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MiddleBrook Pharmaceuticals agar 7h10 medium
Phenotypic characterization of BCG.HIVA 2auxo.int strain. We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA 2auxo.int strain (A) BCG lysine auxotroph strain plated on non-lysine supplemented <t>7H10;</t> (B) BCG lysine auxotroph strain plated on lysine supplemented 7H10; (C) BCG.HIVA 2auxo.int plated on 7H10 without lysine and kanamycin supplementation; (D) BCG.HIVA 2auxo.int plated on 7H10 without lysine supplementation and with kanamycin.
Agar 7h10 Medium, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Preclinical development of BCG.HIVA2auxo.int, harboring an integrative expression vector, for a HIV-TB Pediatric vaccine. Enhancement of stability and specific HIV-1 T-cell immunity"

Article Title: Preclinical development of BCG.HIVA2auxo.int, harboring an integrative expression vector, for a HIV-TB Pediatric vaccine. Enhancement of stability and specific HIV-1 T-cell immunity

Journal: Human Vaccines & Immunotherapeutics

doi: 10.1080/21645515.2017.1316911

Phenotypic characterization of BCG.HIVA 2auxo.int strain. We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA 2auxo.int strain (A) BCG lysine auxotroph strain plated on non-lysine supplemented 7H10; (B) BCG lysine auxotroph strain plated on lysine supplemented 7H10; (C) BCG.HIVA 2auxo.int plated on 7H10 without lysine and kanamycin supplementation; (D) BCG.HIVA 2auxo.int plated on 7H10 without lysine supplementation and with kanamycin.
Figure Legend Snippet: Phenotypic characterization of BCG.HIVA 2auxo.int strain. We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA 2auxo.int strain (A) BCG lysine auxotroph strain plated on non-lysine supplemented 7H10; (B) BCG lysine auxotroph strain plated on lysine supplemented 7H10; (C) BCG.HIVA 2auxo.int plated on 7H10 without lysine and kanamycin supplementation; (D) BCG.HIVA 2auxo.int plated on 7H10 without lysine supplementation and with kanamycin.

Techniques Used:

2) Product Images from "Preclinical development of BCG.HIVA2auxo.int, harboring an integrative expression vector, for a HIV-TB Pediatric vaccine. Enhancement of stability and specific HIV-1 T-cell immunity"

Article Title: Preclinical development of BCG.HIVA2auxo.int, harboring an integrative expression vector, for a HIV-TB Pediatric vaccine. Enhancement of stability and specific HIV-1 T-cell immunity

Journal: Human Vaccines & Immunotherapeutics

doi: 10.1080/21645515.2017.1316911

Phenotypic characterization of BCG.HIVA 2auxo.int strain. We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA 2auxo.int strain (A) BCG lysine auxotroph strain plated on non-lysine supplemented 7H10; (B) BCG lysine auxotroph strain plated on lysine supplemented 7H10; (C) BCG.HIVA 2auxo.int plated on 7H10 without lysine and kanamycin supplementation; (D) BCG.HIVA 2auxo.int plated on 7H10 without lysine supplementation and with kanamycin.
Figure Legend Snippet: Phenotypic characterization of BCG.HIVA 2auxo.int strain. We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA 2auxo.int strain (A) BCG lysine auxotroph strain plated on non-lysine supplemented 7H10; (B) BCG lysine auxotroph strain plated on lysine supplemented 7H10; (C) BCG.HIVA 2auxo.int plated on 7H10 without lysine and kanamycin supplementation; (D) BCG.HIVA 2auxo.int plated on 7H10 without lysine supplementation and with kanamycin.

Techniques Used:

3) Product Images from "Pre-Clinical Development of BCG.HIVACAT, an Antibiotic-Free Selection Strain, for HIV-TB Pediatric Vaccine Vectored by Lysine Auxotroph of BCG"

Article Title: Pre-Clinical Development of BCG.HIVACAT, an Antibiotic-Free Selection Strain, for HIV-TB Pediatric Vaccine Vectored by Lysine Auxotroph of BCG

Journal: PLoS ONE

doi: 10.1371/journal.pone.0042559

Phenotypic characterization of the BCG.HIVA CAT . We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA CAT strain. ( A ) BCG lysine auxotroph strain plated on non-lysine supplemented 7H10; ( B ) BCG lysine auxotroph strain plated on lysine supplemented 7H10; ( C ) BCG.HIVA CAT plated on 7H10 without lysine and kanamycin supplementation; ( D ) BCG.HIVA CAT plated on 7H10 without lysine supplementation and with kanamycin.
Figure Legend Snippet: Phenotypic characterization of the BCG.HIVA CAT . We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA CAT strain. ( A ) BCG lysine auxotroph strain plated on non-lysine supplemented 7H10; ( B ) BCG lysine auxotroph strain plated on lysine supplemented 7H10; ( C ) BCG.HIVA CAT plated on 7H10 without lysine and kanamycin supplementation; ( D ) BCG.HIVA CAT plated on 7H10 without lysine supplementation and with kanamycin.

Techniques Used:

4) Product Images from "MTBVAC-Based TB-HIV Vaccine Is Safe, Elicits HIV-T Cell Responses, and Protects against Mycobacterium tuberculosis in Mice"

Article Title: MTBVAC-Based TB-HIV Vaccine Is Safe, Elicits HIV-T Cell Responses, and Protects against Mycobacterium tuberculosis in Mice

Journal: Molecular Therapy. Methods & Clinical Development

doi: 10.1016/j.omtm.2019.01.014

Construction of MTBVAC.HIVA 2auxo (A) The HIVA gene was fused to the region encoding the 19-kDa lipoprotein signal sequence of the episomal p2auxo.Ø E. coli -mycobacterial shuttle plasmid to obtain p2auxo.HIVA plasmid. The BALB/c mouse T cell and Mab-Pk epitopes used in this study are depicted. P α-Ag ( M. tuberculosis α-antigen promoter), PHSP60 (heat shock protein 60 gene promoter), and glyA - and lysA -complementing genes are used as markers for selection and maintenance in E. coli M15Δ Gly and MTBVACΔ lys , respectively. (B) Phenotypic characterization of the lysine auxotrophy and plasmid complementation of MTBVACΔ lys and MTBVAC.HIVA 2auxo (MTBVACΔ lys plated on lysine-supplemented 7H10, left; MTBVACΔ lys plated on non-lysine-supplemented 7H10, center; and MTBVAC.HIVA 2auxo plated on non-lysine-supplemented 7H10, right). (C) Western blot of MTBVACHIVA 2auxo lysates. Lanes 1 and 2, MTBVAC.Ø 2auxo clones 1 and 2; lanes 3 and 5, MTBVAC.HIVA 2auxo ; lane 6, BCG wild-type lysate (negative control). HIVA immunogen was detected using the anti-Pk monoclonal antibodies (mAbs) followed by horseradish peroxidase-protein A and enhanced chemiluminescence detection.
Figure Legend Snippet: Construction of MTBVAC.HIVA 2auxo (A) The HIVA gene was fused to the region encoding the 19-kDa lipoprotein signal sequence of the episomal p2auxo.Ø E. coli -mycobacterial shuttle plasmid to obtain p2auxo.HIVA plasmid. The BALB/c mouse T cell and Mab-Pk epitopes used in this study are depicted. P α-Ag ( M. tuberculosis α-antigen promoter), PHSP60 (heat shock protein 60 gene promoter), and glyA - and lysA -complementing genes are used as markers for selection and maintenance in E. coli M15Δ Gly and MTBVACΔ lys , respectively. (B) Phenotypic characterization of the lysine auxotrophy and plasmid complementation of MTBVACΔ lys and MTBVAC.HIVA 2auxo (MTBVACΔ lys plated on lysine-supplemented 7H10, left; MTBVACΔ lys plated on non-lysine-supplemented 7H10, center; and MTBVAC.HIVA 2auxo plated on non-lysine-supplemented 7H10, right). (C) Western blot of MTBVACHIVA 2auxo lysates. Lanes 1 and 2, MTBVAC.Ø 2auxo clones 1 and 2; lanes 3 and 5, MTBVAC.HIVA 2auxo ; lane 6, BCG wild-type lysate (negative control). HIVA immunogen was detected using the anti-Pk monoclonal antibodies (mAbs) followed by horseradish peroxidase-protein A and enhanced chemiluminescence detection.

Techniques Used: Sequencing, Plasmid Preparation, Selection, Western Blot, Clone Assay, Negative Control

Genetic Stability of p2auxo.HIVA Plasmid DNA (A) In vitro . Serial passages of the working vaccine stock (WVS) were performed weekly (+1 to +6), and HIVA PCRs were used to check stability of the plasmid DNA. Lane 1, WVS MTBVAC.HIVA 2auxo ; lanes 2–4, passages +4, +5, and +6 WVS MTBVAC.HIVA 2auxo ; lane 5, H 2 0 (negative control); lane 6, positive control; lane 7, molecular weight marker. (B) In vivo . Spleens from SCID mice inoculated with 10 6 CFU MTBVAC.HIVA 2auxo and used for safety experiments were harvested and plated on complete 7H10 supplemented with Lys and Km. The presence of p2auxo.HIVA plasmid in the colonies from these mice was analyzed by specific PCR using the pairs of primers to detect HIVA (19kDss-fw/HIVA-rv). Each number represents one colony and numbers with # symbol indicate colonies from the same animal. Minus and plus symbols indicate negative and positive controls of PCR, respectively. Plasmid maintained in vivo was calculated as the percent of positive colonies with respect to total colonies analyzed.
Figure Legend Snippet: Genetic Stability of p2auxo.HIVA Plasmid DNA (A) In vitro . Serial passages of the working vaccine stock (WVS) were performed weekly (+1 to +6), and HIVA PCRs were used to check stability of the plasmid DNA. Lane 1, WVS MTBVAC.HIVA 2auxo ; lanes 2–4, passages +4, +5, and +6 WVS MTBVAC.HIVA 2auxo ; lane 5, H 2 0 (negative control); lane 6, positive control; lane 7, molecular weight marker. (B) In vivo . Spleens from SCID mice inoculated with 10 6 CFU MTBVAC.HIVA 2auxo and used for safety experiments were harvested and plated on complete 7H10 supplemented with Lys and Km. The presence of p2auxo.HIVA plasmid in the colonies from these mice was analyzed by specific PCR using the pairs of primers to detect HIVA (19kDss-fw/HIVA-rv). Each number represents one colony and numbers with # symbol indicate colonies from the same animal. Minus and plus symbols indicate negative and positive controls of PCR, respectively. Plasmid maintained in vivo was calculated as the percent of positive colonies with respect to total colonies analyzed.

Techniques Used: Plasmid Preparation, In Vitro, Negative Control, Positive Control, Molecular Weight, Marker, In Vivo, Mouse Assay, Polymerase Chain Reaction

Construction and Characterization of MTBVACΔ lys Strain (A) lysA gene (gray arrow) from MTBVAC was inactivated using homologous recombination techniques by introducing a kanamycin resistance cassette (gray rectangle), flanked by two resolvase sites (white arrowheads) in order to allow the release of the resistance cassette. The inactivated phoP and fadD2 6 genes in the MTBVAC parental strain are also illustrated. (B) Genotypic characterization of the lysA gene inactivation by the kanamycin cassette (km) insertion, primers used, and expected sizes of the PCR products are indicated. MTBVAC sample was used in lanes 3, 5, and 7 and MTBVACΔ lys samples in lanes 4, 6, and 8. Lane 1, molecular weight marker; lane 2, negative control; lanes 3 and 4, PCR product of the lysA gene using Lys-fw and Lys-rv primers; lanes 5 and 6, PCR of the 5′ insertion point of km expression cassette using Lys-fw and km-OUT-rv primers; and lanes 7 and 8, PCR of the 3′ insertion point of km expression cassette using km-OUT-fw and Lys-rv primers. Genes are represented as gray arrows; gray rectangles illustrate antibiotic resistance markers and white arrowheads depict resolvase recognition sequences or res sites. (C) Phenotypic lysine auxotrophic verification by plating MTBVACΔ lys strain in 7H10-ADC with and without lysine supplementation.
Figure Legend Snippet: Construction and Characterization of MTBVACΔ lys Strain (A) lysA gene (gray arrow) from MTBVAC was inactivated using homologous recombination techniques by introducing a kanamycin resistance cassette (gray rectangle), flanked by two resolvase sites (white arrowheads) in order to allow the release of the resistance cassette. The inactivated phoP and fadD2 6 genes in the MTBVAC parental strain are also illustrated. (B) Genotypic characterization of the lysA gene inactivation by the kanamycin cassette (km) insertion, primers used, and expected sizes of the PCR products are indicated. MTBVAC sample was used in lanes 3, 5, and 7 and MTBVACΔ lys samples in lanes 4, 6, and 8. Lane 1, molecular weight marker; lane 2, negative control; lanes 3 and 4, PCR product of the lysA gene using Lys-fw and Lys-rv primers; lanes 5 and 6, PCR of the 5′ insertion point of km expression cassette using Lys-fw and km-OUT-rv primers; and lanes 7 and 8, PCR of the 3′ insertion point of km expression cassette using km-OUT-fw and Lys-rv primers. Genes are represented as gray arrows; gray rectangles illustrate antibiotic resistance markers and white arrowheads depict resolvase recognition sequences or res sites. (C) Phenotypic lysine auxotrophic verification by plating MTBVACΔ lys strain in 7H10-ADC with and without lysine supplementation.

Techniques Used: Homologous Recombination, Polymerase Chain Reaction, Molecular Weight, Marker, Negative Control, Expressing

5) Product Images from "Pre-Clinical Development of BCG.HIVACAT, an Antibiotic-Free Selection Strain, for HIV-TB Pediatric Vaccine Vectored by Lysine Auxotroph of BCG"

Article Title: Pre-Clinical Development of BCG.HIVACAT, an Antibiotic-Free Selection Strain, for HIV-TB Pediatric Vaccine Vectored by Lysine Auxotroph of BCG

Journal: PLoS ONE

doi: 10.1371/journal.pone.0042559

Phenotypic characterization of the BCG.HIVA CAT . We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA CAT strain. ( A ) BCG lysine auxotroph strain plated on non-lysine supplemented 7H10; ( B ) BCG lysine auxotroph strain plated on lysine supplemented 7H10; ( C ) BCG.HIVA CAT plated on 7H10 without lysine and kanamycin supplementation; ( D ) BCG.HIVA CAT plated on 7H10 without lysine supplementation and with kanamycin.
Figure Legend Snippet: Phenotypic characterization of the BCG.HIVA CAT . We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA CAT strain. ( A ) BCG lysine auxotroph strain plated on non-lysine supplemented 7H10; ( B ) BCG lysine auxotroph strain plated on lysine supplemented 7H10; ( C ) BCG.HIVA CAT plated on 7H10 without lysine and kanamycin supplementation; ( D ) BCG.HIVA CAT plated on 7H10 without lysine supplementation and with kanamycin.

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    MiddleBrook Pharmaceuticals agar 7h10 medium
    Phenotypic characterization of BCG.HIVA 2auxo.int strain. We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA 2auxo.int strain (A) BCG lysine auxotroph strain plated on non-lysine supplemented <t>7H10;</t> (B) BCG lysine auxotroph strain plated on lysine supplemented 7H10; (C) BCG.HIVA 2auxo.int plated on 7H10 without lysine and kanamycin supplementation; (D) BCG.HIVA 2auxo.int plated on 7H10 without lysine supplementation and with kanamycin.
    Agar 7h10 Medium, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/agar 7h10 medium/product/MiddleBrook Pharmaceuticals
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    agar 7h10 medium - by Bioz Stars, 2022-05
    86/100 stars
      Buy from Supplier

    86
    MiddleBrook Pharmaceuticals 7h10 agar medium
    GreA is required for resistance to adverse stress. (A–E, H–J) Survival of M. smegmatis strains during adverse stress. The strain symbols listed in the (J) . WT, Δ greA and comp- Δ greA strains were treated for at the indicated time with varying concentrations of SDS (A) , 5mM H 2 O 2 (B) , PBS + 0.05% Tween 80 (C) , low pH (pH4.5) (D) , heat shock (48°C) (E) , 1 μg/ml of streptomycin (H) , 100 μg/ml of rifampicin (I) , or 2.5 μg/ml of bedaquiline (J) . After treatment, dilutions were plated on <t>7H10</t> and survival is expressed as a ratio of CFUs compared to untreated cultures. (F) M. tuberculosis H37Rv strains were spotted on the 7H10 plates after they were treated under 0.5% SDS for 24 h. (G) WT, Δ greA , and comp- Δ greA strains from M. tuberculosis H37Ra were subjected to 0.5% SDS for 8 h. CFU enumeration was performed after incubation for the indicated time. These experiments were performed three times with similar results. Data are shown as mean ± SEM of three independent experiments. * p
    7h10 Agar Medium, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/7h10 agar medium/product/MiddleBrook Pharmaceuticals
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    7h10 agar medium - by Bioz Stars, 2022-05
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    MiddleBrook Pharmaceuticals 7h9 broth medium
    PE_PGRS41 expression in M. smegmatis results in increased cell wall permeability. ( A ) Mid-log-phase cultures of Ms_Vec and Ms_PE_PGRS41 were incubated in <t>7H9</t> containing 2 μg/ml ethidium bromide for indicated time. ( B ) Mid-log-phase cultures of Ms_Vec and Ms_PE_PGRS41 were incubated in 7H9 with 2 μM Nile red stain for indicated time. ( C ) The growth of Ms_Vec and Ms_PE_PGRS41 after treatment with different pH gradient for indicated time. The Ms_Vec and Ms_PE_PGRS41 strains were centrifuged, re-suspended to 5 ml MB 7H9 at an OD 600 of 0.5, 10-fold serial dilutions of Ms_Vec and Ms_PE_PGRS41 were spotted on MB 7H10. ( D ) Mid-log-phase cultures of Ms_Vec and Ms_PE_PGRS41 were incubated in MB 7H9 supplemented with 0.05% SDS for indicated time. And then the recombinant strains were plated onto 7H10 plates by serially ten-fold dilution, the bacterial numbers were counted after 3–4 days of cultivation.
    7h9 Broth Medium, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/7h9 broth medium/product/MiddleBrook Pharmaceuticals
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    Phenotypic characterization of BCG.HIVA 2auxo.int strain. We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA 2auxo.int strain (A) BCG lysine auxotroph strain plated on non-lysine supplemented 7H10; (B) BCG lysine auxotroph strain plated on lysine supplemented 7H10; (C) BCG.HIVA 2auxo.int plated on 7H10 without lysine and kanamycin supplementation; (D) BCG.HIVA 2auxo.int plated on 7H10 without lysine supplementation and with kanamycin.

    Journal: Human Vaccines & Immunotherapeutics

    Article Title: Preclinical development of BCG.HIVA2auxo.int, harboring an integrative expression vector, for a HIV-TB Pediatric vaccine. Enhancement of stability and specific HIV-1 T-cell immunity

    doi: 10.1080/21645515.2017.1316911

    Figure Lengend Snippet: Phenotypic characterization of BCG.HIVA 2auxo.int strain. We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA 2auxo.int strain (A) BCG lysine auxotroph strain plated on non-lysine supplemented 7H10; (B) BCG lysine auxotroph strain plated on lysine supplemented 7H10; (C) BCG.HIVA 2auxo.int plated on 7H10 without lysine and kanamycin supplementation; (D) BCG.HIVA 2auxo.int plated on 7H10 without lysine supplementation and with kanamycin.

    Article Snippet: Bloom, and T. Hsu was transformed with the p2auxo.HIVAint plasmid DNA without the kanamycin resistance gene by electroporation.. Mycobacterial cultures were grown in Middlebrook 7H9 broth medium or on Middlebrook agar 7H10 medium supplemented with albumin-dextrose-catalase (ADC; Difco) containing 0.05% Tween 80.

    Techniques:

    GreA is required for resistance to adverse stress. (A–E, H–J) Survival of M. smegmatis strains during adverse stress. The strain symbols listed in the (J) . WT, Δ greA and comp- Δ greA strains were treated for at the indicated time with varying concentrations of SDS (A) , 5mM H 2 O 2 (B) , PBS + 0.05% Tween 80 (C) , low pH (pH4.5) (D) , heat shock (48°C) (E) , 1 μg/ml of streptomycin (H) , 100 μg/ml of rifampicin (I) , or 2.5 μg/ml of bedaquiline (J) . After treatment, dilutions were plated on 7H10 and survival is expressed as a ratio of CFUs compared to untreated cultures. (F) M. tuberculosis H37Rv strains were spotted on the 7H10 plates after they were treated under 0.5% SDS for 24 h. (G) WT, Δ greA , and comp- Δ greA strains from M. tuberculosis H37Ra were subjected to 0.5% SDS for 8 h. CFU enumeration was performed after incubation for the indicated time. These experiments were performed three times with similar results. Data are shown as mean ± SEM of three independent experiments. * p

    Journal: Frontiers in Microbiology

    Article Title: Involvement of Transcription Elongation Factor GreA in Mycobacterium Viability, Antibiotic Susceptibility, and Intracellular Fitness

    doi: 10.3389/fmicb.2020.00413

    Figure Lengend Snippet: GreA is required for resistance to adverse stress. (A–E, H–J) Survival of M. smegmatis strains during adverse stress. The strain symbols listed in the (J) . WT, Δ greA and comp- Δ greA strains were treated for at the indicated time with varying concentrations of SDS (A) , 5mM H 2 O 2 (B) , PBS + 0.05% Tween 80 (C) , low pH (pH4.5) (D) , heat shock (48°C) (E) , 1 μg/ml of streptomycin (H) , 100 μg/ml of rifampicin (I) , or 2.5 μg/ml of bedaquiline (J) . After treatment, dilutions were plated on 7H10 and survival is expressed as a ratio of CFUs compared to untreated cultures. (F) M. tuberculosis H37Rv strains were spotted on the 7H10 plates after they were treated under 0.5% SDS for 24 h. (G) WT, Δ greA , and comp- Δ greA strains from M. tuberculosis H37Ra were subjected to 0.5% SDS for 8 h. CFU enumeration was performed after incubation for the indicated time. These experiments were performed three times with similar results. Data are shown as mean ± SEM of three independent experiments. * p

    Article Snippet: The comp- ΔgreA strains were selected on Middlebrook 7H10 agar medium (complemented with 10% OADC) containing 30 μg/ml kanamycin.

    Techniques: Incubation

    Intracellular fitness of Δ greA mutant strain was evaluated in vivo and in vitro . RAW264.7 cells were seeded at 3 × 10 5 cells per well in 24-well culture plates. After adhesion, cells were infected with bacteria at MOI of 10. The extracellular bacteria were removed 2 h post infection by washing with PBS. The cells of M. smegmatis (A) , M. tuberculosis H37Ra (B) , and M. tuberculosis H37Rv (C) were further incubated for 24 and 48 h at 37°C. Then serially diluted (10-fold) cells lysates were spotted on a 7H10-OADC agar and cultured in 37°C. CFUs were counted after incubation for the indicated time for M. smegmatis and M. tuberculosis , respectively. (D) Mice were infected i.p. (1 × 10 7 ) with WT, Δ greA , or comp- Δ greA strains of M. tuberculosis H37Ra. The number of surviving bacilli by CFU counting at the indicated time. Data are shown as mean ± SEM of two independent experiments. * p

    Journal: Frontiers in Microbiology

    Article Title: Involvement of Transcription Elongation Factor GreA in Mycobacterium Viability, Antibiotic Susceptibility, and Intracellular Fitness

    doi: 10.3389/fmicb.2020.00413

    Figure Lengend Snippet: Intracellular fitness of Δ greA mutant strain was evaluated in vivo and in vitro . RAW264.7 cells were seeded at 3 × 10 5 cells per well in 24-well culture plates. After adhesion, cells were infected with bacteria at MOI of 10. The extracellular bacteria were removed 2 h post infection by washing with PBS. The cells of M. smegmatis (A) , M. tuberculosis H37Ra (B) , and M. tuberculosis H37Rv (C) were further incubated for 24 and 48 h at 37°C. Then serially diluted (10-fold) cells lysates were spotted on a 7H10-OADC agar and cultured in 37°C. CFUs were counted after incubation for the indicated time for M. smegmatis and M. tuberculosis , respectively. (D) Mice were infected i.p. (1 × 10 7 ) with WT, Δ greA , or comp- Δ greA strains of M. tuberculosis H37Ra. The number of surviving bacilli by CFU counting at the indicated time. Data are shown as mean ± SEM of two independent experiments. * p

    Article Snippet: The comp- ΔgreA strains were selected on Middlebrook 7H10 agar medium (complemented with 10% OADC) containing 30 μg/ml kanamycin.

    Techniques: Mutagenesis, In Vivo, In Vitro, Infection, Incubation, Cell Culture, Mouse Assay

    PE_PGRS41 expression in M. smegmatis results in increased cell wall permeability. ( A ) Mid-log-phase cultures of Ms_Vec and Ms_PE_PGRS41 were incubated in 7H9 containing 2 μg/ml ethidium bromide for indicated time. ( B ) Mid-log-phase cultures of Ms_Vec and Ms_PE_PGRS41 were incubated in 7H9 with 2 μM Nile red stain for indicated time. ( C ) The growth of Ms_Vec and Ms_PE_PGRS41 after treatment with different pH gradient for indicated time. The Ms_Vec and Ms_PE_PGRS41 strains were centrifuged, re-suspended to 5 ml MB 7H9 at an OD 600 of 0.5, 10-fold serial dilutions of Ms_Vec and Ms_PE_PGRS41 were spotted on MB 7H10. ( D ) Mid-log-phase cultures of Ms_Vec and Ms_PE_PGRS41 were incubated in MB 7H9 supplemented with 0.05% SDS for indicated time. And then the recombinant strains were plated onto 7H10 plates by serially ten-fold dilution, the bacterial numbers were counted after 3–4 days of cultivation.

    Journal: Scientific Reports

    Article Title: Mycobacterium tuberculosis PE_PGRS41 Enhances the Intracellular Survival of M. smegmatis within Macrophages Via Blocking Innate Immunity and Inhibition of Host Defense

    doi: 10.1038/srep46716

    Figure Lengend Snippet: PE_PGRS41 expression in M. smegmatis results in increased cell wall permeability. ( A ) Mid-log-phase cultures of Ms_Vec and Ms_PE_PGRS41 were incubated in 7H9 containing 2 μg/ml ethidium bromide for indicated time. ( B ) Mid-log-phase cultures of Ms_Vec and Ms_PE_PGRS41 were incubated in 7H9 with 2 μM Nile red stain for indicated time. ( C ) The growth of Ms_Vec and Ms_PE_PGRS41 after treatment with different pH gradient for indicated time. The Ms_Vec and Ms_PE_PGRS41 strains were centrifuged, re-suspended to 5 ml MB 7H9 at an OD 600 of 0.5, 10-fold serial dilutions of Ms_Vec and Ms_PE_PGRS41 were spotted on MB 7H10. ( D ) Mid-log-phase cultures of Ms_Vec and Ms_PE_PGRS41 were incubated in MB 7H9 supplemented with 0.05% SDS for indicated time. And then the recombinant strains were plated onto 7H10 plates by serially ten-fold dilution, the bacterial numbers were counted after 3–4 days of cultivation.

    Article Snippet: Reagents Middlebrook 7H10 agar and Middlebrook 7H9 broth medium were purchased from BD Difco Laboratories.

    Techniques: Expressing, Permeability, Mass Spectrometry, Incubation, Staining, Recombinant

    M. tuberculosis PE_PGRS41 expression in M. smegmatis decreased bacterial survival following exposure to various antibiotics, including vancomycin ( A ), norfloxacin ( B ), isoniazid ( C ), and ciprofloxacin ( D ). Ms_Vec and Ms_PE_PGRS41 were exposed to different concentration of vancomycin, norfloxacin, isoniazid, and ciprofloxacin for 5 h, then ten-fold dilution spotted the bacteria on MB 7H9 supplemented with 1% agar, the bacterial number of Ms_Vec and Ms_PE_PGRS41 were counted after 3 days cultivation.

    Journal: Scientific Reports

    Article Title: Mycobacterium tuberculosis PE_PGRS41 Enhances the Intracellular Survival of M. smegmatis within Macrophages Via Blocking Innate Immunity and Inhibition of Host Defense

    doi: 10.1038/srep46716

    Figure Lengend Snippet: M. tuberculosis PE_PGRS41 expression in M. smegmatis decreased bacterial survival following exposure to various antibiotics, including vancomycin ( A ), norfloxacin ( B ), isoniazid ( C ), and ciprofloxacin ( D ). Ms_Vec and Ms_PE_PGRS41 were exposed to different concentration of vancomycin, norfloxacin, isoniazid, and ciprofloxacin for 5 h, then ten-fold dilution spotted the bacteria on MB 7H9 supplemented with 1% agar, the bacterial number of Ms_Vec and Ms_PE_PGRS41 were counted after 3 days cultivation.

    Article Snippet: Reagents Middlebrook 7H10 agar and Middlebrook 7H9 broth medium were purchased from BD Difco Laboratories.

    Techniques: Expressing, Mass Spectrometry, Concentration Assay

    The effect of PE_PGRS41 on the growth of M. smegmatis . ( A ) PCR amplification of PE_PGRS41 encoding sequence from M. tuberculosis genome approximately 1086 bp. ( B ) M. tuberculosis PE_PGRS41 was expressed in M. smegmatis and detected using Western blotting. Cell lysates of Ms_Vec and Ms_PE_PGRS41 were subjected to Western blot to determine the expression of His-tagged PE_PGRS41 protein in M. smegmatis by anti-His antibody. ( C ) Cell fractionation experiments were performed to determine the sub-cellular localization of PE_PGRS41, GroEL2 protein serves as a cytoplasm marker of M. smegmatis , CW represents cell wall; CP represents cytoplasm. ( D ) The morphology of Ms_Vec and Ms_PE_PGRS41 were detected after induction by acetamide in the presence of hygromycin. ( E ) Ms_Vec and Ms_PE_PGRS41 were grown in Middlebrook 7H9 medium supplemented with 0.05% Tween 80, 1% acetamide and 0.2% glycerinum, with hygromycin (100 μg/ml). The OD 600 was determined at an interval of 4 h.

    Journal: Scientific Reports

    Article Title: Mycobacterium tuberculosis PE_PGRS41 Enhances the Intracellular Survival of M. smegmatis within Macrophages Via Blocking Innate Immunity and Inhibition of Host Defense

    doi: 10.1038/srep46716

    Figure Lengend Snippet: The effect of PE_PGRS41 on the growth of M. smegmatis . ( A ) PCR amplification of PE_PGRS41 encoding sequence from M. tuberculosis genome approximately 1086 bp. ( B ) M. tuberculosis PE_PGRS41 was expressed in M. smegmatis and detected using Western blotting. Cell lysates of Ms_Vec and Ms_PE_PGRS41 were subjected to Western blot to determine the expression of His-tagged PE_PGRS41 protein in M. smegmatis by anti-His antibody. ( C ) Cell fractionation experiments were performed to determine the sub-cellular localization of PE_PGRS41, GroEL2 protein serves as a cytoplasm marker of M. smegmatis , CW represents cell wall; CP represents cytoplasm. ( D ) The morphology of Ms_Vec and Ms_PE_PGRS41 were detected after induction by acetamide in the presence of hygromycin. ( E ) Ms_Vec and Ms_PE_PGRS41 were grown in Middlebrook 7H9 medium supplemented with 0.05% Tween 80, 1% acetamide and 0.2% glycerinum, with hygromycin (100 μg/ml). The OD 600 was determined at an interval of 4 h.

    Article Snippet: Reagents Middlebrook 7H10 agar and Middlebrook 7H9 broth medium were purchased from BD Difco Laboratories.

    Techniques: Polymerase Chain Reaction, Amplification, Sequencing, Western Blot, Mass Spectrometry, Expressing, Cell Fractionation, Marker