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Bacterial Intracellular Sodium Ion Measurement using CoroNa Green
使用CoroNa绿测定细菌细胞内钠离子   

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Abstract

The bacterial flagellar type III export apparatus consists of a cytoplasmic ATPase complex and a transmembrane export gate complex, which are powered by ATP and proton motive force (PMF) across the cytoplasmic membrane, respectively, and transports flagellar component proteins from the cytoplasm to the distal end of the growing flagellar structure where their assembly occurs (Minamino, 2014). The export gate complex can utilize sodium motive force in addition to PMF when the cytoplasmic ATPase complex does not work properly. A transmembrane export gate protein FlhA acts as a dual ion channel to conduct both H+ and Na+ (Minamino et al., 2016). Here, we describe how to measure the intracellular Na+ concentrations in living Escherichia coli cells using a sodium-sensitive fluorescent dye, CoroNa Green (Minamino et al., 2016). Fluorescence intensity measurements of CoroNa Green by epi-fluorescence microscopy allows us to measure the intracellular Na+ concentration quantitatively.

Keywords: Bacteria(细菌), Bacterial Flagellum(细菌鞭毛), FlhA(FlhA), Fluorescence microscopy(荧光显微镜检查), Proton motive force(质子动力), Sodium ion channel(钠离子通道), PomAB complex(PomAB复合物), Type III protein export(III型蛋白输出)

Background

Measurements of intracellular Na+ concentrations by fluorescence imaging techniques are able to be more accurately and quantitatively performed at single cell levels, because background noise of each cell can be removed by image analysis procedures. Lo et al. have established a protocol for measurement of the cytoplasmic Na+ concentrations in living E. coli cells using a sodium-sensitive fluorescent dye, Sodium Green and have shown that the cytoplasmic Na+ concentration maintains around 10 mM in E. coli over a wide range of 0 to 100 mM of the external Na+ concentrations (Lo et al., 2006). Because CoroNa Green, which is a sodium-sensitive fluorescent dye too, shows much higher cell permeability than Sodium Green, we have developed a CoroNa Green-based protocol to measure the intracellular Na+ concentrations in E. coli. (Minamino et al., 2016). This protocol allows us to quite easily and reproducibly measure the intracellular Na+ concentration of E. coli cells overexpressing FlhA or PomAB complex, both of which have the Na+ channel activity.

Materials and Reagents

  1. 1.5 ml Eppendorf tubes
  2. Aluminum foil
  3. Slide
  4. 24 x 32 mm coverslip (thickness: 0.12-0.17 mm) (Matsunami Glass, catalog number: C024321 )
  5. 18 x 18 mm coverslip (thickness: 0.12-0.17 mm) (Matsunami Glass, catalog number: C018181 )
  6. Double-sided tape (NICHIBAN, catalog number: NW-5 )
  7. Pipette tips   
  8. Filter paper
  9. E. coli BL21(DE3) cell (Novagen)
  10. pBAD24 expression vector (Guzman et al., 1995)
  11. pNH319 (pBAD24/ N-His-FLAG-FlhA) (Minamino et al., 2016)
  12. pBAD-PomΔplug (pBAD24/ PomA + PomB[∆41-120]) (Minamino et al., 2016)
  13. Ampicillin sodium (Wako Pure Chemical Industries, catalog number: 014-23302 )
  14. L-arabinose (Wako Pure Chemical Industries, catalog number: 010-04582 )
  15. CoroNa Green-AM (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C36676 )
  16. Ethylenediamine-N, N, N', N'-tetraacetic acid, dipotassium salt, dihydrate (EDTA·2K) (Dojindo, catalog number: 340-01511 )
  17. Sodium chloride (Wako Pure Chemical Industries, catalog number: 192-13925 )
  18. Gramicidin (Thermo Fisher Scientific, catalog number: G6888 )
  19. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma-Aldrich, catalog number: C2759 )
  20. Bacto tryptone (BD, catalog number: 211705 )
  21. Potassium dihydrogenphosphate (Wako Pure Chemical Industries, catalog number: 164-22635 )
  22. Dipotassium hydrogenphosphate (Wako Pure Chemical Industries, catalog number: 164-04295 )
  23. T-broth (see Recipes)

Equipment

  1. haking incubator (30 °C, at 200 rpm) (TAITEC, model: BR-40LF )
  2. Centrifuge (able to hold 1.5 ml tube, spin at 6,000 x g) (TOMY SEIKO, model: MX-305 )
  3. Tube rotator (able to hold 1.5 ml tubes, rotate at 5 rpm) (WAKENBTECH, model: WKN-2210 )
  4. Single channel pipettes (1,000 µl, 100 µl) (Gilson, model: P-1000 , P-100 )
  5. Spectrophotometer (able to measure OD600) (Shimadzu, model: UV-1800 )
  6. Inverted fluorescence microscope (Olympus, model: IX-73 )
    1. 100x oil immersion objective lens (Olympus, model: UPLSAPO100XO , NA 1.4)
    2. sCMOS camera (Andor Technology, model: Zyla4.2 )
    3. Mercury light source system (Olympus, model: U-HGLGPS )
    4. Fluorescence mirror unit (Olympus, model: U-FGFP [Excitation BP 460-480; Emission BP 495-540])

Software

  1. Image J (National Institutes of Health, https://imagej.nih.gov/ij/)
  2. KaleidaGraph (Synergy Software)
  3. Microsoft Excel (Microsoft)

Procedure

Note: Carry out procedures at ca. 23 °C unless otherwise specified.

  1. Transform competent cells of the E. coli BL21(DE3) strain, which are treated with calcium chloride method, with a pBAD24-based plasmid encoding FlhA or the PomAB∆plug, which is a Na+ channel complex of the marine Vibrio flagellar motor (Morimoto and Minamino, 2014).
  2. Grow the resulting transformants overnight in duplicate in 5 ml T-broth containing 100 µg/ml ampicillin at 30 °C.
  3. Inoculate 50 µl of overnight culture of the E. coli BL21 (DE3) cells carrying the pBAD24-based plasmid into 5 ml of fresh T-broth containing 100 µg/ml ampicillin and incubate at 30 °C for 4 h with shaking at 150 rpm. (The cell density reaches an OD600 of ca. 1.0.)
  4. Add 0.2% arabinose (final concentration, w/v) to induce over-expression of FlhA or PomAB∆plug derived by a pBAD promotor on the pBAD24 vector and keep shaking for another 1 h.
  5. After 1 h: Transfer 200 µl of each culture to a 1.5 ml Eppendorf tube and then collect the cells by centrifugation (6,000 x g, 2 min).
  6. Suspend the cell pellet gently in 1.0 ml of fresh TB using a pipette (P-1000).
  7. Centrifuge at 6,000 x g for 2 min.
  8. Discard supernatant.
  9. Repeat steps 6-8 (2 x).
  10. Resuspend the cells in 100 µl of T-broth containing 40 µM CoroNa Green (Invitrogen) and 10 mM EDTA.
  11. Cover the tubes with aluminum foil to keep them in the dark.
  12. Rotate the tubes at 5 rpm for 60 min using a tube rotator.
  13. Centrifuge at 6,000 x g for 2 min.
  14. Discard supernatant.
  15. Suspend the cell pellet in 1.0 ml of fresh T-broth.
  16. Centrifuge at 6,000 x g for 2 min.
  17. Discard supernatant.
  18. Repeat steps 14-16 (3 x).
  19. Resuspend the cells in 500 µl of fresh T-broth.
  20. Make a tunnel slide by sandwiching double-sided tape between 24 x 32 mm coverslip (bottom side) and 18 x 18 mm coverslip (top side) (see Video 1).

    Video 1. Preparation for a tunnel slide

  21. Add the cell suspension to the tunnel slide and leave for 5-10 min to attach the cells onto the coverslip surface.
  22. Wash out unbound cells by supplying 100 µl of fresh T-broth using a pipette (P-100). Absorb the excess amount of the medium with a piece of filter paper.
  23. Put the sample on an inverted fluorescence microscope.
  24. Select a 100x objective.
  25. Capture fluorescence images of CoroNa Green using a mirror unit for GFP (U-FGFP, Ex: 460–480, Em: 495–540, Olympus) by an sCMOS camera every 100 msec.
  26. Measure the fluorescence intensity of the cells stained with CoroNa Green in T-broth with 0 mM, 5 mM, 10 mM, 20 mM, 50 mM or 100 mM NaCl in the presence of 20 µM gramicidin and 5 µM CCCP.

Data analysis

Analyze fluorescent images by an image analysis software ImageJ (National Institutes of Health).

  1. Apply a rectangular mask for the fluorescent image of the bacterial cell body of 8 x 8 pixels to the ROI (region of interest).
  2. Subtract the total background intensity from each pixel value. The instrumental background intensity is defined as the mean pixel intensity within the ROI of a nearby cell-less region.
  3. Measure the intensity of CoroNa Green from more than 100 cells at each condition.
  4. Make the calibration curve from the data acquired at various external Na+ concentrations in the presence of 20 µM gramicidin and 5 µM CCCP, both of which equilibrate Na+ concentrations inside and outside E. coli cells (Figure 1).


    Figure 1. Calibration curve for intracellular Na+ concentrations using CoroNa Green. The fluorescence intensity of the cells stained with CoroNa Green were measured in T-broth containing 20 µM gramicidin and 5 µM CCCP for 30 min at various Na+ concentrations (5 mM, 10 mM, 20 mM, 50 mM and 100 mM NaCl). The fluorescent intensities of intracellular CoroNa Green were plotted against external Na+ concentrations and then were fitted by the Hill equation (black line). Vertical bars indicate standard deviations. Because the staining efficiency with CoroNa Green is quite variable among E. coli cells, the standard deviations of fluorescence intensity become large at high Na+ concentrations.

  5. Fit the calibration curve to the Hill equation by KaleidaGraph (Synergy Software).

  6. Estimate the intracellular Na+ concentration through the fluorescent intensity of CoroNa Green of each cell using the calibration curve.
  7. Calculate the average of the intracellular Na+ concentration and standard error of the mean from the data of more than 100 cells using Microsoft Excel (Microsoft).

Representative data



Figure 2. Measurement of intracellular Na+ concentrations using CoroNa Green. Effect of overexpression of FlhA or PomAB∆plug on intracellular Na+ concentrations in E. coli cells. Intracellular Na+ concentrations were measured with CoroNa Green in the presence and absence of 100 mM NaCl at an external pH of 7.0. The E. coli BL21(DE3) strain was transformed with pBAD24 (Vector, V), pNH319 (FlhA) or pBAD-Pom∆plug (PomAB∆plug). For each transformant, 200 cells were measured. Vertical bars indicate standard errors. (Modified from Minamino et al., 2016)

Notes

  1. To investigate the effect of extracellular sodium concentration, whole experiments are done using T-broth with or without 100 mM NaCl.
  2. 10 mM EDTA is required to allow CoroNa Green to be incorporated into the E. coli cells.
  3. To avoid contamination of sodium, EDTA·2K must be used (Do not use EDTA·2Na).

Recipes

  1. T-broth
    1% Bacto tryptone, 10 mM potassium phosphate, pH 7.0 with or without 100 mM NaCl

Acknowledgments

This protocol was modified from a previous work (Lo et al., 2006). This research has been supported in part by JSPS KAKENHI Grant Numbers JP15K14498 and JP15H05593 to YVM, JP21227006 and JP25000013 to KN and JP26293097 to TM and MEXT KAKENHI Grant Numbers JP26115720 and JP15H01335 to YVM and JP23115008, JP24117004, JP25121718 and JP15H01640 to TM.

References

  1. Guzman, L. M., Belin, D., Carson, M. J., Beckwith. J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177(14): 4121-4130.
  2. Lo, C. J., Leake, M. C., Berry, R. M. (2006). Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells. Biophys J 90(1): 357-365.
  3. Minamino, T. (2014).  Protein export through the bacterial flagellar type III export pathway. Biochim Biophys Acta 1843(8): 1642-1648.
  4. Minamino, T., Morimoto, Y. V., Hara, N., Aldridge, P. D. and Namba, K. (2016). The bacterial flagellar type III export gate complex is a dual fuel engine that can use both H+ and Na+ for flagellar protein export. PLoS Pathog 12(3): e1005495.
  5. Morimoto, Y. V. and Minamino, T. (2014). Structure and function of the bi-directional bacterial flagellar motor. Biomolecules 4(1): 217-234.

简介

细菌鞭毛III型出口设备由细胞质ATP酶复合物和跨膜出口门复合物组成,分别由ATP和质子动力(PMF)驱动跨越细胞质膜,并将鞭毛成分蛋白从细胞质转运到远端结束它们的组装发生的鞭毛结构(Minamino,2014)。当细胞质ATPase复合物不能正常工作时,出口门复合物可以利用除PMF之外的钠动力。跨膜出口门蛋白FlhA充当双离子通道,以进行H + 和Na + (Minamino等人,2016)。在这里,我们描述如何使用钠敏感荧光染料CoroNa Green(Minamino等人)测量活细胞大肠杆菌细胞中的细胞内Na+浓度,。,2016)。通过荧光显微镜检测CoroNa Green的荧光强度,可以定量测定细胞内Na +的浓度。

背景 通过荧光成像技术测量细胞内Na +浓度能够在单细胞水平上更精确和定量地进行,因为每个细胞的背景噪声可以通过图像分析程序去除。 Lo <等等。已经建立了用于测量活体中的细胞质Na +浓度的方案。使用钠敏感荧光染料钠绿,并显示细胞质Na +浓度维持在10mM左右。大肠杆菌在0至100mM的外部Na +浓度范围的宽范围内(Lo et al。,2006)。因为CoroNa Green也是钠敏感荧光染料,它比钠绿显示出更高的细胞渗透性,因此我们开发了一种基于CoroNa Green的方案来测量E细胞内Na +浓度。大肠杆菌。 (Minamino等人,2016)。该方案允许我们非常容易且可重复地测量E的细胞内Na +浓度。大肠杆菌细胞过表达FlhA或PomAB复合物,两者均具有Na +通道活性。

关键字:细菌, 细菌鞭毛, FlhA, 荧光显微镜检查, 质子动力, 钠离子通道, PomAB复合物, III型蛋白输出

材料和试剂

  1. 1.5 ml Eppendorf管
  2. 铝箔
  3. 幻灯片
  4. 24 x 32毫米盖玻片(厚度:0.12-0.17毫米)(松本玻璃,目录号:C024321)
  5. 18 x 18毫米盖玻片(厚度:0.12-0.17毫米)(松本玻璃,目录号:C018181)
  6. 双面胶带(NICHIBAN,目录号:NW-5)
  7. 移液器提示
  8. 滤纸
  9. E。 大肠杆菌BL21(DE3)细胞(Novagen)
  10. pBAD24表达载体(Guzman等人,1995)
  11. pNH319(pBAD24/N-His-FLAG-FlhA)(Minamino等人,2016)
  12. pBAD-PomΔplug(pBAD24/PomA + PomB [Δ41-120])(Minamino等人,2016)
  13. 氨苄青霉素钠(和光纯药工业公司,目录号:014-23302)
  14. L-阿拉伯糖(Wako Pure Chemical Industries,目录号:010-04582)
  15. CoroNa Green-AM(Thermo Fisher Scientific,Molecular Probes TM,目录号:C36676)
  16. 乙二胺-N,N,N',N'-四乙酸,二钾盐,二水合物(EDTA·2K)(Dojindo,目录号:340-01511)
  17. 氯化钠(和光纯药,目录号:192-13925)
  18. 革兰定酸(Thermo Fisher Scientific,目录号:G6888)
  19. 羰基氰化物3-氯苯腙(CCCP)(Sigma-Aldrich,目录号:C2759)
  20. Bacto胰蛋白胨(BD,目录号:211705)
  21. 磷酸二氢钾(Wako Pure Chemical Industries,目录号:164-22635)
  22. 磷酸氢二钾(Wako Pure Chemical Industries,目录号:164-04295)
  23. T肉汤(见食谱)

设备

  1. (30°C,200 rpm)(TAITEC,型号:BR-40LF)
  2. 离心机(能够容纳1.5ml管,以6,000 x g旋转)(TOMY SEIKO,型号:MX-305)
  3. 管旋转器(能够容纳1.5ml管,以5rpm旋转)(WAKENBTECH,型号:WKN-2210)
  4. 单通道移液管(1,000μl,100μl)(Gilson,型号:P-1000,P-100)
  5. 分光光度计(能够测量OD 600)(Shimadzu,型号:UV-1800)
  6. 倒置荧光显微镜(Olympus,型号:IX-73)
    1. 100x油浸物镜(Olympus,型号:UPLSAPO100XO,NA 1.4)
    2. sCMOS相机(安道尔科技,型号:Zyla4.2)
    3. 水银光源系统(奥林巴斯,型号:U-HGLGPS)
    4. 荧光镜单元(Olympus,型号:U-FGFP [Excitation BP 460-480; Emission BP 495-540])

软件

  1. Image J(National Institutes of Health, https://imagej.nih.gov/ij/
  2. KaleidaGraph(Synergy Software)
  3. Microsoft Excel(Microsoft)

程序

注意:执行程序在ca. 23°C,除非另有说明

  1. 转换E的能力细胞。用氯化钙处理的大肠杆菌BL21(DE3)菌株与编码FlhA的pBAD24基质粒或PomABΔplug(其是海洋中的Na +)通道复合物鞭毛虫电动机(Morimoto and Minamino,2014)。
  2. 在30℃下在含有100μg/ml氨苄青霉素的5ml T-broth中一式两份生成所得转化体一夜。
  3. 接种50微升E过夜培养物。将含有pBAD24的质粒的大肠杆菌BL21(DE3)细胞加入5ml含有100μg/ml氨苄青霉素的新鲜T培养液中,并在30℃下以150rpm摇动孵育4小时。 (细胞密度达到约1.0的OD 600)
  4. 加入0.2%阿拉伯糖(终浓度,w/v)以诱导由pBAD启动子在pBAD24载体上衍生的FlhA或PomABΔplug的过量表达,并保持另外1小时的振荡。
  5. 1小时后:将200μl每种培养物转移到1.5ml Eppendorf管中,然后通过离心(6,000 x g,2分钟)收集细胞。
  6. 使用移液管(P-1000)将细胞沉淀物轻轻悬浮于1.0 ml新鲜结核菌中
  7. 以6,000 x g离心2分钟。
  8. 弃去上清液。
  9. 重复步骤6-8(2 x)。
  10. 将细胞重悬于含有40μMCoroNa Green(Invitrogen)和10mM EDTA的100μlT培养液中。
  11. 用铝箔盖住管子,使其保持在黑暗中。
  12. 使用管旋转器以5rpm旋转管子60分钟。
  13. 以6,000 x g离心2分钟。
  14. 弃去上清液。
  15. 将细胞沉淀悬浮于1.0ml新鲜的T-broth中
  16. 以6,000 x g离心2分钟。
  17. 弃去上清液。
  18. 重复步骤14-16(3 x)。
  19. 将细胞重新悬浮在500μl新鲜的T-broth中
  20. 通过将双面胶带夹在24 x 32毫米盖玻片(底侧)和18 x 18毫米盖玻片(上侧)之间,形成隧道滑轨(见视频1)。

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  21. 将细胞悬浮液添加到隧道载玻片上,放置5-10分钟,将细胞附着在盖玻片表面上
  22. 通过使用移液管(P-100)提供100μl新鲜的T-肉汤,清洗未结合的细胞。用一张滤纸吸收多余的介质。
  23. 将样品置于倒置荧光显微镜上
  24. 选择一个100x的目标。
  25. 使用sCMOS相机每100毫秒,使用GFP(U-FGFP,Ex:460-480,Em:495-540,Olympus)的镜像单元捕获CoroNa Green的荧光图像。
  26. 在20μM短杆菌肽和5μMCCCP存在下,测量在含有0mM,5mM,10mM,20mM,50mM或100mM NaCl的T-broth中用CoroNa Green染色的细胞的荧光强度。

数据分析

通过图像分析软件ImageJ(National Institutes of Health)分析荧光图像。

  1. 将8 x 8像素的细菌细胞体的荧光图像应用于ROI(感兴趣区域)的矩形掩模。
  2. 从每个像素值减去总背景强度。工具背景强度被定义为附近无细胞区域的ROI内的平均像素强度
  3. 在每个条件下测量超过100个细胞的CoroNa Green的强度。
  4. 在存在20μM短杆菌肽和5μMCCCP的情况下,从各种外部Na + 浓度获得的数据进行校准曲线,两者都平衡了内部和外部的Na + E。大肠杆菌细胞(图1)。


    图1.使用CoroNa Green的细胞内Na +浓度的校准曲线使用CoroNa Green染色的细胞的荧光强度在含有20μM短杆菌肽的T-broth中测量,5 μMCCCP在各种Na +浓度(5mM,10mM,20mM,50mM和100mM NaCl)下30分钟。将细胞内CoroNa Green的荧光强度与外部Na +浓度作图,然后通过Hill方程(黑线)拟合。垂直条表示标准偏差。因为CoroNa Green的染色效率在E之间变化很大。大肠杆菌细胞,荧光强度的标准偏差在高Na +浓度时变大。

  5. 通过KaleidaGraph(Synergy Software)将校准曲线拟合到Hill方程式。

  6. 使用校准曲线,通过每个细胞的CoroNa Green的荧光强度估算细胞内Na +浓度。
  7. 使用Microsoft Excel(Microsoft)从100多个细胞的数据计算细胞内Na + 浓度的平均值和平均值的标准误差。

代表数据



图2.使用CoroNa Green测量细胞内Na +浓度。将FlhA或PomABΔplug的过表达对细胞内Na +浓度的影响< E> E。大肠杆菌细胞。在存在和不存在100mM NaCl的情况下,在7.0的外部pH下,用CoroNa Green测量细胞内Na +浓度。 E。用pBAD24(Vector,V),pNH319(FlhA)或pBAD-PomΔplug(PomABΔplug)转化大肠杆菌BL21(DE3)菌株。对于每个转化体,测量200个细胞。垂直条表示标准错误。 (从Minamino等人修改,2016)

笔记

  1. 为了研究细胞外钠浓度的影响,使用含有或不含100mM NaCl的T-肉汤进行全部实验
  2. 需要10 mM EDTA才能将CoroNa Green加入到E中。大肠杆菌细胞。
  3. 为了避免钠的污染,必须使用EDTA·2K(不要使用EDTA·2Na)。

食谱

  1. T-broth
    1%Bacto胰蛋白胨,10mM磷酸钾,pH 7.0,含或不含100mM NaCl

致谢

该协议是从以前的工作中修改的(Lo et al。,2006)。该研究部分由JSS KAKENHI Grant Numbers JP15K14498和JP15H05593授予YVM,JP21227006和JP25000013至KN和JP26293097以及TMXT KAKENHI Grant Numbers JP26115720和JP15H01335授予YVM和JP23115008,JP24117004,JP25121718和JP15H01640。

参考文献

  1. Guzman,L.M.,Belin,D.,Carson,M.J.,Beckwith。 J.(1995)。严格的规管,调制和通过含有阿拉伯糖PBAD启动子的载体的高水平表达.J1细菌177(14):4121-4130。
  2. Lo,CJ,Leake,MC,Berry,RM(2006)。  通过细菌鞭毛III型出口途径的蛋白质出口。/a>  Biochim Biophys Acta 1843(8):1642-1648。
  3. Minamino,T.,Morimoto,YV,Hara,N.,Aldridge,PD和Namba,K。(2016)。细菌鞭毛III型出口门复合体是双燃料发动机,可以同时使用H +和Na +进行鞭毛蛋白质输出。 em> PLoS Pathog 12(3):e1005495。
  4. Morimoto,YV和Minamino,T。(2014)。双向细菌鞭毛马达的结构和功能生物分子 4(1):217-234。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Morimoto, Y. V., Namba, K. and Minamino, T. (2017). Bacterial Intracellular Sodium Ion Measurement using CoroNa Green. Bio-protocol 7(1): e2092. DOI: 10.21769/BioProtoc.2092.
  2. Minamino, T., Morimoto, Y. V., Hara, N., Aldridge, P. D. and Namba, K. (2016). The bacterial flagellar type III export gate complex is a dual fuel engine that can use both H+ and Na+ for flagellar protein export. PLoS Pathog 12(3): e1005495
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