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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.

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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

微生物学 > 微生物生物化学 > 其它化合物
作者: Joris van der Heijden
Joris van der HeijdenAffiliation 1: Michael Smith Laboratories, University of British Columbia, Vancouver, Canada
Affiliation 2: Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada
Bio-protocol author page: a2518
 and B. Brett Finlay
B. Brett FinlayAffiliation 1: Michael Smith Laboratories, University of British Columbia, Vancouver, Canada
Affiliation 2: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada
Affiliation 3: Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada
For correspondence: bfinlay@interchange.ubc.ca
Bio-protocol author page: a2519
Vol 5, Iss 17, 9/5/2015, 1778 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1579

[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.

[Abstract]

Materials and Reagents

  1. LB broth (Miller) (Sigma-Aldrich, catalog number: MKBS8229V )
  2. Salmonella typhimurium (SL1344)
  3. Carbenicillin (Goldbio, catalog number: 4800-94-6 ) [stock solution 100 mg/ml in 50% water/methanol (v/v) kept at -20 °C]
  4. Saline (sodium chloride) (Sigma-Aldrich, catalog number: S9888 ) [0.9% sodium chloride in sterile water (w/w)]
  5. Hydrogen peroxide [30% (w/w)] (Sigma-Aldrich, catalog number: 026K3770 )
  6. Dithiothreitol (AMRESCO, catalog number: 0363C45 ) (stock solution 1 M in sterile water kept at -20 °C)
  7. pfpv25-roGFP2 vector (available by contacting bfinlay@interchange.ubc.ca) (van der Heijden et al., 2015)
  8. 10% glycerol in sterile water (v/v) (glycerol) (Sigma-Aldrich, catalog number: G5516 )
  9. Agar (Sigma-Aldrich, catalog number: A5306 )
  10. 125 ml flasks (VWR International, catalog number: 89090-316 )
  11. 1.5 ml sterile Eppendorf tubes (VWR International, catalog number: 89000-028 )
  12. 10% glycerol in sterile water
  13. Luria Broth (see Recipes)

Equipment

  1. Black, 96-well assay plate with flat clear bottom (Corning, Costar®, catalog number: 3631 )
  2. 37 °C incubator/shaker (New Brunswick Scientific, model: M1025-0000 )
  3. Electroporator (Bio-Rad Laboratories, catalog number: 165-2100 )
  4. 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
  5. Spectrophotometer (Thermo Fisher Scientific, model: spectronic 200 )
  6. 20-200 μl Multichannel pipet (Rainin, model: L12 200R )
  7. Electroporation cuvettes (gap size 1 mm) (VWR International, catalog number: 89047-206 )
  8. Microcentrifuge (Thermo Fisher Scientific, catalog number: 75003424 )
  9. 10 ml glass culture tube (VWR International, catalog number: 47729-574 )

Software

  1. 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

  1. Create roGFP2-expressing strain
    Day 0
    In the afternoon, streak bacterial target strain from -80 °C glycerol stock [25% (v/v) glycerol in LB] on 1.5% LB agar plates and incubate overnight at 37 °C (appropriate antibiotics added).
    Day 1
    In the afternoon, pick one single colony using a sterile pipette tip from the LB plate. Use the pipette tip to inoculate 5 ml of LB medium in a glass culture tube and grow overnight at 37 °C while shaking at 225 rpm (appropriate antibiotics added).
    Day 2
    1. In the morning, take 150 μl from the overnight culture and inoculate 5 ml of fresh LB medium in a glass culture tube. Grow for 3 h at 37 °C and put culture on ice for 5 min.
    2. Spin down 5 ml of the culture at 4,000 x g for 15 min and dispose of supernatant.
    3. Wash the pellet once by adding 1 ml of sterile water and resuspension of the bacterial pellet by pipetting up and down several times. Transfer bacterial suspension to sterile Eppendorf tube and spin down at 14,000 x g for 1 min. Discard the supernatant.
    4. Wash the pellet by adding 1 ml sterile glycerol solution [10% (v/v) glycerol in water] and spin down at 14,000 x g for 1 min. Discard the supernatant. Repeat step A4.
    5. Resuspend the pellet in 50 μl of glycerol solution and add 2 μl of pfpv25-roGFP2 plasmid DNA (~50 ng/μl total plasmid DNA).
    6. Transfer bacterial solution to an electro-cuvette and transform the bacteria using electroporation (V = 2.5 kV). Transfer bacterial solution to 1 ml of fresh LB medium and incubate for 1 h at 37 °C while shaking at 225 rpm prior to plating on LB plates containing 100 μg/ml carbenicillin.
    Day 3
    Pick one colony and grow at 37 °C overnight in 5 ml LB in a glass culture tube containing 100 μg/ml carbenicillin before making a freezer stock the next day by mixing 1 ml of bacterial culture with 1 ml of 50% glycerol/LB (v/v) for a final concentration of 25% glycerol and freeze down at -80 °C.

  2. Preparing bacterial cultures for measurement
    Day 0
    In the afternoon, streak the bacterial strain that contains the pfpv25-roGFP2 plasmid and the original target strain (without the plasmid and therefore non-fluorescent) from -80 °C glycerol stock on LB agar plates containing appropriate antibiotics and incubate overnight at 37 °C.
    Day 1
    In the afternoon, pick a single colony from each plate and grow the bacteria in 5 ml of LB in a glass culture tube at containing the appropriate antibiotics overnight at 37 °C while shaking (225 rpm).
    Day 2
    1. In the morning, take 300 μl from each of the overnight cultures and inoculate 10 ml of fresh LB medium for each bacterial culture in a 125 ml Erlenmeyer flask (no antibiotic added).
    2. Grow for 3 h (or until OD600 ~1.0) while shaking at 225 rpm. Spin down the cultures at 2,500 x g for 15 min and wash the pellets once in 5 ml saline solution (0.9% w/w in water).
    3. Resuspend the pellet in 1 ml of saline solution and determine the OD600 of the solution in a spectrometer.
    4. Dilute the bacterial solution to OD600=2.0 using saline solution. Transfer 100 μl of bacterial saline solution per well of a black 96-well clear bottom plate.
    5. For each fluorescent strain, also transfer 100 μl of the identical non-fluorescent strain (at OD600=2.0) to determine background fluorescence. In Figure 1 an example of a plate layout is shown.

  3. Setting up the plate reader
    1. Pre-warm the Tecan fluorescent plate reader to 37 °C. The 405/480 ratio will be obtained by two subsequent measurements in which the bacteria are first excited at wavelength 405 nm while emission is measured at 510 nm. Immediately after the first measurement, the cells are excited again at 480 nm while emission is measured at 510 nm. Both fluorescent intensities are used for calculation of the normalized 405/480 nm ratio and the corresponding intra-bacterial redox potential.
    2. These measurements can be repeated for extended periods of time to follow the intra-bacterial redox potential in real-time.
    3. Program the plate reader with the following settings:
      1. Temperature 37 °C.
      2. Before each round of measurement shake for a duration of 2 sec.
      3. Excitation at 405 nm and 480 nm.
      4. Emission at 510 nm.
      5. Reading from bottom.
      6. Gain determined from well (65% of maximum gain for roGFP2-expressing bacteria).
      7. Measurements for 30 min continuously.
      8. Automatically generate excel sheet with fluorescence intensities.
    4. Put the loaded black 96-well clear bottom plate containing bacterial solutions in the Tecan plate reader and measure for 30 min continuously. This data will be used to determine that all bacterial solutions have identical intra-bacterial redox potentials prior to challenge with oxidizing agents.
    5. While the Tecan plate reader is measuring, prepare saline solutions with varying amounts of hydrogen peroxide (2 mM, 4 mM, 6 mM, 8 mM, 10 mM and 12 mM). Also prepare a saline solution with 100 mM hydrogen peroxide and a saline solution with 2 mM DTT. These solutions are used to determine the maximum oxidized 405/480 ratio and maximum reduced 405/480 ratio respectively. Always prepare the hydrogen peroxide solutions fresh.
    6. After the 30 min of continuous measurements are finished, set up the machine again so that it can be immediately restarted to measure for 90 min continuously. Only when the Tecan plate reader is fully prepared and programmed, quickly add 100 μl of the pre-made hydrogen peroxide solutions from step C5 to each well. For this step use a multichannel pipet for rapid sample handling. Use the same hydrogen peroxide concentration in wells adjacent to each other (e.g. A1 and A2, B1 and B2 etc.). Add 100 μl of the 100 mM hydrogen peroxide solution to wells G1 and G2 and add 100 μl of the 2 mM DTT solution to wells H1 and H2 that were also pre-made in step C5. Immediately after the hydrogen peroxide and DTT are added start the measurement. The bacteria will detoxify hydrogen peroxide rapidly and after 90 min (or earlier depending on the experimental set up) the addition of hydrogen peroxide can be repeated for subsequent challenges.


      Figure 1. Example layout of a 96-well imaging plate. In lane 1 all bacteria express roGFP2 while in lane 2 contains well with empty-vector containing bacteria to determine the background fluorescence (BG) for each condition. The two adjacent well e.g. A1 and A2 undergo an identical challenge with oxidizing agents. In row G, 100 mM of hydrogen peroxide is added and these results are used as maximum oxidized values for normalization. In row H, 10 mM DTT is added and these results are used as maximum reduced values for normalization.

  4. Data analysis
    First, we calculate the R (ratio 405/480 nm) of S. typhimurium using Equation 1 for each time point. To better explain this calculation we have used the layout from Figure 1. For each fluorescence intensity obtained from well in column 1, background fluorescence is subtracted from the corresponding well in column 2. In Equation 1 we have indicated the corresponding well from Figure 1 between brackets to better explain the calculation.



    After R is calculated using Equation 1, it will be normalized to the maximum oxidized and maximum reduced 405/480 values for better comparability using Equation 2. The maximum oxidized ratio (Rox) is calculated from fluorescence of well G1 and the maximum reduced ratio (Rred) is calculated from fluorescence of well H1 by using Equation 1 as described above. After normalization, the upper limit for oxidation given by Rnormalized is 1.0 whereas the lower limit for reduction given by Rnormalized is 0.1.



    The Rnormalized for each time point can be plotted in a graph to obtain a real-time report on changes to the intra-bacterial redox potential. A typical result can be seen in Figure 2. In this figure, the upward arrows indicate a challenge with 1 mM H2O2.
    Using the midpoint potential of roGFP2 (-280mV), the R, Rred and Rox can be used to calculate the actual EroGFP2 inside S. typhimurium. This calculation has previously been described (Gutscher et al., 2008; van der Heijden et al., 2015). In addition to the R, Rred and Rox we need the fluorescence intensity at 480 nm under reduced or oxidized conditions (I480max and I480min respectively). With these values we calculated the degree of roGFP2 oxidation (OxDroGFP2) given by Equation 3.



    The degree of oxidation can then be used to calculate the intracellular sensor redox potential EroGFP2 by using Equation 4.



    In which R is the gas constant (8.315 J K-1 mol-1), T is the absolute temperature (310.15 K), z is the number of transferred electrons (Gutscher et al., 2008) and F is the Faraday constant (96,485 C mol-1). The midpoint potential of roGFP2 (E0’roGFP2) is -280 mV (Hanson et al., 2004).

Representative data



Figure 2. Real-time intra-bacterial redox potential of S. typhimurium after two subsequent challenges with 1 mM hydrogen peroxide. Each upward arrow indicates a challenge with hydrogen peroxide.

Notes

  1. It is important to first plate bacteria on LB instead of starting liquid cultures straight from -80 °C glycerol stocks. This can substantially alter the fitness of the bacterial population and therefore influence your final results.
  2. In this protocol bacteria in late exponential growth phase are used however bacteria in any other growth phase can be used as well. Within one experiment, the control strains should be grown to the same growth phase.
  3. To keep subsequent fluorescence reads frequent enough to accurately monitor the intra-bacterial redox potential, it is advised to never read more than 36 wells per experiment.
  4. To measure the intra-bacterial redox potential in other Gram-negative bacteria, the roGFP2 gene can be cloned into an appropriate vector for the bacteria of choice under a constitutive promotor. In the pfpv25-roGFP2 vector, the roGFP2 gene is under the control of RpsM ribosomal promotor (Salmonella promotor). Promotors of highly expressed genes are recommended to allow for high expression and significant fluorescent signal. For different target strains a promotor has to be selected that allows for high level constitutive expression in the respective target strain.
  5. Since 100 μl of the hydrogen peroxide solutions will be added to the bacterial solutions in the black 96-well clear bottom plate, the final concentration of hydrogen peroxide will be half the concentration of the hydrogen peroxide solutions.

Recipes

  1. Luria Broth
    25 g of LB broth (Miller) per L of sterile water

Acknowledgements

This work was supported by operating grants from the Canadian Institutes of Health Research (CIHR). B.B.F. is the University of British Columbia Peter Wall Distinguished Professor. No competing interests exist for this work. This protocol was adapted from previous work by our laboratory (van der Heijden et al., 2015). We sincerely thank Dr. James Remington from the University of Oregon for providing the original pRSETB roGFP2 construct.

References

  1. Gutscher, M., Pauleau, A. L., Marty, L., Brach, T., Wabnitz, G. H., Samstag, Y., Meyer, A. J. and Dick, T. P. (2008). Real-time imaging of the intracellular glutathione redox potential. Nat Methods 5(6): 553-559.
  2. Hanson, G. T., Aggeler, R., Oglesbee, D., Cannon, M., Capaldi, R. A., Tsien, R. Y. and Remington, S. J. (2004). Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem 279(13): 13044-13053.
  3. Hoiseth, S. K. and Stocker, B. A. (1981). Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291(5812): 238-239.
  4. Imlay, J. A. (2013). The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium. Nat Rev Microbiol 11(7): 443-454.
  5. van der Heijden, J., Bosman, E. S., Reynolds, L. A. and Finlay, B. B. (2015). Direct measurement of oxidative and nitrosative stress dynamics in Salmonella inside macrophages. Proc Natl Acad Sci U S A 112(2): 560-565.

材料和试剂

  1. LB肉汤(Miller)(Sigma-Aldrich,目录号:MKBS8229V)
  2. 沙门氏菌(SL1344)
  3. 羧苄青霉素(Goldbio,目录号:4800-94-6)[保存于-20℃的50%水/甲醇(v/v)中的储备溶液100mg/ml]
  4. 盐水(氯化钠)(Sigma-Aldrich,目录号:S9888)[0.9%氯化钠的无菌水(w/w)]
  5. 过氧化氢[30%(w/w)](Sigma-Aldrich,目录号:026K3770)
  6. 二硫苏糖醇(AMRESCO,目录号:0363C45)(储备溶液1M,保存在-20℃的无菌水中)
  7. pfpv25-roGFP2载体(可通过联系bfinlay@interchange.ubc.ca获得)(van der Heijden等人,2015)
  8. 10%甘油的无菌水(v/v)(甘油)(Sigma-Aldrich,目录号:G5516)
  9. 琼脂(Sigma-Aldrich,目录号:A5306)
  10. 125ml烧瓶(VWR International,目录号:89090-316)
  11. 1.5ml无菌Eppendorf管(VWR International,目录号:89000-028)
  12. 10%甘油的无菌水溶液
  13. Luria Broth(请参阅食谱)

设备

  1. 具有平底透明的黑色96孔测定板(Corning,Costar ,目录号:3631)
  2. 37℃培养箱/摇床(New Brunswick Scientific,型号:M1025-0000)
  3. 电穿孔仪(Bio-Rad Laboratories,目录号:165-2100)
  4. 荧光平板读数器(Tecan Trading AG,型号:Infinite M200)或能够在37℃测量96孔板的类似装置,在405nm和480nm激发,在510nm发射
  5. 分光光度计(Thermo Fisher Scientific,型号:spectronic 200)
  6. 20-200μl多通道移液管(Rainin,型号:L12 200R)
  7. 电穿孔杯(间隙尺寸1mm)(VWR International,目录号:89047-206)
  8. 微量离心机(Thermo Fisher Scientific,目录号:75003424)
  9. 10ml玻璃培养管(VWR International,目录号:47729-574)

软件

  1. 对于荧光板读取器(Magellan 7)
    注意:此软件随附有Tecan荧光板读取器。 如果使用不同的平板读数器,与Magellan 7相当的软件可用于荧光测量的数据采集。

程序

  1. 创建roGFP2表达菌株
    第0天
    下午,在1.5%LB琼脂平板上从-80℃甘油储液[25%(v/v)甘油]中条纹细菌靶菌株,并在37℃下孵育过夜(加入适当的抗生素)。
    第1天
    在下午,从LB平板使用无菌移液器吸头挑选一个单一的殖民地。 使用移液器吸头接种玻璃培养管中的5ml LB培养基,并在37℃下生长过夜,同时以225rpm振荡(适当的 抗生素)。
    第2天
    1. 在早晨,从过夜培养物中取150μl,接种5 ml的新鲜LB培养基。 在37℃下生长3小时   将培养物置于冰上5分钟。
    2. 在4,000×g下旋转5ml培养物15分钟,并处理上清液。
    3. 通过加入1ml无菌水和悬浮液洗涤沉淀一次 的细菌沉淀通过吸移上下数次。 转让   细菌悬浮液至无菌Eppendorf管中并在14,000×下离心   g 1分钟。 弃去上清液。
    4. 通过加入1洗涤沉淀 ml无菌甘油溶液[10%(v/v)甘油水溶液]并旋转   以14,000in x g 搅拌1分钟。 弃去上清液。 重复步骤A4。
    5. 重悬在50μl甘油溶液中的沉淀,加入2μl的pfpv25-roGFP2质粒DNA(〜50ng /μl总质粒DNA)。
    6. 转移细菌溶液到电子比色皿,并转化 细菌使用电穿孔(V = 2.5kV)。 转移细菌溶液   加入到1ml新鲜LB培养基中,并在37℃下振荡孵育1小时 在225rpm下接种在含有100μg/ml的LB平板上 羧苄青霉素。
    第3天
    挑取一个菌落并在37℃下在含有100μg/ml羧苄青霉素的玻璃培养管中的5ml LB中生长过夜,然后在第二天通过将1ml细菌培养物与1ml 50%甘油/LB v/v),终浓度为25%甘油并在-80℃下冷冻。

  2. 准备测量细菌培养物
    第0天
    下午,将含有pfpv25-roGFP2质粒和原始靶菌株(不含质粒,因此非荧光)的细菌菌株从含有适当抗生素的LB琼脂平板上的-80℃甘油储液中划线,并在37℃下孵育过夜C。
    第1天
    在下午,从每个板中挑取单个菌落,并在含有合适抗生素的玻璃培养管中的5ml LB中在37℃振荡(225rpm)下培养细菌过夜。
    第2天
    1. 在早晨,从每个过夜培养物中取300μl 在125℃对每种细菌培养物接种10ml新鲜LB培养基 ml锥形瓶(未加入抗生素)。
    2. 生长3小时(或直到 同时在225rpm下摇动。在2,500xg下旋转培养物15分钟,并在5ml盐溶液(0.9%w/w)中洗涤沉淀一次 在水里)。
    3. 将沉淀重悬在1ml盐水溶液中,并在光谱仪中测定溶液的OD 600。
    4. 使用盐水溶液稀释细菌溶液至OD 600 = 2.0。 转移100微升细菌生理盐水溶液每孔黑色96孔   透明底板。
    5. 对于每个荧光菌株,也转移100   μl相同的非荧光菌株(在OD 600 = 2.0),以确定 背景荧光。 在图1中,板布局的示例是 显示。

  3. 设置读板器
    1. 将Tecan荧光板读数器预温至37℃。 405/480比例 将通过两次后续测量获得,其中细菌 首先在波长405nm激发,而在510测量发射   nm。 在第一次测量后,立即激发细胞 再次在480nm处测量,而在510nm处测量发射。 两者荧光 强度用于计算归一化的405/480nm比率 和相应的细菌内氧化还原电位。
    2. 这些测量可以重复延长的时间段以实时跟踪细菌内氧化还原电位。
    3. 使用以下设置对读板器进行编程:
      1. 温度37℃。
      2. 在每轮测量前振动持续2秒。
      3. 在405nm和480nm下的激发
      4. 在510nm处发射。
      5. 从底部读取。
      6. 由孔确定的增益(表达roGFP2的细菌的最大增殖的65%)
      7. 连续测量30分钟。
      8. 自动生成具有荧光强度的Excel表。
    4. 装上装有黑色96孔透明底板的细菌 溶液,并连续测量30分钟。   该数据将用于确定所有细菌溶液具有 相同的细菌内氧化还原电位 氧化剂。
    5. 当Tecan读板仪测量时, 制备含有不同量的过氧化氢的盐溶液 4mM,6mM,8mM,10mM和12mM)。 也准备盐溶液 用100mM过氧化氢和含2mM DTT的盐溶液。 这些   溶液用于确定最大氧化405/480比率和 最大降低405/480比例。 总是准备氢 过氧化物溶液。
    6. 30分钟后连续 测量完成后,再次设置机器,以便它可以 立即重新开始连续测量90分钟。只有当 Tecan板读数器是完全准备和编程,快速添加100微升 的来自步骤C5的预制的过氧化氢溶液加入到每个孔中。 对于此步骤,使用多通道移液器快速样品处理。使用 在彼此相邻的孔中相同的过氧化氢浓度  (例如A1和A2,B1和B2 等)。加入100微升的100毫米氢 过氧化物溶液加入孔G1和G2,并加入100μl的2mM DTT 溶液加入也在步骤C5中预制备的孔H1和H2中。 在加入过氧化氢和DTT后立即开始 测量。细菌将迅速解毒过氧化氢 90分钟后(或更早取决于实验设置) 可以重复加入过氧化氢用于随后的挑战。


      图1. 96孔成像板的示例布局。在泳道1中 细菌表达roGFP2,而泳道2包含空载体  以确定背景荧光(BG) 每个条件。两个相邻的井,例如A1和A2经历 用氧化剂进行相同的攻击。在G行中,100mM的氢 加入过氧化物,这些结果用作最大氧化值 用于归一化。在H行中,加入10mM DTT,这些结果是 用作归一化的最大减小值。

  4. 数据分析
    首先,我们计算S的比率(405/480nm)。 typhimurium 使用公式1为每个时间点。为了更好地解释这种计算,我们使用了图1的布局。对于从第1列孔中获得的每个荧光强度,从第2列的相应孔中减去背景荧光。在方程1中,我们已经从图1中指示了相应的孔括号以更好地解释计算。



    在使用等式1计算R em之后,使用等式2将其归一化为最大氧化值和最大值减小的405/480值以获得更好的可比性。最大氧化比( R ox ) 通过使用如上所述的等式1从孔H1的荧光计算从孔G1的荧光计算的最大减小的比率( R red 。在归一化之后,由归一化的 标准化的氧化物的上限为1.0,而由 R 给出的归约的下限> 正常化 为0.1


    可以在图中绘制每个时间点的 R 标准化 ,以获得关于细菌内氧化还原电位变化的实时报告。典型的结果可以在图2中看到。在该图中,向上的箭头表示用1mM H 2 O 2 Sub 2的挑战。
    使用roGFP2(-280mV)的中点电位, R , /em> ox 可用于计算 S内的实际E roGFP2 。 typhimurium 。该计算先前已经描述过(Gutscher等人,2008; van der Heijden等人,2015)。除了 R , R 红色 和 R ox 我们需要在还原或氧化条件下在480nm处的荧光强度( max 480分钟)。使用这些值,我们计算由等式3给出的roGFP 2氧化的程度(OxD roGFP2 )。



    然后可以使用氧化度来通过使用等式4计算细胞内传感器氧化还原电位E subGFP2。



    其中R是气体常数(8.315JK < - sup-mol -1 ),T是绝对温度(310.15K),z是转移电子数 ,2008),F是法拉第常数(96,485℃ mol -1 )。 roGFP2的中点电位(E roGFP2 )为-280mV(Hanson等人,2004)。

代表数据



图2.实时细菌内氧化还原电位。 鼠伤寒沙门氏菌。每个向上箭头表示用过氧化氢攻击。

笔记

  1. 重要的是先在LB上平板细菌,而不是直接从-80℃甘油原液开始液体培养。 这可以基本上改变细菌群体的适应性,并因此影响你的最终结果。
  2. 在该方案中,使用晚指数生长期的细菌,但是也可以使用任何其他生长期的细菌。在一个实验中,对照菌株应生长至相同的生长期
  3. 为了保持随后的荧光读数足够频繁以精确监测细菌内氧化还原电位,建议从不读取超过36个孔每个实验。
  4. 为了测量其它革兰氏阴性细菌中的细菌内氧化还原电位,可以将roGFP2基因克隆到用于组成型启动子下选择的细菌的合适载体中。在pfpv25-roGFP2载体中,roGFP2基因处于RpsM核糖体启动子(沙门氏菌启动子)的控制下。推荐高度表达的基因的启动子允许高表达和显着的荧光信号。对于不同的靶菌株,必须选择允许在各自的靶菌株中高水平组成型表达的启动子
  5. 由于将100μl的过氧化氢溶液加入黑色96孔透明底板中的细菌溶液中,过氧化氢的终浓度将是过氧化氢溶液浓度的一半。

食谱

  1. Luria肉汤
    25g LB肉汤(Miller)/L无菌水

致谢

这项工作得到了加拿大卫生研究院(CIHR)的经营资助。 B.B.F. 是不列颠哥伦比亚大学Peter Wall杰出教授。 这项工作没有竞争利益。 该协议改编自我们实验室的先前工作(van der Heijden等人,2015)。 我们真诚地感谢俄勒冈大学的James Remington博士提供原始的pRSETB roGFP2构建体。

参考文献

  1. Gutscher,M.,Pauleau,A.L.,Marty,L.,Brach,T.,Wabnitz,G.H.,Samstag,Y.,Meyer,A.J.and Dick,T.P。(2008)。 细胞内谷胱甘肽氧化还原电位的实时成像。/em> 5(6):553-559。
  2. Hanson,G.T.,Aggeler,R.,Oglesbee,D.,Cannon,M.,Capaldi,R.A.,Tsien,R.Y。和Remington,S.J。(2004)。 用氧化还原敏感性绿色荧光蛋白指示剂调查线粒体氧化还原电位。 Biol Chem 279(13):13044-13053
  3. Hoiseth,S.K。和Stocker,B.A。(1981)。 芳香依赖型沙门氏菌typhimurium 是非毒性的,有效的活疫苗。 Nature 291(5812):238-239。
  4. Imlay,J.A。(2013)。 氧化应激的分子机制和生理后果:来自模型细菌的教训 < em> Nat Rev Microbiol 11(7):443-454。
  5. van der Heijden,J.,Bosman,E.S.,Reynolds,L.A。和Finlay,B.B。(2015)。 直接测量巨噬细胞内沙门氏菌的氧化和亚硝化胁迫动力学。 Natl Acad Sci USA 111(2):560-565。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. 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; Full Text
  2. van der Heijden, J., Bosman, E. S.,Reynolds, L. A. and Finlay, B. B. (2015). Directmeasurement of oxidative and nitrosative stress dynamics in Salmonella insidemacrophages. Proc Natl Acad Sci U S A 112(2): 560-565.




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