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Quantification of Bacterial Polyhydroxybutyrate Content by Flow Cytometry
流式细胞术定量分析细菌聚羟基丁酸酯含量   

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Abstract

We describe here a detailed protocol for the quantification of the intracellular content of polyhydroxybutyrate (PHB) in a population of bacterial cells by flow cytometry, which is based on a consensus of several previously reported works.

Keywords: Polyhydroxybutyrate(聚羟基丁酸酯), PHB(PHB), Bacteria(细菌), Flow cytometry(流式细胞术), Nile red(尼罗红)

Background

Upon nutrient exhaustion and in the presence of an available carbon source, several bacterial species (e.g., Cupriavidus necator, Sinorhizobium meliloti, species of Pseudomonas) switch on the accumulation of the neutral polymer of β-hydroxybutyrate, polyhydroxybutyrate (PHB), for storage of carbon and reducing power (Jendrossek and Pfeiffer, 2014). Classical methods for quantification of bacterial PHB content are based on its initial depolymerization, followed by the quantitative detection of specific chemical derivatives of β-hydroxybutyrate by UV spectrophotometry or gas chromatography (Law and Slepecky, 1961; Braunegg et al., 1978). More recently, the application of the fluorochrome Nile red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) (Greenspan and Fowler, 1985) to stain intracellular PHB granules (Müller et al., 1993) led to the development of novel rapid fluorometric methods to quantify the content of the polymer in bacterial samples. Subsequently, coupling of Nile red–based PHB staining to flow cytometry made possible the quantification of the polymer content at a single-cell level within bacterial populations (Gorenflo et al., 1999; Kacmar et al., 2006; Tyo et al., 2006; Alves et al., 2017; Lagares Jr. et al., 2017). We report here a detailed protocol that summarizes the consensus steps for the quantification of PHB in bacterial cells by Nile red staining coupled to flow cytometry.

Materials and Reagents

  1. Pipette tips (2-200 µl and 100-1,000 µl tips)
  2. 1.5 ml Eppendorf tubes
  3. 12 x 75-mm Falcon® 5 ml uncapped Round Bottom Polystyrene Test tubes (Corning, catalog number: 352052 )
  4. Bacterial samples for analysis
    Note: The bacteria can be harvested from multiple sources (e.g., liquid cultures, colonies on semisolid agar plates, natural environments). However, it must be taken into account that adaptations in the steps for cell preparation should be considered, depending on the specific source of the cells.
  5. Absolute ethanol (e.g., Sigma-Aldrich, catalog number: E7023 )
  6. Sterile deionized water (e.g., Sigma-Aldrich, catalog number: W3500 )
  7. Sodium chloride (NaCl) (e.g., Sigma-Aldrich, catalog number: S9888 )
  8. Potassium chloride (KCl) (e.g., Sigma-Aldrich, catalog number: P3911 )
  9. Sodium phosphate dibasic (Na2HPO4) (e.g., Sigma-Aldrich, catalog number: S9763 )
  10. Potassium phosphate monobasic (KH2PO4) (e.g., Sigma-Aldrich, catalog number: P0662 )
  11. Dimethyl sulfoxide (DMSO) (e.g., Sigma-Aldrich, catalog number: D8418 )
  12. Nile red stain (e.g., Sigma-Aldrich, catalog number: N3013 )
  13. 1 N HCl and 1 N NaOH solutions for pH adjustments
  14. Cytometer sheath fluid (e.g., BD FACSFlowTM solution, BD, catalog number: 342003 )
  15. Full-strength bleach (for waste disposal)
  16. Phosphate-buffered saline (PBS; see Recipes for 10x and 1x solution preparation)
  17. Permeabilization solution (see Recipes)
  18. Nile red stock solution (see Recipes)

Equipment

  1. Pipets (e.g., Gilson Pipetman Classic) (Gilson, models: P20 , P200 , P1000 )
  2. Centrifuge for 1.5 ml Eppendorf tubes (e.g., Eppendorf, model: 5424 , catalog number: 5424000010)
  3. pH meter
  4. Spectrophotometer suitable for measurement of optical density at 600 nm (OD600)
  5. FACSCalibur flow cytometer (BD, model: FACSCALIBUR ) equipped with an excitation laser of 488-nm-wavelength and an emission filter with bandwidth of 585 ± 45 nm
    Note: The suitability of other flow cytometers than FACSCalibur (BD, USA) for this assay is subjected to the availability of a proper excitation laser source and of the filter for the emitted fluorescence, as indicated above.

Software

  1. Flow cytometer acquisition software
  2. FlowJo [FlowJo, USA]

Procedure

  1. Bacterial PHB staining
    1. Pellet down ca. 5 x 108 bacterial cells by centrifugation at 7,500 x g for 5 min at room temperature (ca. equivalent to 1 ml of an Escherichia coli liquid culture at an OD600 of 0.5).
      Notes:
      1. Note that lower amounts of cells will be subjected to the flow cytometric analysis, however, higher initial bacterial amounts are easier to handle during the centrifugation steps prior to staining.
      2. Include as a negative control for PHB measurements a bacterial population (ideally of the same species) grown under conditions that are not permissive for PHB accumulation (here referred to as PHB- bacteria), and a population of bacteria loaded with PHB (PHB+ bacteria) that has not been stained with Nile red, as a negative control for non-specific fluorescence.
      3. Centrifugation speed and time need to be adjusted in each particular case to optimize bacterial collection at the bottom of the tube, mainly depending on the viscosity of the supernatant.
    2. Wash the bacterial pellet once with 500 µl 1x PBS (see Recipes), and resuspend cells in 500 µl of permeabilization solution (see Recipes). Incubate the suspension at room temperature (RT) for 15 min.
    3. Collect the cells by centrifugation at 7,500 x g for 5 min at RT.
    4. Resuspend the pellet in 1 ml 1x PBS and dilute the bacterial suspension 1:100 with 1x PBS to a final volume of 1 ml (i.e., to reach a bacterial concentration of ca. 5 x 106 cells per milliliter). Mix well by vortexing for a few seconds.
    5. Add 20 µl of Nile red stock solution (see Recipes) to the diluted bacterial suspension. Mix well by vortexing for a few seconds and incubate protected from light at RT for 30 min.
      Note: Please note that, as reviewed by Alves et al. (2017), the concentration of Nile red that is required to optimally stain the bacterial PHB granules largely varies among species (i.e., from 0.032 µM for C. necator to 94.2 µM for E. coli). The concentration reported in this protocol has been shown to properly stain PHB in Sinorhizobium meliloti (Lagares Jr. et al., 2017). Nile red concentration and exposure time should be optimized for each bacterial species.
    6. Transfer samples to 12 x 75-mm 5 ml Falcon® test tubes and proceed to analyze them by flow cytometry as indicated in the following steps.
      Note: Since Nile red fluorescence starts to decay shortly after its addition (Alves et al., 2017), it is critical to measure all the samples at an equal time after they are stained.

  2. Flow cytometry
    1. Set the flow cytometer for cell excitation by the 488-nm-wavelength argon laser and Nile red fluorescence acquisition by the filter with bandwidth centered at 585 nm (on FL2 channel if using FACScalibur flow cytometer). Register Forward (FSC)- and side (SSC)-scattered light as well. Adjust FSC, SSC and FL2 detector voltages, if necessary. Set the sample flow rate at the low flow position (e.g., ca. 12 µl/sec).
      Notes:
      1. Carefully follow flow cytometer facility use guidelines (e.g., warm-up instructions, sheath and waste management).
      2. In general, setting FSC and SSC channel voltages to E02 and 300, respectively, would allow to clearly identify bacterial populations in FSCxSSC dot plots. FL2 channel voltage should be adjusted to maximize the difference of the median values corresponding to PHB+ and PHB- bacterial populations (e.g., FL2 channel voltage was set to 500 during acquisition of the data plotted in Figure 1).
    2. Analyze samples and controls with the flow cytometer. Record at least 100,000 events per sample.

Data analysis

Analyze data with a suitable software (e.g., FlowJo [FlowJo, USA]). The median fluorescence intensity (MFI; expressed in AFU, Arbitrary Fluorescence Units) from histograms of FL2-H has been reported to linearly correlate well with the bacterial PHB content (Gorenflo et al., 1999; Kacmar et al., 2006; Tyo et al., 2006; Ratcliff et al., 2008; Alves et al., 2017). The population of bacteria loaded with PHB in each sample could be identified by comparing the corresponding MFI with that of the bacteria that were analyzed as a negative control (see Figure 1). In bacterial samples displaying phenotypic heterogeneity, the percentage of PHB+ cells could be calculated. At least three independent samples must be processed as biological replicates.


Figure 1. Example of dot plots (upper panels) and histograms (lower panels) from a sample of Sinorhizobium meliloti cells grown under a nitrogen-limiting and carbon-excess condition that enables PHB accumulation (right panels, PHB+ bacteria), and from a sample of the same bacterial species grown under nitrogen sufficient conditions as a negative control for PHB accumulation (left panels, PHB- bacteria) (Lagares Jr. et al., 2017). The x-axis represents the cellular PHB content estimated by the fluorescence of Nile red (expressed in AFU, Arbitrary Fluorescence Units) that was registered by the channel FL2. The median fluorescence intensity (MFI) values for each bacterial population are indicated in the corresponding histogram insets.

Recipes

  1. Phosphate-buffered saline (PBS) (10x concentrated)
    8 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    Adjust pH to 7.2 with 1 N NaOH or 1 N HCl and make up to 100 ml with water (usually less than 1 ml of 1 N NaOH or 1 N HCl stocks would be enough)
    Autoclave and store at RT
  2. Permeabilization solution (ethanol 35% in PBS [vol/vol])
    350 µl absolute ethanol
    100 µl 10x PBS
    550 µl sterile deionized water
  3.  Nile red stock solution
    1 mg Nile red
    Make up to 1 ml with DMSO
    Store at 4 °C

Acknowledgments

This work was supported by the National Scientific and Technical Research Council (CONICET), the National Agency for Promotion of Science and Technology (ANPCyT), and Universidad Nacional de Quilmes. A.L. was supported by CONICET fellowships. C.V. is a researcher of CONICET. The current protocol was adapted from (Lagares Jr. et al., 2017), which was mainly based on several previously reported works (Kacmar et al., 2006; Ratcliff et al., 2008). The authors declare no conflicts of interest.

References

  1. Alves, L. P., Almeida, A. T., Cruz, L. M., Pedrosa, F. O., de Souza, E. M., Chubatsu, L. S., Muller-Santos, M. and Valdameri, G. (2017). A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry. Braz J Med Biol Res 50(1): e5492.
  2. Braunegg, G., Sonnleitner, B. and Lafferty, R. M. (1978). A rapid gas chromatographic method for the determination of poly-β-hydroxybutyric acid in microbial biomass. Appl Microbiol Biotechnol 6: 29-37.
  3. Gorenflo, V., Steinbuchel, A., Marose, S., Rieseberg, M. and Scheper, T. (1999). Quantification of bacterial polyhydroxyalkanoic acids by Nile red staining. Appl Microbiol Biotechnol 51(6): 765-772.
  4. Greenspan, P. and Fowler, S. D. (1985). Spectrofluorometric studies of the lipid probe, nile red. J Lipid Res 26(7): 781-789.
  5. Jendrossek, D. and Pfeiffer, D. (2014). New insights in the formation of polyhydroxyalkanoate granules (carbonosomes) and novel functions of poly(3-hydroxybutyrate). Environ Microbiol 16(8): 2357-2373.
  6. Kacmar, J., Carlson, R., Balogh, S. J. and Srienc, F. (2006). Staining and quantification of poly-3-hydroxybutyrate in Saccharomyces cerevisiae and Cupriavidus necator cell populations using automated flow cytometry. Cytometry A 69(1): 27-35.
  7. Lagares, A., Jr., Ceizel Borella, G., Linne, U., Becker, A. and Valverde, C. (2017). Regulation of polyhydroxybutyrate accumulation in Sinorhizobium meliloti by the trans-encoded small RNA MmgR. J Bacteriol 199(8).
  8. Law, J. H. and Slepecky, R. A. (1961). Assay of poly-β-hydroxybutyric acid. J Bacteriol 82: 33-36.
  9. Müller, S., Lösche, A. and Bley, T. (1993). Staining procedures for flow cytometric monitoring of bacterial populations. Acta Biotechnol 13: 289-297.
  10. Ratcliff, W. C., Kadam, S. V. and Denison, R. F. (2008). Poly-3-hydroxybutyrate (PHB) supports survival and reproduction in starving rhizobia. FEMS Microbiol Ecol 65(3): 391-399.
  11. Tyo, K. E., Zhou, H. and Stephanopoulos, G. N. (2006). High-throughput screen for poly-3-hydroxybutyrate in Escherichia coli and Synechocystis sp. strain PCC6803. Appl Environ Microbiol 72(5): 3412-3417.

简介

我们在这里描述了通过流式细胞术定量细菌细胞群中的聚羟基丁酸酯(PHB)的细胞内含量的详细方案,其基于几个先前报道的作品的共识。

【背景】在营养物质耗尽和存在可利用的碳源的情况下,可以使用几种细菌(例如,Cupriavidus necator,中华根瘤菌Sinorhizobium meliloti, >假单胞菌(Pseudomonas)开启了聚羟基丁酸酯(PHB)中性聚合物的积累,用于储存碳和还原能力(Jendrossek和Pfeiffer,2014)。定量细菌PHB含量的经典方法基于其初始解聚,随后通过紫外分光光度法或气相色谱定量检测β-羟基丁酸的特定化学衍生物(Law and Slepecky,1961; Braunegg等人, 1978)。最近,荧光染料尼罗红(9-二乙氨基-5H-苯并[α]吩恶嗪-5-酮)(Greenspan和Fowler,1985)将PHB颗粒染色(Müller等人,1993)导致了开发新的快速荧光方法来定量细菌样品中聚合物的含量。随后,将基于尼罗红的PHB染色与流式细胞术偶联使得可以定量细菌群体内单细胞水平的聚合物含量(Gorenflo等人,1999; Kacmar等人2006; Tyo等人,2006; Alves等人,2017; Lagares Jr.等人,, ,2017)。我们在这里报告了一个详细的协议,总结了与尼罗红染色联合流式细胞术定量细菌细胞PHB的共识步骤。

关键字:聚羟基丁酸酯, PHB, 细菌, 流式细胞术, 尼罗红

材料和试剂

  1. 移液器吸头(2-200μl和100-1,000μl吸头)
  2. 1.5 ml Eppendorf管
  3. 12 x 75毫米猎鹰®5毫升无盖圆底聚苯乙烯试管(Corning,目录号:352052)
  4. 细菌样本进行分析
    注意:细菌可以从多种来源(例如,液体培养物,半固体琼脂平板上的菌落,自然环境)中收获。但是,必须考虑到细胞制备步骤的适应性,这取决于细胞的具体来源。
  5. 无水乙醇(例如,Sigma-Aldrich,目录号:E7023)
  6. 无菌去离子水(例如,Sigma-Aldrich,目录号:W3500)
  7. 氯化钠(NaCl)(例如,Sigma-Aldrich,目录号:S9888)
  8. 氯化钾(KCl)(例如,Sigma-Aldrich,目录号:P3911)
  9. 磷酸二氢钠(Na 2 HPO 4)(例如,Sigma-Aldrich,目录号:S9763)。
  10. 磷酸二氢钾(KH 2 PO 4)(例如,Sigma-Aldrich,目录号:P0662)
  11. 二甲基亚砜(DMSO)(例如,Sigma-Aldrich,目录号:D8418)
  12. 尼罗红染色(例如,Sigma-Aldrich,目录号:N3013)
  13. 1 N HCl和1 N NaOH溶液进行pH调节
  14. 血细胞计数器鞘液(例如,BD FACSFlow TM溶液,BD,目录号:342003)
  15. 全强度漂白剂(废物处理)
  16. 磷酸盐缓冲盐水(PBS;参见10x和1x溶液制备食谱)
  17. 透化溶液(见食谱)
  18. 尼罗河红储备液(见食谱)

设备

  1. 吸管(例如,Gilson Pipetman Classic)(Gilson,型号:P20,P200,P1000)
  2. 对于1.5ml Eppendorf管(例如Eppendorf,型号:5424,目录号:5424000010)进行离心分离。
  3. pH计
  4. 分光光度计适用于测量在600nm(OD 600)的光密度
  5. FACSCalibur流式细胞仪(BD,型号:FACSCALIBUR),配备有488nm波长的激发激光器和585±45nm带宽的发射滤光片。 注意:如上所述,其他流式细胞仪的适用性比FACSCalibur(BD,USA)用于该测定受到适当的激发激光源和发射荧光的过滤器的可用性。 br />

软件

  1. 流式细胞仪采集软件
  2. FlowJo [FlowJo,美国]

程序

  1. 细菌PHB染色
    1. 沉淀下来 ca。 5×10 8个细菌细胞,在室温下7,500×g离心5分钟(相当于1毫升在0.5的OD 600处的大肠杆菌液体培养物)。
      注意:
      1. 注意,较低量的细胞将进行流式细胞术分析,然而,较高的初始细菌量在染色前的离心步骤期间更容易处理。
      2. 作为PHB测量的阴性对照,包括在不允许PHB积累的条件下生长的细菌群体(理想地为相同物种)(以下称为PHB-细菌)和装载有PHB的细菌群体( PHB +细菌)没有被尼罗红染色,作为非特异性荧光的阴性对照。
      3. 离心速度和时间需要在每种情况下进行调整,以优化管底部的细菌收集,主要取决于上清液的粘度。
    2. 用500μl1x PBS清洗细菌沉淀一次(见食谱),并将细胞重悬于500μl透化溶液中(见食谱)。
      室温(RT)孵育15分钟

    3. 在7500×g离心5分钟收集细胞
    4. 在1ml 1x PBS中重悬沉淀,用1x PBS稀释细菌悬浮液1:100至终体积为1ml(即,达到细菌浓度 > 5×10 6个细胞/毫升)。搅拌几秒钟。
    5. 添加20μL的尼罗红储备液(见食谱)稀释的细菌悬液。
      涡旋混合几秒钟,在室温孵育30分钟 注:请注意,正如Alves等人所述。 (2017)中,为了最佳地对细菌PHB颗粒进行染色所需的尼罗红浓度在很大程度上随着物种的不同而不同(即从C. necator的0.032μM到大肠杆菌的94.2μM)。已经显示在该方案中报道的浓度在苜蓿中华根瘤菌中适当地染色PHB(Lagares Jr.等,2017)。尼罗红浓度和接触时间应该针对每种细菌进行优化。
    6. 将样品转移至12×75mm 5ml Falcon试管并按照以下步骤通过流式细胞仪进行分析。
      注意:由于尼罗红荧光在加入后不久即开始衰减(Alves等,2017),所以在染色后同时测量所有样品是至关重要的。

  2. 流式细胞仪
    1. 用488nm波长的氩激光设置流式细胞仪进行细胞激发,通过带宽集中在585nm的滤波器(如果使用FACScalibur流式细胞仪,在FL2通道上)获取尼罗红色荧光。寄存器正向(FSC)和侧面(SSC)散射光。如有必要,调整FSC,SSC和FL2检测器电压。将流速设置在低流量位置(例如, ca。 12μl/ sec)。
      注意:
      1. 小心遵循流式细胞仪设施使用指南(例如,预热指示,护套和废物管理)。
      2. 通常,将FSC和SSC通道电压分别设置为E02和300,将允许在FSCxSSC点图中清楚地识别细菌种群。应调整FL2通道电压以使对应于PHB + 和PHB 的中值的差值最大化> - 细菌种群(例如,在获取图1中绘制的数据期间,FL2通道电压设置为500)。
    2. 用流式细胞仪分析样本和对照。每个样本记录至少100,000个事件。

数据分析

使用合适的软件(如,FlowJo [FlowJo,USA])分析数据。已经报道了来自FL2-H的直方图的中值荧光强度(MFI;以AFU,任意荧光单位表示)与细菌PHB含量线性相关(Gorenflo等人,1999; Kacmar等人, 2006; Tyo等人,2006; Ratcliff等人,2008; Alves等人,, >,2017)。通过比较相应的MFI和作为阴性对照分析的细菌的MFI,可以鉴定每个样品中装载有PHB的细菌的群体(参见图1)。在显示表型异质性的细菌样品中,可以计算PHB <+>细胞的百分比。
至少有三个独立的样本必须作为生物样本进行处理


图1.来自苜蓿中华根瘤菌生长的样品的点样图(上图)和直方图(下图)的示例一个能够使PHB积累的氮限制和碳过量条件(右图,PHB细菌),以及来自相同样品的样品在充足氮条件下生长的细菌种类作为PHB积累的阴性对照(左图,PHB-细菌)(Lagares Jr.等人,2017) 即可。 X轴表示由通道FL2登记的尼罗红(以AFU,任意荧光单位表示)的荧光估计的细胞PHB含量。每个细菌群体的中值荧光强度(MFI)值在对应的直方图插图中指示。

食谱

  1. 磷酸盐缓冲盐水(PBS)(10倍浓缩)
    8克NaCl
    0.2克KCl
    1.44克Na 2 HPO 4 4 0.24克KH 2 PO 4 4 用1N NaOH或1N HCl将pH值调节至7.2,用水补足至100ml(通常少于1ml的1N NaOH或1N HCl储备就足够了)
    在RT高压灭菌器和商店
  2. 透化溶液(乙醇中35%的PBS [体积/体积])
    350微升无水乙醇
    100μl10x PBS
    550μl无菌去离子水
  3. 尼罗河红股票解决方案
    1毫克尼罗红
    用DMSO补足1毫升
    在4°C储存

致谢

这项工作得到了国家科学和技术研究委员会(CONICET),国家科学技术促进局(ANPCyT)和国立奎尔梅斯大学的支持。 A.L.得到CONICET奖学金的支持。简历。是CONICET的研究员。目前的方案改编自(Lagares Jr.等人,2017),其主要基于若干先前报道的作品(Kacmar等人,2006; Ratcliff等人,等人,2008)。作者宣称没有利益冲突。

参考

  1. Alves,A.P.,Almeida,A.T.,Cruz,L.M.,Pedrosa,F.O.,de Souza,E.M.,Chubatsu,L.S.,Muller-Santos,M.and Valdameri,G。(2017)。 使用流式细胞术在5分钟内在固氮菌中定量聚-3-羟基丁酸的简单而有效的方法。 Braz J Med Biol Res 50(1):e5492。
  2. Braunegg,G.,Sonnleitner,B.和Lafferty,R. M.(1978)。 快速气相色谱法测定微生物生物量中的聚-β-羟基丁酸
    应用微生物生物技术 6:29-37
  3. Gorenflo,V.,Steinbuchel,A.,Marose,S.,Rieseberg,M。和Scheper,T。(1999)。 通过尼罗红染色定量细菌聚羟基链烷酸应用微生物生物技术< / em> 51(6):765-772。
  4. Greenspan,P。和Fowler,S.D。(1985)。 脂质探针的荧光光谱研究,尼罗红。 26(7):781-789。
  5. Jendrossek,D。和Pfeiffer,D。(2014)。 聚羟基链烷酸酯颗粒(碳纳米颗粒)形成的新见解和聚(3-羟基丁酸酯) 。Environ Microbiol 16(8):2357-2373。
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引用:Lagares Jr., A. and Valverde, C. (2017). Quantification of Bacterial Polyhydroxybutyrate Content by Flow Cytometry. Bio-protocol 7(23): e2638. DOI: 10.21769/BioProtoc.2638.
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