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M. tuberculosis VapC30 regulates cellular growth through both Mg 2+ and Mn 2+ <t>-dependent</t> <t>ribonuclease</t> activity. ( A ) Expression of the VapC30 or VapBC30 complex was induced at hour 0, and cell growth was monitored by measuring the optical density at 600 nm (OD 600 ). Red curves indicate cells expressing the VapC30, and the blue curves indicate cells co-expressing the VapC30 and VapB30. The green curves indicate cells carrying empty pCOLD1 vector and pGro7 plasmids. The data (OD 600 ) represent the average of experiments performed in triplicate, with standard deviations indicated by error bars. ( B and C ) Fluorescence quenching assay. In this assay system, fluorescence increases when the substrate RNA is cleaved. For the assay, 10 μM VapC30 (or VapBC30), 0.5 M NaCl, 20 mM Tris-HCl buffer at pH 8.0, 40 units of RiboLock TM <t>RNase</t> inhibitor (Thermo Scientific), and various concentrations of ( B ) MgCl 2 or ( C ) MnCl 2 were included in a 50 μl reaction volume and incubated at 37°C. For the assay, possible bound metal ions were removed before the assay (see main text for details). Control contained 10 mM MgCl 2 (or 10 mM MnCl 2 ), 0.5 M NaCl, 50 mM Tris-HCl buffer pH 8.0, and 40 units of RiboLock TM (Thermo scientific) RNase inhibitor. Each experiment was performed in triplicate. ( D ) M. tuberculosis tRNA fMET (1 μM) was used to analyze the ribonuclease activity of VapC30 (2, 4, and 6 μM, respectively). For the assay, 0.5 M NaCl, 20 mM Tris-HCl buffer at pH 8.0, and 40 units of RiboLock TM RNase inhibitor (Thermo Scientific) and 1 mM MgCl 2 were included in a 10 μl reaction volume and incubated at 37°C for 60 minutes. Digested RNA fragments were analyzed by denaturing 15% acrylamide gel electrophoresis in the presence of 8 M urea.
Rnase Alert Kit, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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M. tuberculosis VapC30 regulates cellular growth through both Mg 2+ and Mn 2+ -dependent ribonuclease activity. ( A ) Expression of the VapC30 or VapBC30 complex was induced at hour 0, and cell growth was monitored by measuring the optical density at 600 nm (OD 600 ). Red curves indicate cells expressing the VapC30, and the blue curves indicate cells co-expressing the VapC30 and VapB30. The green curves indicate cells carrying empty pCOLD1 vector and pGro7 plasmids. The data (OD 600 ) represent the average of experiments performed in triplicate, with standard deviations indicated by error bars. ( B and C ) Fluorescence quenching assay. In this assay system, fluorescence increases when the substrate RNA is cleaved. For the assay, 10 μM VapC30 (or VapBC30), 0.5 M NaCl, 20 mM Tris-HCl buffer at pH 8.0, 40 units of RiboLock TM RNase inhibitor (Thermo Scientific), and various concentrations of ( B ) MgCl 2 or ( C ) MnCl 2 were included in a 50 μl reaction volume and incubated at 37°C. For the assay, possible bound metal ions were removed before the assay (see main text for details). Control contained 10 mM MgCl 2 (or 10 mM MnCl 2 ), 0.5 M NaCl, 50 mM Tris-HCl buffer pH 8.0, and 40 units of RiboLock TM (Thermo scientific) RNase inhibitor. Each experiment was performed in triplicate. ( D ) M. tuberculosis tRNA fMET (1 μM) was used to analyze the ribonuclease activity of VapC30 (2, 4, and 6 μM, respectively). For the assay, 0.5 M NaCl, 20 mM Tris-HCl buffer at pH 8.0, and 40 units of RiboLock TM RNase inhibitor (Thermo Scientific) and 1 mM MgCl 2 were included in a 10 μl reaction volume and incubated at 37°C for 60 minutes. Digested RNA fragments were analyzed by denaturing 15% acrylamide gel electrophoresis in the presence of 8 M urea.

Journal: Nucleic Acids Research

Article Title: Structural and functional studies of the Mycobacterium tuberculosis VapBC30 toxin-antitoxin system: implications for the design of novel antimicrobial peptides

doi: 10.1093/nar/gkv689

Figure Lengend Snippet: M. tuberculosis VapC30 regulates cellular growth through both Mg 2+ and Mn 2+ -dependent ribonuclease activity. ( A ) Expression of the VapC30 or VapBC30 complex was induced at hour 0, and cell growth was monitored by measuring the optical density at 600 nm (OD 600 ). Red curves indicate cells expressing the VapC30, and the blue curves indicate cells co-expressing the VapC30 and VapB30. The green curves indicate cells carrying empty pCOLD1 vector and pGro7 plasmids. The data (OD 600 ) represent the average of experiments performed in triplicate, with standard deviations indicated by error bars. ( B and C ) Fluorescence quenching assay. In this assay system, fluorescence increases when the substrate RNA is cleaved. For the assay, 10 μM VapC30 (or VapBC30), 0.5 M NaCl, 20 mM Tris-HCl buffer at pH 8.0, 40 units of RiboLock TM RNase inhibitor (Thermo Scientific), and various concentrations of ( B ) MgCl 2 or ( C ) MnCl 2 were included in a 50 μl reaction volume and incubated at 37°C. For the assay, possible bound metal ions were removed before the assay (see main text for details). Control contained 10 mM MgCl 2 (or 10 mM MnCl 2 ), 0.5 M NaCl, 50 mM Tris-HCl buffer pH 8.0, and 40 units of RiboLock TM (Thermo scientific) RNase inhibitor. Each experiment was performed in triplicate. ( D ) M. tuberculosis tRNA fMET (1 μM) was used to analyze the ribonuclease activity of VapC30 (2, 4, and 6 μM, respectively). For the assay, 0.5 M NaCl, 20 mM Tris-HCl buffer at pH 8.0, and 40 units of RiboLock TM RNase inhibitor (Thermo Scientific) and 1 mM MgCl 2 were included in a 10 μl reaction volume and incubated at 37°C for 60 minutes. Digested RNA fragments were analyzed by denaturing 15% acrylamide gel electrophoresis in the presence of 8 M urea.

Article Snippet: Ribonuclease activity was also measured by fluorescence quenching assay using an RNase Alert kit (IDT).

Techniques: Activity Assay, Expressing, Plasmid Preparation, Fluorescence, Incubation, Acrylamide Gel Assay, Electrophoresis

Fluorescence quenching assays with 10 μM VapC30 (or VapBC30 complex) and various concentrations of the peptides [10 μM (blue), 25 μM (orange), and 100 μM (red)]. VapBC30 complex and the peptides were incubated at 37°C for 60 min before measuring the fluorescence. Fluorescence (RFU) obtained with the 10 μM VapC30 was taken as 100% and fluorescence (RFU) obtained with the 10 μM VapBC30 complex was taken as 0%. For the assay, 0.5 M NaCl, 20 mM Tris–HCl buffer at pH 8.0, 1 mM MgCl 2 , and 40 units of RiboLock™ RNase inhibitor (Thermo Scientific) were included in a 50 μl reaction volume. Error bars represent the standard deviation of three replicate reactions.

Journal: Nucleic Acids Research

Article Title: Structural and functional studies of the Mycobacterium tuberculosis VapBC30 toxin-antitoxin system: implications for the design of novel antimicrobial peptides

doi: 10.1093/nar/gkv689

Figure Lengend Snippet: Fluorescence quenching assays with 10 μM VapC30 (or VapBC30 complex) and various concentrations of the peptides [10 μM (blue), 25 μM (orange), and 100 μM (red)]. VapBC30 complex and the peptides were incubated at 37°C for 60 min before measuring the fluorescence. Fluorescence (RFU) obtained with the 10 μM VapC30 was taken as 100% and fluorescence (RFU) obtained with the 10 μM VapBC30 complex was taken as 0%. For the assay, 0.5 M NaCl, 20 mM Tris–HCl buffer at pH 8.0, 1 mM MgCl 2 , and 40 units of RiboLock™ RNase inhibitor (Thermo Scientific) were included in a 50 μl reaction volume. Error bars represent the standard deviation of three replicate reactions.

Article Snippet: Ribonuclease activity was also measured by fluorescence quenching assay using an RNase Alert kit (IDT).

Techniques: Fluorescence, Incubation, Standard Deviation