGGTCCGCGCGCGGCACTCTCGTCGATCACGCTGGGTATGC-3′—for the L176P mutation) or (5′-GGCGGGCTCGCAGCCAGACTTCTACATGATGTGGGCCGAGGGTCTGGCCCGGATCTGGCCGCCGTGGGAG-3′—for the T313A mutation) and pYUB412 (50 ng) followed by plating on 7H10 agar plates containing 50 μg ml−1 hygromycin. .. SNPs in hygromycin-resistant clones were confirmed by PCR and sequencing using oligos 5′-TGCTGATCACCGGCGTGTAT-3′ and 5′-AAGATGATCCCCGGCAACAG-3′ for L176P or 5′-TTCAAGTCCGGCGCATTTTT-3′ and 5′-TAGACGAACGGCGGCAGAAT-3′ for T313A.

Article Title: The Chromosomal parDE2 Toxin–Antitoxin System of Mycobacterium tuberculosis H37Rv: Genetic and Functional Characterization
Article Snippet: Paragraph title: Bacterial Strain, Plasmid, and Culture Conditions ... M. tuberculosis and M. smegmatis strains were grown in Middlebrook 7H10 agar (Difco) containing 10% (v/v) oleic acid, albumin, dextrose and catalase (OADC; Becton Dickinson) enrichment and 0.5% (v/v) glycerol, or in 7H9 broth (Difco) supplemented with 10% (v/v) albumin, dextrose and catalase (ADC; Becton Dickinson), 0.2% (v/v) glycerol and 0.05% (v/v) Tween 80 ( ).

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  • 99
    Difco middlebrook 7h9 broth
    Growth curve of the fadD32 conditional mutant TB47 in the presence of ATc and pristinamycin I. Bacteria were grown in <t>Middlebrook</t> <t>7H9</t> without ATc (diamonds) or with 200 ng/ml ATc (all the others). Forty-eight hours after the beginning of the experiment cultures were supplemented with 20 (filled triangles), 200 (circles) or 2000 ng/ml (open triangles) of pristinamycin I. As a control, one culture grown in 200 ng/ml ATc did not receive pristinamycin I (squares).
    Middlebrook 7h9 Broth, supplied by Difco, used in various techniques. Bioz Stars score: 99/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/middlebrook 7h9 broth/product/Difco
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    middlebrook 7h9 broth - by Bioz Stars, 2020-01
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    78
    Difco mb 7h9 broth
    MALDI-TOF mass spectra of MAMEs from various strains of M. tuberculosis. M. tuberculosis , MtbΔ virS , Mtb mym :: hyg , and MtbΔ virS _ virS strains were grown in MB <t>7H9</t> medium at pH 7.0 or exposed to pH 5.0 for 12 h. MAMEs were extracted and subjected to MALDI-TOF analysis as described in Materials and Methods. MALDI-TOF mass spectra of MAMEs from M. tuberculosis (A and E), MtbΔ virS (B and F), Mtb mym :: hyg (C and G), and MtbΔ virS _ virS (D and H) are shown. The psuedomolecular masses of mycolates are indicated on each mass spectrum. Arrows and brackets indicate the major differences observed in MALDI-TOF analysis of MAMEs from various strains.
    Mb 7h9 Broth, supplied by Difco, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mb 7h9 broth/product/Difco
    Average 78 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mb 7h9 broth - by Bioz Stars, 2020-01
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    78
    Difco middlebrook 7h9 oadc broth
    Effect of AU1235 on mycolic acid biosynthesis and transfer in M. tb M. tb H37Ra cultured in <t>7H9-OADC-Tween</t> 80 broth was treated for 5 hr at 37°C with either no inhibitor or with AU1235 at a concentration of 0.05 μg ml −1 , 0.5 μg ml −1 , or 1 μg ml −1 (0.5x to 10x MIC). [ 14 C]-acetate was added to the cultures at the same time as the inhibitor. ( a ) Bacterial cells (B) and culture filtrates (CF) were collected and the lipids contained in each of these fractions extracted as described under the Methods section. The same volume of samples was loaded per lane. The TLC was developed in the solvent system [chloroform:methanol:water] (20:4:0.5, by vol.) and revealed by autoradiography. PE, phosphatidylethanolamine; CL, cardiolipin. ( b ) Mycolic acid methyl esters were prepared from delipidated cells as described 48 . α–, methoxy- and keto- denote the three forms of mycolic acids produced by M. tb . The same volume of samples was loaded per lane. The TLC was developed thrice in the solvent system [ n -hexanes:ethyl acetate] (95:5, by vol.) and revealed by autoradiography. The amount of radioactivity incorporated in the products of interest was semi-quantified using a PhosphoImager and the results (expressed as a % of the value measured in the untreated control) are presented as histograms under their corresponding autoradiograms.
    Middlebrook 7h9 Oadc Broth, supplied by Difco, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/middlebrook 7h9 oadc broth/product/Difco
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    Image Search Results


    Growth curve of the fadD32 conditional mutant TB47 in the presence of ATc and pristinamycin I. Bacteria were grown in Middlebrook 7H9 without ATc (diamonds) or with 200 ng/ml ATc (all the others). Forty-eight hours after the beginning of the experiment cultures were supplemented with 20 (filled triangles), 200 (circles) or 2000 ng/ml (open triangles) of pristinamycin I. As a control, one culture grown in 200 ng/ml ATc did not receive pristinamycin I (squares).

    Journal: Nucleic Acids Research

    Article Title: Development of a repressible mycobacterial promoter system based on two transcriptional repressors

    doi: 10.1093/nar/gkq235

    Figure Lengend Snippet: Growth curve of the fadD32 conditional mutant TB47 in the presence of ATc and pristinamycin I. Bacteria were grown in Middlebrook 7H9 without ATc (diamonds) or with 200 ng/ml ATc (all the others). Forty-eight hours after the beginning of the experiment cultures were supplemented with 20 (filled triangles), 200 (circles) or 2000 ng/ml (open triangles) of pristinamycin I. As a control, one culture grown in 200 ng/ml ATc did not receive pristinamycin I (squares).

    Article Snippet: Mycobacterial strains were grown at 37°C in Middlebrook 7H9 broth (Difco) in 150 ml roller bottles with slow rotation (3 rmp) or 7H10 agar plates (Difco), supplemented with 0.2% glycerol and 0.05% Tween-80.

    Techniques: Mutagenesis

    Characterization of the M. smegmatis ftsZ mutant MS98. ( A ) MS98 was plated on Middlebrook 7H10 medium with or without 50 ng/ml ATc. ( B ) MS98 and its parental wild-type strain were grown in Middlebrook 7H9 with or without ATc.

    Journal: Nucleic Acids Research

    Article Title: Development of a repressible mycobacterial promoter system based on two transcriptional repressors

    doi: 10.1093/nar/gkq235

    Figure Lengend Snippet: Characterization of the M. smegmatis ftsZ mutant MS98. ( A ) MS98 was plated on Middlebrook 7H10 medium with or without 50 ng/ml ATc. ( B ) MS98 and its parental wild-type strain were grown in Middlebrook 7H9 with or without ATc.

    Article Snippet: Mycobacterial strains were grown at 37°C in Middlebrook 7H9 broth (Difco) in 150 ml roller bottles with slow rotation (3 rmp) or 7H10 agar plates (Difco), supplemented with 0.2% glycerol and 0.05% Tween-80.

    Techniques: Mutagenesis

    β-Galactosidase assay in liquid media. ( A ) M. smegmatis MS82 was pre-grown in Middlebrook 7H9 containing 50 ng/ml ATc, and diluted into fresh media containing different ATc concentrations. Every 24 h and for 3 days, one aliquot of each culture was used to measure β-galactosidase activity and one aliquot was diluted into fresh medium containing the same amount of ATc as before. Black bars: 24 h; gray bar: 48 h; white bars: 72 h. ( B ) TB38.2 was pre-grown in Middlebrook 7H9 containing 200 ng/ml ATc, and diluted into fresh media containing different ATc concentrations. Every 48 h and for 6 days, one aliquot of each culture was used to measure β-galactosidase activity and one aliquot was diluted into fresh medium containing the same amount of ATc as before. Black bars: 48 h; grey bar: 96 h; white bars: 144 h. Values are indicated as percentages of maximal activity.

    Journal: Nucleic Acids Research

    Article Title: Development of a repressible mycobacterial promoter system based on two transcriptional repressors

    doi: 10.1093/nar/gkq235

    Figure Lengend Snippet: β-Galactosidase assay in liquid media. ( A ) M. smegmatis MS82 was pre-grown in Middlebrook 7H9 containing 50 ng/ml ATc, and diluted into fresh media containing different ATc concentrations. Every 24 h and for 3 days, one aliquot of each culture was used to measure β-galactosidase activity and one aliquot was diluted into fresh medium containing the same amount of ATc as before. Black bars: 24 h; gray bar: 48 h; white bars: 72 h. ( B ) TB38.2 was pre-grown in Middlebrook 7H9 containing 200 ng/ml ATc, and diluted into fresh media containing different ATc concentrations. Every 48 h and for 6 days, one aliquot of each culture was used to measure β-galactosidase activity and one aliquot was diluted into fresh medium containing the same amount of ATc as before. Black bars: 48 h; grey bar: 96 h; white bars: 144 h. Values are indicated as percentages of maximal activity.

    Article Snippet: Mycobacterial strains were grown at 37°C in Middlebrook 7H9 broth (Difco) in 150 ml roller bottles with slow rotation (3 rmp) or 7H10 agar plates (Difco), supplemented with 0.2% glycerol and 0.05% Tween-80.

    Techniques: Activity Assay

    Characterization of the TetR/Pip OFF system. ( A ) β-galactosidase assay in M. smegmatis in liquid media: MS82 and MS83 were grown in Middlebrook 7H9 medium with or without 50 ng/ml ATc. White bars: bacteria grown without ATc; grey bars: bacteria exposed to ATc for 6 h; black bars: bacteria exposed to ATc for 18 h. ( B ) β-galactosidase assay in M. tuberculosis in liquid media: TB38.1 and TB38.2 were grown in Middlebrook 7H9 medium with or without 200 ng/ml ATc. White bars: bacteria grown without ATc; black bars: bacteria exposed to ATc for 48 h. Results are expressed in Miller units.

    Journal: Nucleic Acids Research

    Article Title: Development of a repressible mycobacterial promoter system based on two transcriptional repressors

    doi: 10.1093/nar/gkq235

    Figure Lengend Snippet: Characterization of the TetR/Pip OFF system. ( A ) β-galactosidase assay in M. smegmatis in liquid media: MS82 and MS83 were grown in Middlebrook 7H9 medium with or without 50 ng/ml ATc. White bars: bacteria grown without ATc; grey bars: bacteria exposed to ATc for 6 h; black bars: bacteria exposed to ATc for 18 h. ( B ) β-galactosidase assay in M. tuberculosis in liquid media: TB38.1 and TB38.2 were grown in Middlebrook 7H9 medium with or without 200 ng/ml ATc. White bars: bacteria grown without ATc; black bars: bacteria exposed to ATc for 48 h. Results are expressed in Miller units.

    Article Snippet: Mycobacterial strains were grown at 37°C in Middlebrook 7H9 broth (Difco) in 150 ml roller bottles with slow rotation (3 rmp) or 7H10 agar plates (Difco), supplemented with 0.2% glycerol and 0.05% Tween-80.

    Techniques:

    Characterization of the M. tuberculosis fadD32 conditional mutant TB47. ( A ) Growth curve in the presence of different concentrations of ATc. TB47 was grown in Middlebrook 7H9 medium containing 200 ng/ml ATc (empty triangles), 100 ng/ml ATc (crosses), 50 ng/ml ATc (filled triangles) or No ATc (empty squares). ( B ) Viable counts variation during growth inhibition due to fadD32 depletion. TB47 was grown in Middlebrook 7H9 medium containing 200 ng/ml ATc. An aliquot of the bacterial culture was collected at different time points starting from 48 h after the beginning of the experiment, diluted and plated for cfu determination. Squares: cfu/ml; diamonds:optical density at 540 nm.

    Journal: Nucleic Acids Research

    Article Title: Development of a repressible mycobacterial promoter system based on two transcriptional repressors

    doi: 10.1093/nar/gkq235

    Figure Lengend Snippet: Characterization of the M. tuberculosis fadD32 conditional mutant TB47. ( A ) Growth curve in the presence of different concentrations of ATc. TB47 was grown in Middlebrook 7H9 medium containing 200 ng/ml ATc (empty triangles), 100 ng/ml ATc (crosses), 50 ng/ml ATc (filled triangles) or No ATc (empty squares). ( B ) Viable counts variation during growth inhibition due to fadD32 depletion. TB47 was grown in Middlebrook 7H9 medium containing 200 ng/ml ATc. An aliquot of the bacterial culture was collected at different time points starting from 48 h after the beginning of the experiment, diluted and plated for cfu determination. Squares: cfu/ml; diamonds:optical density at 540 nm.

    Article Snippet: Mycobacterial strains were grown at 37°C in Middlebrook 7H9 broth (Difco) in 150 ml roller bottles with slow rotation (3 rmp) or 7H10 agar plates (Difco), supplemented with 0.2% glycerol and 0.05% Tween-80.

    Techniques: Mutagenesis, Inhibition

    Real-time PCR of MAC strain 104 incubated in single metals. MAC was exposed to Middlebrook 7H9 broth containing the indicated volume of a 1 M concentration of the indicated metal per 500 ml of Middlebrook 7H9 broth for 24 h, and then RNA was extracted

    Journal: Infection and Immunity

    Article Title: Mimicry of the Pathogenic Mycobacterium Vacuole In Vitro Elicits the Bacterial Intracellular Phenotype, Including Early-Onset Macrophage Death ▿

    doi: 10.1128/IAI.01120-10

    Figure Lengend Snippet: Real-time PCR of MAC strain 104 incubated in single metals. MAC was exposed to Middlebrook 7H9 broth containing the indicated volume of a 1 M concentration of the indicated metal per 500 ml of Middlebrook 7H9 broth for 24 h, and then RNA was extracted

    Article Snippet: Five hundred milliliters of Middlebrook 7H9 broth (Difco, Becton-Dickinson, Sparks, MD) was prepared as usual, and then the amounts of the chemicals or supplements listed in were added to make a mixture that mimicked the vacuole 1 h after infection (1-h elemental mixture) and a mixture matching the vacuole 1 day after infection (24-h elemental mixture).

    Techniques: Real-time Polymerase Chain Reaction, Incubation, Concentration Assay

    Uptake of Mycobacterium avium complex (MAC) incubated in the elemental mixture by macrophages. (A) MAC was incubated in Middlebrook 7H9 broth, the 1-h elemental mixture, and the 24-h elemental mixture for 1 h and 24 h, respectively, and then used to infect

    Journal: Infection and Immunity

    Article Title: Mimicry of the Pathogenic Mycobacterium Vacuole In Vitro Elicits the Bacterial Intracellular Phenotype, Including Early-Onset Macrophage Death ▿

    doi: 10.1128/IAI.01120-10

    Figure Lengend Snippet: Uptake of Mycobacterium avium complex (MAC) incubated in the elemental mixture by macrophages. (A) MAC was incubated in Middlebrook 7H9 broth, the 1-h elemental mixture, and the 24-h elemental mixture for 1 h and 24 h, respectively, and then used to infect

    Article Snippet: Five hundred milliliters of Middlebrook 7H9 broth (Difco, Becton-Dickinson, Sparks, MD) was prepared as usual, and then the amounts of the chemicals or supplements listed in were added to make a mixture that mimicked the vacuole 1 h after infection (1-h elemental mixture) and a mixture matching the vacuole 1 day after infection (24-h elemental mixture).

    Techniques: Incubation

    Real-time PCR of MAC strain 104 in the subtracted 24-h elemental mixtures. Total bacterial RNA was extracted from bacteria exposed to the indicated mixture for 24 h and bacteria exposed to Middlebrook 7H9 broth as a reference. cDNA was made from the RNA

    Journal: Infection and Immunity

    Article Title: Mimicry of the Pathogenic Mycobacterium Vacuole In Vitro Elicits the Bacterial Intracellular Phenotype, Including Early-Onset Macrophage Death ▿

    doi: 10.1128/IAI.01120-10

    Figure Lengend Snippet: Real-time PCR of MAC strain 104 in the subtracted 24-h elemental mixtures. Total bacterial RNA was extracted from bacteria exposed to the indicated mixture for 24 h and bacteria exposed to Middlebrook 7H9 broth as a reference. cDNA was made from the RNA

    Article Snippet: Five hundred milliliters of Middlebrook 7H9 broth (Difco, Becton-Dickinson, Sparks, MD) was prepared as usual, and then the amounts of the chemicals or supplements listed in were added to make a mixture that mimicked the vacuole 1 h after infection (1-h elemental mixture) and a mixture matching the vacuole 1 day after infection (24-h elemental mixture).

    Techniques: Real-time Polymerase Chain Reaction

    Real-time PCR of MAC strain 104 exposed to the 24-h elemental mixture. Total bacterial RNA was extracted from MAC strain 104 exposed to the 24-h elemental mixture for 24 h and bacteria exposed to Middlebrook 7H9 broth as a control. cDNA was made from

    Journal: Infection and Immunity

    Article Title: Mimicry of the Pathogenic Mycobacterium Vacuole In Vitro Elicits the Bacterial Intracellular Phenotype, Including Early-Onset Macrophage Death ▿

    doi: 10.1128/IAI.01120-10

    Figure Lengend Snippet: Real-time PCR of MAC strain 104 exposed to the 24-h elemental mixture. Total bacterial RNA was extracted from MAC strain 104 exposed to the 24-h elemental mixture for 24 h and bacteria exposed to Middlebrook 7H9 broth as a control. cDNA was made from

    Article Snippet: Five hundred milliliters of Middlebrook 7H9 broth (Difco, Becton-Dickinson, Sparks, MD) was prepared as usual, and then the amounts of the chemicals or supplements listed in were added to make a mixture that mimicked the vacuole 1 h after infection (1-h elemental mixture) and a mixture matching the vacuole 1 day after infection (24-h elemental mixture).

    Techniques: Real-time Polymerase Chain Reaction

    MALDI-TOF mass spectra of MAMEs from various strains of M. tuberculosis. M. tuberculosis , MtbΔ virS , Mtb mym :: hyg , and MtbΔ virS _ virS strains were grown in MB 7H9 medium at pH 7.0 or exposed to pH 5.0 for 12 h. MAMEs were extracted and subjected to MALDI-TOF analysis as described in Materials and Methods. MALDI-TOF mass spectra of MAMEs from M. tuberculosis (A and E), MtbΔ virS (B and F), Mtb mym :: hyg (C and G), and MtbΔ virS _ virS (D and H) are shown. The psuedomolecular masses of mycolates are indicated on each mass spectrum. Arrows and brackets indicate the major differences observed in MALDI-TOF analysis of MAMEs from various strains.

    Journal: Journal of Bacteriology

    Article Title: Requirement of the mymA Operon for Appropriate Cell Wall Ultrastructure and Persistence of Mycobacterium tuberculosis in the Spleens of Guinea Pigs

    doi: 10.1128/JB.187.12.4173-4186.2005

    Figure Lengend Snippet: MALDI-TOF mass spectra of MAMEs from various strains of M. tuberculosis. M. tuberculosis , MtbΔ virS , Mtb mym :: hyg , and MtbΔ virS _ virS strains were grown in MB 7H9 medium at pH 7.0 or exposed to pH 5.0 for 12 h. MAMEs were extracted and subjected to MALDI-TOF analysis as described in Materials and Methods. MALDI-TOF mass spectra of MAMEs from M. tuberculosis (A and E), MtbΔ virS (B and F), Mtb mym :: hyg (C and G), and MtbΔ virS _ virS (D and H) are shown. The psuedomolecular masses of mycolates are indicated on each mass spectrum. Arrows and brackets indicate the major differences observed in MALDI-TOF analysis of MAMEs from various strains.

    Article Snippet: M. tuberculosis Erdman, the parental strain, mutant strains (MtbΔ virS and Mtb mym :: hyg ) and a virS -complemented strain (MtbΔ virS _ virS ) were grown in Middlebrook (MB) 7H9 broth (Difco Laboratories) supplemented with 0.5% glycerol, 0.2% Tween 80, and 1× ADC (albumin-dextrose complex; Difco Laboratories) or MB 7H10 medium (Difco Laboratories) supplemented with 1× OADC (oleic acid-albumin-dextrose complex; Difco Laboratories).

    Techniques:

    Effect of mymA disruption on the cell wall ultrastructure of M. tuberculosis . The cell wall of M. tuberculosis and Mtb mym :: hyg was examined by transmission electron microscopy. Cells were cultured in MB 7H9 medium to an A 600 ). Shown is electron microscopic analysis (×44,000) of (A) M. tuberculosis and (B) Mtb mym :: hyg . The photomicrographs shown are typical of the population as a whole, as judged by viewing many independent fields.

    Journal: Journal of Bacteriology

    Article Title: Requirement of the mymA Operon for Appropriate Cell Wall Ultrastructure and Persistence of Mycobacterium tuberculosis in the Spleens of Guinea Pigs

    doi: 10.1128/JB.187.12.4173-4186.2005

    Figure Lengend Snippet: Effect of mymA disruption on the cell wall ultrastructure of M. tuberculosis . The cell wall of M. tuberculosis and Mtb mym :: hyg was examined by transmission electron microscopy. Cells were cultured in MB 7H9 medium to an A 600 ). Shown is electron microscopic analysis (×44,000) of (A) M. tuberculosis and (B) Mtb mym :: hyg . The photomicrographs shown are typical of the population as a whole, as judged by viewing many independent fields.

    Article Snippet: M. tuberculosis Erdman, the parental strain, mutant strains (MtbΔ virS and Mtb mym :: hyg ) and a virS -complemented strain (MtbΔ virS _ virS ) were grown in Middlebrook (MB) 7H9 broth (Difco Laboratories) supplemented with 0.5% glycerol, 0.2% Tween 80, and 1× ADC (albumin-dextrose complex; Difco Laboratories) or MB 7H10 medium (Difco Laboratories) supplemented with 1× OADC (oleic acid-albumin-dextrose complex; Difco Laboratories).

    Techniques: Transmission Assay, Electron Microscopy, Cell Culture

    Representative HPLC chromatograms of mycolic acids from various strains of M. tuberculosis. M. tuberculosis MtbΔ virS , Mtb mym :: hyg , and MtbΔ virS _ virS strains were inoculated separately in MB 7H9 medium and grown to an A 600 of 1.5. Total fatty acids were extracted, derivatized to UV-absorbing p -bromophenacyl esters, and separated by HPLC as described in Materials and Methods. Shown are mycolic acid profiles of (A) M. tuberculosis , (B) MtbΔ virS , (C) Mtb mym :: hyg , and (D) MtbΔ virS _ virS from HPLC analysis. Peaks were labeled according to their RRTs. IS, internal high-molecular-weight standard. Arrows indicate the significant differences in the mycolic peaks between various strains.

    Journal: Journal of Bacteriology

    Article Title: Requirement of the mymA Operon for Appropriate Cell Wall Ultrastructure and Persistence of Mycobacterium tuberculosis in the Spleens of Guinea Pigs

    doi: 10.1128/JB.187.12.4173-4186.2005

    Figure Lengend Snippet: Representative HPLC chromatograms of mycolic acids from various strains of M. tuberculosis. M. tuberculosis MtbΔ virS , Mtb mym :: hyg , and MtbΔ virS _ virS strains were inoculated separately in MB 7H9 medium and grown to an A 600 of 1.5. Total fatty acids were extracted, derivatized to UV-absorbing p -bromophenacyl esters, and separated by HPLC as described in Materials and Methods. Shown are mycolic acid profiles of (A) M. tuberculosis , (B) MtbΔ virS , (C) Mtb mym :: hyg , and (D) MtbΔ virS _ virS from HPLC analysis. Peaks were labeled according to their RRTs. IS, internal high-molecular-weight standard. Arrows indicate the significant differences in the mycolic peaks between various strains.

    Article Snippet: M. tuberculosis Erdman, the parental strain, mutant strains (MtbΔ virS and Mtb mym :: hyg ) and a virS -complemented strain (MtbΔ virS _ virS ) were grown in Middlebrook (MB) 7H9 broth (Difco Laboratories) supplemented with 0.5% glycerol, 0.2% Tween 80, and 1× ADC (albumin-dextrose complex; Difco Laboratories) or MB 7H10 medium (Difco Laboratories) supplemented with 1× OADC (oleic acid-albumin-dextrose complex; Difco Laboratories).

    Techniques: High Performance Liquid Chromatography, Labeling, Molecular Weight

    Thin-layer chromatography of FAMEs and MAMEs from various strains of M. tuberculosis. M. tuberculosis , MtbΔ virS , Mtb mym :: hyg , and MtbΔ virS _ virS strains were grown to an A 600 of 1.5 in MB 7H9 medium, adjusted either to pH 7.0 or to pH 5.0, and labeled with 1 μCi/ml [1,2- 14 C]acetate for 12 h. Labeled lipids were extracted, derivatized to methyl esters, and resolved by normal phase TLC using petroleum ether/diethyl ether (17:3). Equal counts (200,000 cpm) were loaded in each lane, and a phosphorimager was used to quantify the proportion of FAMEs and MAMEs derived from various strains of M. tuberculosis . Shown are TLC profiles of total fatty acid esters (FAMEs and MAMEs) extracted from M. tuberculosis , MtbΔ virS , Mtb mym :: hyg , and MtbΔ virS _ virS strains growing at (A) neutral pH (7.0) or (B) acidic pH (5.0). These experiments were repeated four times, and similar results were obtained.

    Journal: Journal of Bacteriology

    Article Title: Requirement of the mymA Operon for Appropriate Cell Wall Ultrastructure and Persistence of Mycobacterium tuberculosis in the Spleens of Guinea Pigs

    doi: 10.1128/JB.187.12.4173-4186.2005

    Figure Lengend Snippet: Thin-layer chromatography of FAMEs and MAMEs from various strains of M. tuberculosis. M. tuberculosis , MtbΔ virS , Mtb mym :: hyg , and MtbΔ virS _ virS strains were grown to an A 600 of 1.5 in MB 7H9 medium, adjusted either to pH 7.0 or to pH 5.0, and labeled with 1 μCi/ml [1,2- 14 C]acetate for 12 h. Labeled lipids were extracted, derivatized to methyl esters, and resolved by normal phase TLC using petroleum ether/diethyl ether (17:3). Equal counts (200,000 cpm) were loaded in each lane, and a phosphorimager was used to quantify the proportion of FAMEs and MAMEs derived from various strains of M. tuberculosis . Shown are TLC profiles of total fatty acid esters (FAMEs and MAMEs) extracted from M. tuberculosis , MtbΔ virS , Mtb mym :: hyg , and MtbΔ virS _ virS strains growing at (A) neutral pH (7.0) or (B) acidic pH (5.0). These experiments were repeated four times, and similar results were obtained.

    Article Snippet: M. tuberculosis Erdman, the parental strain, mutant strains (MtbΔ virS and Mtb mym :: hyg ) and a virS -complemented strain (MtbΔ virS _ virS ) were grown in Middlebrook (MB) 7H9 broth (Difco Laboratories) supplemented with 0.5% glycerol, 0.2% Tween 80, and 1× ADC (albumin-dextrose complex; Difco Laboratories) or MB 7H10 medium (Difco Laboratories) supplemented with 1× OADC (oleic acid-albumin-dextrose complex; Difco Laboratories).

    Techniques: Thin Layer Chromatography, Labeling, Derivative Assay

    Effect of AU1235 on mycolic acid biosynthesis and transfer in M. tb M. tb H37Ra cultured in 7H9-OADC-Tween 80 broth was treated for 5 hr at 37°C with either no inhibitor or with AU1235 at a concentration of 0.05 μg ml −1 , 0.5 μg ml −1 , or 1 μg ml −1 (0.5x to 10x MIC). [ 14 C]-acetate was added to the cultures at the same time as the inhibitor. ( a ) Bacterial cells (B) and culture filtrates (CF) were collected and the lipids contained in each of these fractions extracted as described under the Methods section. The same volume of samples was loaded per lane. The TLC was developed in the solvent system [chloroform:methanol:water] (20:4:0.5, by vol.) and revealed by autoradiography. PE, phosphatidylethanolamine; CL, cardiolipin. ( b ) Mycolic acid methyl esters were prepared from delipidated cells as described 48 . α–, methoxy- and keto- denote the three forms of mycolic acids produced by M. tb . The same volume of samples was loaded per lane. The TLC was developed thrice in the solvent system [ n -hexanes:ethyl acetate] (95:5, by vol.) and revealed by autoradiography. The amount of radioactivity incorporated in the products of interest was semi-quantified using a PhosphoImager and the results (expressed as a % of the value measured in the untreated control) are presented as histograms under their corresponding autoradiograms.

    Journal: Nature chemical biology

    Article Title: INHIBITION OF MYCOLIC ACID TRANSPORT ACROSS THE MYCOBACTERIUM TUBERCULOSIS PLASMA MEMBRANE

    doi: 10.1038/nchembio.794

    Figure Lengend Snippet: Effect of AU1235 on mycolic acid biosynthesis and transfer in M. tb M. tb H37Ra cultured in 7H9-OADC-Tween 80 broth was treated for 5 hr at 37°C with either no inhibitor or with AU1235 at a concentration of 0.05 μg ml −1 , 0.5 μg ml −1 , or 1 μg ml −1 (0.5x to 10x MIC). [ 14 C]-acetate was added to the cultures at the same time as the inhibitor. ( a ) Bacterial cells (B) and culture filtrates (CF) were collected and the lipids contained in each of these fractions extracted as described under the Methods section. The same volume of samples was loaded per lane. The TLC was developed in the solvent system [chloroform:methanol:water] (20:4:0.5, by vol.) and revealed by autoradiography. PE, phosphatidylethanolamine; CL, cardiolipin. ( b ) Mycolic acid methyl esters were prepared from delipidated cells as described 48 . α–, methoxy- and keto- denote the three forms of mycolic acids produced by M. tb . The same volume of samples was loaded per lane. The TLC was developed thrice in the solvent system [ n -hexanes:ethyl acetate] (95:5, by vol.) and revealed by autoradiography. The amount of radioactivity incorporated in the products of interest was semi-quantified using a PhosphoImager and the results (expressed as a % of the value measured in the untreated control) are presented as histograms under their corresponding autoradiograms.

    Article Snippet: Strains and growth conditions M. tb H37Rv TMC102 and ATCC 25618 and M. tb H37Ra ATCC 25177 were grown in Middlebrook 7H9-OADC broth (Difco) supplemented with 0.05% Tween 80 and on 7H11-OADC agar (Difco) at 37°C.

    Techniques: Cell Culture, Concentration Assay, Thin Layer Chromatography, Autoradiography, Produced, Radioactivity

    Structure and bactericidal activity of AU1235 ( a ) Structure of AU1235 ( 1 ) [1-(2-adamantyl)-3-(2,3,4-trifluorophenyl)urea] ( b ) Kill-kinetics showing decrease in viability of M. tb H37Rv in 7H9-OADC-Tween 80 medium containing AU1235 at 0.5 and 1 μg ml −1 (5 and 10 × MIC). The results for each drug concentration are representative of at least two independent experiments.

    Journal: Nature chemical biology

    Article Title: INHIBITION OF MYCOLIC ACID TRANSPORT ACROSS THE MYCOBACTERIUM TUBERCULOSIS PLASMA MEMBRANE

    doi: 10.1038/nchembio.794

    Figure Lengend Snippet: Structure and bactericidal activity of AU1235 ( a ) Structure of AU1235 ( 1 ) [1-(2-adamantyl)-3-(2,3,4-trifluorophenyl)urea] ( b ) Kill-kinetics showing decrease in viability of M. tb H37Rv in 7H9-OADC-Tween 80 medium containing AU1235 at 0.5 and 1 μg ml −1 (5 and 10 × MIC). The results for each drug concentration are representative of at least two independent experiments.

    Article Snippet: Strains and growth conditions M. tb H37Rv TMC102 and ATCC 25618 and M. tb H37Ra ATCC 25177 were grown in Middlebrook 7H9-OADC broth (Difco) supplemented with 0.05% Tween 80 and on 7H11-OADC agar (Difco) at 37°C.

    Techniques: Activity Assay, Concentration Assay

    mmpL3 is an essential gene of M. smegmatis ( a ) Evidence for allelic replacement at the mmpL3 locus of M. smegmatis mc 2 155 in the presence of a rescue copy of mmpL3tb expressed from an episomal plasmid. Allelic exchange mutants were rescued with the mmpL3tb gene from M. tb expressed from either pMVGH1- mmpL3tb , pMVGH1- mmpL3tb-G253E or pSETetR- mmpL3tb . Allelic replacement was confirmed by PCR as described under the Methods section. The wild-type (WT) 3,752-bp amplification signal is replaced by a 3,358-bp fragment in the rescued mutants (r-mut) due to the 1,594-bp NotI deletion in the mmpL3 gene and insertion of a 1.2 kb-kanamycin resistance cassette. Only the profiles of two mc 2 155 ΔmmpL3 /pMVGH1- mmpL3tb strains are shown here as identical profiles were obtained will all rescued mutant forms. See Supplementary Figure 9 for the image of the full uncut gel. ( b ) Growth characteristics of wild-type mc 2 155 (circles) and the conditional mutant, mc 2 155 ΔmmpL3 /pSETetR- mmpL3tb (squares), in 7H9-OADC-Tween 80 broth containing either no inducer (open symbols) or 50 ng ml −1 of anhydro-tetracycline (ATc) (full symbols) at 37°C. Reducing ATc concentration in the medium leads to the repression of mmpL3tb in the conditional mutant.

    Journal: Nature chemical biology

    Article Title: INHIBITION OF MYCOLIC ACID TRANSPORT ACROSS THE MYCOBACTERIUM TUBERCULOSIS PLASMA MEMBRANE

    doi: 10.1038/nchembio.794

    Figure Lengend Snippet: mmpL3 is an essential gene of M. smegmatis ( a ) Evidence for allelic replacement at the mmpL3 locus of M. smegmatis mc 2 155 in the presence of a rescue copy of mmpL3tb expressed from an episomal plasmid. Allelic exchange mutants were rescued with the mmpL3tb gene from M. tb expressed from either pMVGH1- mmpL3tb , pMVGH1- mmpL3tb-G253E or pSETetR- mmpL3tb . Allelic replacement was confirmed by PCR as described under the Methods section. The wild-type (WT) 3,752-bp amplification signal is replaced by a 3,358-bp fragment in the rescued mutants (r-mut) due to the 1,594-bp NotI deletion in the mmpL3 gene and insertion of a 1.2 kb-kanamycin resistance cassette. Only the profiles of two mc 2 155 ΔmmpL3 /pMVGH1- mmpL3tb strains are shown here as identical profiles were obtained will all rescued mutant forms. See Supplementary Figure 9 for the image of the full uncut gel. ( b ) Growth characteristics of wild-type mc 2 155 (circles) and the conditional mutant, mc 2 155 ΔmmpL3 /pSETetR- mmpL3tb (squares), in 7H9-OADC-Tween 80 broth containing either no inducer (open symbols) or 50 ng ml −1 of anhydro-tetracycline (ATc) (full symbols) at 37°C. Reducing ATc concentration in the medium leads to the repression of mmpL3tb in the conditional mutant.

    Article Snippet: Strains and growth conditions M. tb H37Rv TMC102 and ATCC 25618 and M. tb H37Ra ATCC 25177 were grown in Middlebrook 7H9-OADC broth (Difco) supplemented with 0.05% Tween 80 and on 7H11-OADC agar (Difco) at 37°C.

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Mutagenesis, Concentration Assay