In vitro Real-time Measurement of the Intra-bacterial Redox Potential
内部细菌氧化还原电位的体外实时测量
发布: 2015年09月05日第5卷第17期 DOI: 10.21769/BioProtoc.1579 浏览次数: 9508
评审: Arsalan DaudiDaan C. SwartsAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jan 2015
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Abstract
All bacteria that live in oxygenated environments have to deal with oxidative stress caused by some form of exogenous or endogenous reactive oxygen species (ROS) (Imlay, 2013). Large quantities of ROS damage DNA, lipids and proteins which can eventually lead to bacterial cell death (Imlay, 2013). In contrast, smaller quantities of ROS can play more sophisticated roles in cellular signalling pathways affecting almost every process in the bacterial cell e.g. metabolism, stress responses, transcription, protein synthesis, etc. Previously, inadequate analytical methods prevented appropriate analysis of the intra-bacterial redox potential. Herein, we describe a method for the measurement of real-time changes to the intra-bacterial redox potential using redox-sensitive GFP (roGFP2) (van der Heijden et al., 2015). The roGFP2 protein is engineered to contain specific cysteine residues that form an internal disulfide bridge upon oxidation which results in a slight shift in protein conformation (Hanson et al., 2004). This shift results in two distinct protein isoforms with different fluorescence excitation spectra after excitation at 405 nm and 480 nm respectively. Consequently, the corresponding 405/480 nm ratio can be used as a measure for the intra-bacterial redox potential. The ratio-metric analysis excludes variations due to differences in roGFP2 concentrations and since the conformational shift is reversible the system allows for measurement of oxidizing as well as reducing conditions. In this protocol we describe the system by measuring the intra-bacterial redox potential inside Salmonella typhimurium (S. typhimurium) however this system can be adjusted for use in other Gram-negative bacteria.
Materials and Reagents
- LB broth (Miller) (Sigma-Aldrich, catalog number: MKBS8229V )
- Salmonella typhimurium (SL1344)
- Carbenicillin (Goldbio, catalog number: 4800-94-6 ) [stock solution 100 mg/ml in 50% water/methanol (v/v) kept at -20 °C]
- Saline (sodium chloride) (Sigma-Aldrich, catalog number: S9888 ) [0.9% sodium chloride in sterile water (w/w)]
- Hydrogen peroxide [30% (w/w)] (Sigma-Aldrich, catalog number: 026K3770 )
- Dithiothreitol (AMRESCO, catalog number: 0363C45 ) (stock solution 1 M in sterile water kept at -20 °C)
- pfpv25-roGFP2 vector (available by contacting bfinlay@interchange.ubc.ca) (van der Heijden et al., 2015)
- 10% glycerol in sterile water (v/v) (glycerol) (Sigma-Aldrich, catalog number: G5516 )
- Agar (Sigma-Aldrich, catalog number: A5306 )
- 125 ml flasks (VWR International, catalog number: 89090-316 )
- 1.5 ml sterile Eppendorf tubes (VWR International, catalog number: 89000-028 )
- 10% glycerol in sterile water
- Luria Broth (see Recipes)
Equipment
- Black, 96-well assay plate with flat clear bottom (Corning, Costar®, catalog number: 3631 )
- 37 °C incubator/shaker (New Brunswick Scientific, model: M1025-0000 )
- Electroporator (Bio-Rad Laboratories, catalog number: 165-2100 )
- Fluorescent plate reader (Tecan Trading AG, model: Infinite M200 ) or comparable device able to measure 96-well plates at 37 °C, excitation at 405 nm and 480 nm, emission at 510 nm
- Spectrophotometer (Thermo Fisher Scientific, model: spectronic 200 )
- 20-200 μl Multichannel pipet (Rainin, model: L12 200R )
- Electroporation cuvettes (gap size 1 mm) (VWR International, catalog number: 89047-206 )
- Microcentrifuge (Thermo Fisher Scientific, catalog number: 75003424 )
- 10 ml glass culture tube (VWR International, catalog number: 47729-574 )
Software
- For fluorescent plate reader (Magellan 7)
Note: This software comes with the Tecan fluorescent plate reader. If a different plate reader is used, software comparable to Magellan 7 can be used for data acquisition of fluorescence measurements.
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
van der Heijden, J. and Finlay, B. B. (2015). In vitro Real-time Measurement of the Intra-bacterial Redox Potential. Bio-protocol 5(17): e1579. DOI: 10.21769/BioProtoc.1579.
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分类
微生物学 > 微生物生物化学 > 其它化合物
生物化学 > 其它化合物 > 活性氧
生物化学 > 蛋白质 > 荧光
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