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AmTracs are the first example of “activity sensors”, since they report the activity of ammonium transporters by means of fluorescence readout in vivo (De Michele et al., 2013). AmTracs are based on a single fluorescent protein, a circularly permuted GFP (cpGFP), inserted into the cytosolic loop connecting the two pseudosymmetrical halves of the Arabidopsis and yeast plasma membrane ammonium transporters AtAMTs and ScMEP (Figure 1). Recently, FRET-based activity sensors for nitrate and peptide transporters have also been developed (Ho et al., 2014). Since transporter activity directly depends on the availability of substrate, AmTracs measure extracellular ammonium concentrations. Several versions of AmTrac exist, with different fluorescence intensity (FI) responses and affinities for ammonium, and based on different ammonium transporters (AmTrac: AtAMT1;3; AmTrac1;2: At AMT1;2; MepTrac: ScMEP2). Currently, the most useful AmTrac versions are probably AmTrac-GS (bright, with Km of 50 µM) and AmTrac-100 (a high capacity version with Km of 100 µM). The protocol for measuring ammonium concentrations in yeast cells at the fluorimeter is the same for all versions.

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Quantification of Extracellular Ammonium Concentrations and Transporter Activity in Yeast Using AmTrac Fluorescent Sensors
采用AmTrac荧光传感器定量测定酵母细胞的胞外铵浓度和转运蛋白活性

微生物学 > 微生物新陈代谢 > 营养运输
作者: Cindy Ast
Cindy AstAffiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, USA
Bio-protocol author page: a1914
Wolf B. Frommer
Wolf B. FrommerAffiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, USA
Bio-protocol author page: a1915
Guido Grossmann
Guido GrossmannAffiliation: Cell Networks-Cluster of Excellence and Centre for Organismal Studies (COS) Heidelberg Bioresources, Universität Heidelberg, Heidelberg, Germany
Bio-protocol author page: a1916
 and Roberto De Michele
Roberto De MicheleAffiliation: Institute of Biosciences and Bioresources, National Research council of Italy (CNR-IBBR), Palermo, Italy
For correspondence: roberto.demichele@cnr.it
Bio-protocol author page: a1917
Vol 5, Iss 1, 1/5/2015, 2897 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1372

[Abstract] AmTracs are the first example of “activity sensors”, since they report the activity of ammonium transporters by means of fluorescence readout in vivo (De Michele et al., 2013). AmTracs are based on a single fluorescent protein, a circularly permuted GFP (cpGFP), inserted into the cytosolic loop connecting the two pseudosymmetrical halves of the Arabidopsis and yeast plasma membrane ammonium transporters AtAMTs and ScMEP (Figure 1). Recently, FRET-based activity sensors for nitrate and peptide transporters have also been developed (Ho et al., 2014). Since transporter activity directly depends on the availability of substrate, AmTracs measure extracellular ammonium concentrations. Several versions of AmTrac exist, with different fluorescence intensity (FI) responses and affinities for ammonium, and based on different ammonium transporters (AmTrac: AtAMT1;3; AmTrac1;2: At AMT1;2; MepTrac: ScMEP2). Currently, the most useful AmTrac versions are probably AmTrac-GS (bright, with Km of 50 µM) and AmTrac-100 (a high capacity version with Km of 100 µM). The protocol for measuring ammonium concentrations in yeast cells at the fluorimeter is the same for all versions.
Keywords: Sensor(传感器), Ammonium(铵), Transporter(转运), Fluorescence(荧光), Yeast(酵母)

[Abstract]


Figure 1. Model of AmTrac/MepTrac sensors and AMT transport mechanism. We propose that AMT switches between at least two distinct states during transport of ammonium: An outward, open state A and an inward, open state B. The movement of TMH-V (red) and TMH-VI (blue) is transmitted to the connecting loop, affecting the inserted cpGFP (green) and resulting in a change in fluorescence emission.

Materials and Reagents

  1. Yeast strain, with auxotrophic selection marker
    Note: We used ura- strains 23344c: MATa ura3; or 31019b: MATa ura3 mep1Δ mep2 Δ::LEU2 mep3 Δ::KanMX2 (Marini et al., 1997). Strain 31019b lacks the endogenous ammonium transporters MEP1-3 and therefore can grow on ammonium as sole nitrogen source only when transformed with vectors harboring functional ammonium transporters. AmTrac should work in any transformable strain.
  2. Vector pDR-F’-GW containing AmTrac under control of strong PMA promoter and ADH terminator, and with the selection marker URA3
    Note: Vectors with many different AmTrac- and MepTrac versions are available through Addgene (www.addgene.org). An empty vector can be used as control.
    1. pDR-AmTrac (Plasmid, catalog number: 47770 ) (original sensor, Km 55=µM, ΔF/F~30%)
    2. pDR-AmTrac-IS (Plasmid, catalog number: 47766 ) [variant with enhanced fluorescence and response (500% FI with respect to AmTrac), ΔF/F~40%]
    3. pDR-AmTrac1;2 (Plasmid, catalog number: 47765 ) (AmTrac based on AtAMT1;2)
    4. pDR-AmTrac-LS (Plasmid, catalog number: 47767 ) [variant with enhanced fluorescence and response (400% FI with respect to AmTrac), ΔF/F~50%]
    5. pDR-AmTrac-GS (Plasmid, catalog number: 47769 ) [variant with enhanced fluorescence and response (850% FI with respect to AmTrac), ΔF/F~40%]
    6. pDR-AmTrac-100 (Plasmid, catalog number: 47771 ) (high capacity sensor, Km=100 µM)
    7. pDR-Meptrac (Plasmid, catalog number: 47768 ) (sensor based on ScMEP2)
    8. pDR-Meptrac-H194E (Plasmid, catalog number: 47764 ) [high capacity, pH insensitive and pseudohyphal growth-impaired Meptrac variant (Boeckstaens et al. 2008)]
    9. pDf1-GW (Plasmid, catalog number 36026 ) (empty vector)
      Note: It is useful to remove the GW cassette since it contains a ccdB suicidal gene.
  3. Ammonium chloride (EMD Millipore, catalog numer: AX1270-7 )
  4. Yeast nitrogen base w/o amino acids w/o ammonium sulfate (Difco, catalog number: 233520 )
  5. Glucose (Fluka Analytical, catalog number: 49159 )
  6. Proline (L-Proline) (Sigma-Aldrich, catalog number: PO380 )
  7. Agar (Sigma-Aldrich, catalog number: A1296 )
  8. Arginine (L-Arginine monohydrochloride) (Sigma-Aldrich, catalog number: A5131 )
  9. MES (Sigma-Aldrich, catalog number: M2933 )
  10. NaOH (Sigma-Aldrich, catalog number: S5881 )
  11. Glycerol (BDH, catalog number: 1172-1LP )
  12. MilliQ water
  13. 100x proline solution (see Recipes)
  14. 50x arginine solution (see Recipes)
  15. SD medium (see Recipes)
  16. Washing buffer (see Recipes)
  17. Resuspension buffer (see Recipes)

Equipment

  1. Petri dishes (plastic, any size/brand)
  2. Sterile plastic tips for pipettes (1 ml and 200 µl sizes)
  3. 50 ml sterile plastic tubes (like Falcon®, any brand)
  4. Tube rack (any brand)
  5. Surgical tape (3 M Micropore, catalog numer: 1535-5 )
  6. 96 well ELISA microplate (flat bottom) (Greiner Bio-One GmbH, catalog numer: 650 101 )
    Multichannel (8) pipette (for 50 µl) (Sartorious, catalog numer: 725240 )
  7. Reservoir with 12 compartments (VWR international, catalog numer: 80092-466 )
  8. Autoclave (tuttnauer Brinkmann, model: 3850 E )
  9. Orbital shaker, with temperature and velocity control (New Brunswick Scientific, model: innova 44 )
  10. Incubator for 28-30 °C plate incubation (VWR International)
  11. Sterile hood (Heraeus Holding)
  12. Centrifuge with swinging rotor for 50 ml tubes (Beckman Coulter, model: Allegra 25R )
  13. Fluorimeter for 96 well plates (for example: Tecan Trading AG, Safire or M1000 )

Procedure

  1. Transform yeast with the pDR-F’ vector containing the desired AmTrac. We routinely use the lithium acetate method (Gietz et al., 1992). Briefly, we add about 100 ng plasmid to a PCR tube containing 100 µl of the transformation solution (containing PEG, lithium acetate, salmon sperm DNA and yeast cells) and heat-shock at 42 °C for 13 min. The first time the reader might also transform with an empty vector, to measure background fluorescence.
  2. Plate the transformation on selective synthetic dextrose minimal medium (SD), with 1 mM arginine (from a filter-sterilized 50x stock dissolved in water) as sole nitrogen source. Wrap the plate in plastic cling wrap to prevent dehydration.
  3. Incubate the plate (upside down to prevent condensation) at 28-30 °C for 3 days.
  4. In the sterile hood, pick a colony with a sterile 1 ml pipette and place it in a 50 ml tube containing 5 ml SD medium, supplemented with 0.1% proline (from a filter-sterilized 100x stock dissolved in water).
  5. Tape the opening of the tube with surgical tape to prevent contamination, still allowing gas exchange.
  6. Place the tube in a rack in the orbital shaker.
  7. Grow for 36-48 h at 30 °C, 230 rpm. The culture should at least be turbid (OD600 >0.5). Even at saturation, the sensor still works. Avoid overgrowth though, since cells will start to die and degrade the sensors. In that case, start a new inoculum.
  8. Remove the tip with forceps, and centrifuge at 3,000 rcf at RT for 5 min to pellet the cells.
  9. Discard the supernatant by inversion. The reader should see a whitish pellet.
  10. Resuspend the pellet in 20 ml washing buffer, RT. The reader can ease resuspension by pipetting up and down, or by pouring a few ml first, shaking vigorously, and then bringing to volume.
  11. Centrifuge as above (step 8).
  12. Wash the pellet a second time as in step 10, to remove any trace of growth medium and released ammonium.
  13. Resuspend the pellet in 5 ml resuspension buffer.
  14. Pipette 200 µl of the culture in a well of the microplate and measure OD600 using the spectrofluorometer (mode: absorbance). As blank, use 200 µl of resuspension buffer in another well.
  15. Adjust to OD600 = 0.5 by diluting with resuspension buffer in the 50 ml tube.
  16. Mix well and aliquot in the wells, 200 µl each.
  17. An excitation and emission spectrum should be recorded first to confirm the fluorescence properties of the cpGFP as part of the sensors (parameters: mode: fluorescence; read: bottom reading; excitation: Fix emission to 530 and record spectra from 350 - 510 nm; emission: fix the excitation to 480 nm and record the spectra from 500 - 600 nm; step size 5 nm: bandwidth: 7.5/7.5 nm; gain:100), see Figure 2A. With knowledge of the major excitation maximum the reader can decide on the excitation wavelength for the single point measurements.
  18. Read basal fluorescence. It should be the same for all wells (parameters: mode: fluorescence; read: bottom reading; excitation: 488 nm; emission: 513 nm; bandwidth: 7.5/7.5 nm; gain:100; shake: orbital, medium, 3 sec, initial and between readings).
  19. Since response is saturated above 1 mM for all variants, it can be useful to make a calibration curve with ammonium chloride ranging from 0 to 1 mM final concentration in the wells (0, 1 µM, 10 µM, 100 µM, 1 mM), see Figure 1B. It is useful to include negative controls, e.g. sodium chloride, to exclude artifacts and to include other salts to check for specificity. In that case, use the highest concentration (e.g. 1 or 10 mM), see Figure 2C.
  20. Add the treatment solutions (as 5x concentrated stocks, dissolved in water) to the compartments of the reservoir (Figure 3).
  21. Add 50 µl of each treatment solution or water control to each wells with a multichannel pipette, pipetting up and down for about 5 times for mixing the treatment with the cells. Set up at least three replicates per treatment.
  22. Response is immediate, within the time limits of the microplate reader. With the parameters as above, it takes about 1 min to read a full plate.
  23. Repeat reading as step 18 after 5 min from the treatment. We usually consider these latter responses, as they tend to be a bit stronger.


    Figure 2. A. Excitation and emission spectra of AmTrac-GS treated with indicated ammonium chloride concentration. Data are normalized to the initial value. B. Response of AmTrac and AmTrac-100 to increasing concentrations of ammonium chloride. C. Specificity of AmTrac, the concentration of the indicated salts was 1 mM (De Michele et al., 2013).


    Figure 3. Schematic representation of the treatment procedure for building a calibration curve. Wells in a microplate are filled with 200 µl of yeast culture, resuspended in 50 mM MES buffer with 5% glycerol. Samples can be arranged in columns, with triplicates for each construct. Fluorescence from yeast transformed with the empty vector will be used as background value. After reading basal fluorescence, 50 µl of 5x stock treatment will be added to the wells, by using a multichannel pipette. In this example, we use a solvent control (water), four concentrations of increasing magnitude of NH4Cl, and a chloride control at the highest concentration.

Data analysis

  1. Subtract background fluorescence (from a yeast transformed with an empty vector) to all fluorescence values (spectra as well as single point measurements).
  2. AmTracs respond to ammonium by decreasing cpGFP fluorescence emission. For this reason, fluorescence of the water control shows always the highest value. Calculate ΔF, by subtracting the response to treatment to that of the water control (0 ammonium). Divide ΔF to the response to treatment (ΔF/F). By using ratios instead of absolute values, it is possible to compare different experiments, and in theory even using different dilutions (not just to OD600 = 0.5). However, it is good practice to maintain conditions as similar as possible.
  3. Calculate averages and errors from all replicates, for each treatment.
  4. Plot as histogram or line. Figure 2B shows the response of AmTrac and AmTrac-100 to increasing concentrations of ammonium chloride.
    The reader can then use the calibration curve to quantify unknown ammonium concentration in a solution.

Recipes

  1. 100x proline solution
    10 g L-Proline dissolved in 100 ml MilliQ water
    pH ~ 5.5
  2. 50x arginine solution
    50 mM L-Arginine monohydrochloride dissolved in MilliQ water
    pH ~ 5.5
  3. SD medium
    1.7 g/L yeast nitrogen base w/o amino acids w/o ammonium sulfate
    30 g/L glucose
    Autoclave, 120 °C, 30 min
    When hand-warm, in a sterile hood, add proline from a 100x stock to a final concentration of 0.1%
    The pH of the SD medium is ~ 4.2 and changes minimally upon the addition of proline to pH ~ 4.3
    For solid medium, add 15 g/L agar before autoclaving
    Add 1 mM arginine (final concentration) when medium is hand-warm before pouring plates. In case the SD medium does not solidify well, which may depend on the autoclave used, it is recommended to adjust the pH of the SD medium to pH 5.8 with NaOH before addition of agar and autoclaving.
  4. Washing buffer
    50 mM MES, bring to pH 6.0 with NaOH pellets
  5. Resuspension buffer
    50 mM MES, bring to pH 6.0 with NaOH pellets
    Add 5% glycerol, to delay sedimentation of the cells

Acknowledgments

This work has been supported by grant MCB-1021677 by the National Science Foundation (Wolf B Frommer). The protocol has been adapted from De Michele et al. (2013).

References

  1. Boeckstaens, M., Andre, B. and Marini, A. M. (2008). Distinct transport mechanisms in yeast ammonium transport/sensor proteins of the Mep/Amt/Rh family and impact on filamentation. J Biol Chem 283(31): 21362-21370.    
  2. De Michele, R., Ast, C., Loque, D., Ho, C. H., Andrade, S. L., Lanquar, V., Grossmann, G., Gehne, S., Kumke, M. U. and Frommer, W. B. (2013). Fluorescent sensors reporting the activity of ammonium transceptors in live cells. Elife 2: e00800.
  3. Gietz, D., St Jean, A., Woods, R. A. and Schiestl, R. H. (1992). Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20(6): 1425.    
  4. Ho, C. H. and Frommer, W. B. (2014). Fluorescent sensors for activity and regulation of the nitrate transceptor CHL1/NRT1.1 and oligopeptide transporters. Elife 3: e01917.
  5. Marini, A. M., Soussi-Boudekou, S., Vissers, S. and Andre, B. (1997). A family of ammonium transporters in Saccharomyces cerevisiae. Mol Cell Biol 17(8): 4282-4293.   


图1. AmTrac/MepTrac传感器和 AMT传输机制。我们建议至少在AMT之间切换 在运输铵期间的两个不同的状态:向外,开放状态   A和向内,打开状态B. TMH-V(红色)和TMH-VI的移动 (蓝色)传输到连接环路,影响插入 cpGFP(绿色),并导致荧光发射的变化。

材料和试剂

  1. 酵母菌株,具有营养缺陷型选择标记
    注意:我们使用ura - 菌株23344c:MATa ura3; 或31019b:MATa ura3 mep1Δmep2Δ:: LEU2 mep3Δ:: KanMX2(Marini等人,1997)。 菌株31019b 缺乏内源性铵转运蛋白MEP1-3,因此可以生长   仅在用载体转化时作为唯一氮源 含有功能性铵转运蛋白。 AmTrac应该在任何工作 可变形应变。
  2. 在强PMA启动子和ADH终止子控制下含有AmTrac的载体pDR-F'-GW,以及用选择标记URA3
    注意:具有许多不同AmTrac和MepTrac版本的矢量 可通过Addgene获得( www.addgene.org )。 可以使用空矢量   作为控制。
    1. pDR-AmTrac(质粒,目录号:47770)(原始传感器,Km 55 =μM,ΔF/F〜30%)
    2. pDR-AmTrac-IS(质粒,目录号:47766) 增强的荧光和响应(相对于AmTrac为500%FI), ΔF/F〜40%]
    3. pDR-AmTrac1; 2(质粒,目录号:47765)(基于AtAMT1的AmTrac; 2)
    4. pDR-AmTrac-LS(质粒,目录号:47767) 增强的荧光和响应(相对于AmTrac为400%FI), ΔF/F〜50%]
    5. pDR-AmTrac-GS(质粒,目录号:47769)[变体   具有增强的荧光和响应(850%FI相对于 AmTrac),ΔF/F〜40%]
    6. pDR-AmTrac-100(质粒,目录号:47771)(高容量传感器,Km =100μM)
    7. pDR-Meptrac(质粒,目录号:47768)(基于ScMEP2的传感器)
    8. pDR-Meptrac-H194E(质粒,目录号:47764)[高容量, pH不敏感和假性近视生长受损的Meptrac变体 (Boeckstaens等人 2008)]
    9. pDf1-GW(Plasmid,目录号36026)(空载体)
      注意:删除GW盒是有用的,因为它包含一个ccdB自杀基因。
  3. 氯化铵(EMD Millipore,目录号:AX1270-7)
  4. 酵母氮源w/o氨基酸w/o硫酸铵(Difco,目录号:233520)
  5. 葡萄糖(Fluka Analytical,目录号:49159)
  6. 脯氨酸(L-脯氨酸)(Sigma-Aldrich,目录号:PO380)
  7. 琼脂(Sigma-Aldrich,目录号:A1296)
  8. 精氨酸(L-精氨酸一盐酸盐)(Sigma-Aldrich,目录号:A5131)
  9. MES(Sigma-Aldrich,目录号:M2933)
  10. NaOH(Sigma-Aldrich,目录号:S5881)
  11. 甘油(BDH,目录号:1172-1LP)
  12. MilliQ水
  13. 100x脯氨酸溶液(见配方)
  14. 50x精氨酸溶液(见配方)
  15. SD介质(参见配方)
  16. 洗涤缓冲液(见配方)
  17. 重悬缓冲液(见配方)

设备

  1. 培养皿(塑料,任何尺寸/品牌)
  2. 移液器的无菌塑料吸头(1 ml和200μl尺寸)
  3. 50 ml无菌塑料管(如Falcon ®,任何品牌)
  4. 管架(任何品牌)
  5. 手术带(3M Micropore,目录号:1535-5)
  6. 96孔ELISA微板(平底)(Greiner Bio-One GmbH,目录号:650101) 多通道(8)移液管(50μl)(Sartorious,目录编号:725240)
  7. 12个水库(VWR国际,目录号:80092-466)
  8. 高压灭菌器(tuttnauer Brinkmann,型号:3850 E)
  9. 轨道振动器,具有温度和速度控制(New Brunswick Scientific,型号:innova 44)
  10. 用于28-30℃板培养的孵育器(VWR International)
  11. 无菌罩(Heraeus Holding)
  12. 使用用于50ml管(Beckman Coulter,型号:Allegra 25R)的摆动转子离心,
  13. 用于96孔板(例如:Tecan Trading AG,Safire或M1000)的荧光计

程序

  1. 转变   酵母与含有所需AmTrac的pDR-F'载体。 我们 常规地使用乙酸锂方法(Gietz等人,1992)。 简而言之, 我们向含有100μl的PCR管中加入约100ng质粒 转化溶液(含有PEG,乙酸锂,鲑鱼精子 DNA和酵母细胞)并在42℃热休克13分钟。 第一次 读者也可以用空向量进行变换,以进行测量 背景荧光
  2. 平板变换 选择性合成葡萄糖基本培养基(SD),含1mM精氨酸 (来自溶解在水中的过滤灭菌的50×储备液)作为唯一的氮   资源。 将板子包裹在塑料保护膜中,以防止脱水。
  3. 在28-30℃下孵育平板(上下颠倒以防止冷凝)3天
  4. 在   无菌罩,用无菌1ml移液管挑取菌落并放置 它在含有5ml SD培养基的50ml管中,补充有0.1% 脯氨酸(来自过滤灭菌的100x储备溶解于水中)
  5. 用手术胶带缠绕管的开口,以防止污染,仍然允许气体交换
  6. 将管放在轨道摇床的架子上。
  7. 增长   在30℃,230rpm下36-48小时。 培养物应至少是混浊的 (OD <600> 0.5)。 即使在饱和时,传感器仍然工作。 避免 过度生长,因为细胞会开始死亡并降解 传感器。 在这种情况下,开始新的接种物
  8. 用钳子取出尖端,并在室温下以3,000 rcf离心5分钟以沉淀细胞
  9. 通过倒置弃去上清液。 读者应该看到发白的颗粒。
  10. 重悬   沉淀在20ml洗涤缓冲液中,RT。 读者可以放松 通过上下吸取重悬,或通过首先倒几毫升, 摇动,然后使体积。
  11. 离心机如上(步骤8)。
  12. 按照步骤10第二次洗涤沉淀,以去除任何微量的生长培养基并释放铵
  13. 将沉淀重悬在5ml重悬缓冲液中
  14. 吸管   在微量培养板的孔中的200μl培养物,并使用分光光度计(模式:吸光度)测量OD 600。 作为空白,使用200微升   再悬浮缓冲液在另一个井中
  15. 通过用50ml管中的重悬缓冲液稀释来调节至OD 600 = 0.5。
  16. 混合好,等分在孔中,每个200μl。
  17. 一个   激发和发射光谱应首先记录确认 作为传感器的一部分的cpGFP的荧光性质 (参数:模式:荧光;读数:底部读数;激发:固定 发射至530,并记录350-510nm的光谱; 发射:固定 激发至480nm并记录500-600nm的光谱; 步长   5nm:带宽:7.5/7.5nm; 增益:100),参见图2A。 有知识 的主要激发最大值,读者可以决定激发 波长为单点测量。
  18. 阅读基础 荧光。 它应该是相同的所有水井(参数:模式: 荧光; 阅读: 激发:488nm; 发射:513 nm; 带宽:7.5/7.5nm; 增益:100; 摇动:轨道,中,3秒, 初始和读数之间)。
  19. 因为响应饱和   对于所有变体,高于1mM,进行校准可能是有用的 曲线,其中氯化铵的终浓度为0至1mM 在孔(0,1μM,10μM,100μM,1mM)中,参见图1B。 这是有用的   包括阴性对照,例如氯化钠,以排除 并包括其它盐以检查特异性。 在那里 情况下,使用最高浓度(例如1或10mM),参见图2C。
  20. 将处理溶液(5x浓缩的原液,溶于水中)加入到储层的隔室中(图3)。
  21. 加   每个处理溶液50μl或水对照每孔用a 多通道移液器,上下移液约5次混合   用细胞治疗。 每个设置至少三个重复 治疗
  22. 响应是即时的,在时间限制内   酶标仪。 使用上面的参数,它需要大约1 min读完整板。
  23. 5后重复读取步骤18   min。 我们通常认为这些后面的反应 他们往往有点强。


    图2。 A.激发和 用指定氯化铵处理的AmTrac-GS的发射光谱 浓度。 数据被归一化为初始值。 B.答复 AmTrac和AmTrac-100至增加浓度的氯化铵。   C.AmTrac的特异性,所示盐的浓度为1   mM(De Michele等人,2013)。


    图3.原理图 表示用于建立校准的处理程序 曲线。在微孔板中的孔用200μl酵母培养物填充, 重悬于含有5%甘油的50mM MES缓冲液中。 样品可以 排列成列,每个构建体一式三份。 荧光 来自用空载体转化的酵母将用作背景 值。 读取基础荧光后,50μl5x储备液处理 将通过使用多通道移液器加入到孔中。 在这里 例如,我们使用溶剂对照(水),四种浓度 增加NH 4 Cl的量,并且氯化物对照最高 浓度。

数据分析

  1. 减去   背景荧光(来自用空载体转化的酵母)   到所有荧光值(光谱以及单点 测量)。
  2. AmTracs通过降低对铵的响应 cpGFP荧光发射。 因此,水的荧光 控制显示总是最高值。 计算ΔF,减去   对水处理的响应(0铵)。 划分   ΔF对处理的响应(ΔF/F)。 通过使用ratio而不是 绝对值,可以比较不同的实验,   理论,甚至使用不同的稀释度(不仅仅是OD <600> = 0.5)。 然而,良好的做法是保持类似的条件 可能。
  3. 计算每次处理的所有重复的平均值和误差。
  4. 情节   作为直方图或线。 图2B显示了AmTrac和的反应 AmTrac-100到增加浓度的氯化铵 然后读数器可以使用校准曲线来量化溶液中未知的铵浓度

食谱

  1. 100x脯氨酸溶液
    10g L-脯氨酸溶解在100ml MilliQ水中 pH〜5.5
  2. 50x精氨酸溶液
    50mM L-精氨酸盐酸盐溶解于MilliQ水中 pH〜5.5
  3. SD介质
    1.7g/L酵母氮源w/o氨基酸w/o硫酸铵
    30g/L葡萄糖 高压灭菌,120°C,30分钟
    当在无菌罩中手工加热时,将脯氨酸从100x储液中加至终浓度0.1%
    SD培养基的pH为〜4.2,并且在加入脯氨酸至pH〜4.3时变化最小
    对于固体培养基,在高压灭菌之前加入15g/L琼脂
    加入1mM精氨酸(最终浓度)时,培养基手温   浇注板。 在SD介质不能很好地固化的情况下,这可能   取决于所用的高压釜,建议调节pH值   SD培养基至NaOH 5.8,加入琼脂和高压灭菌之前
  4. 洗涤缓冲液
    50mM MES,用NaOH颗粒使pH为6.0
  5. 重悬缓冲液
    50mM MES,用NaOH颗粒使pH为6.0 加入5%甘油,延缓细胞沉淀

致谢

这个   工作由国家科学基金会MCB-1021677支持 基金会(Wolf B Frommer)。 该协议已经改编自De Michele等人(2013)。

参考文献

  1. Boeckstaens,M.,Andre,B.and Marini,A.M。(2008)。 Mep/Amt/Rh家族的酵母铵转运/传感蛋白中的独特转运机制和影响。 J Biol Chem 283(31):21362-21370。    
  2. De Michele,R.,Ast,C.,Loque,D.,Ho,C.H.,Andrade,S.L.,Lanquar,V.,Grossmann,G.,Gehne,S.,Kumke,M.U.and Frommer, 荧光传感器报告活细胞中铵转移体的活性。 2:e00800
  3. Gietz,D.,St Jean,A.,Woods,R.A。和Schiestl,R.H。(1992)。 改进的完整酵母细胞高效转化方法核酸研究 20(6):1425.    
  4. Ho,C.H。和Frommer,W.B。(2014)。 用于硝酸盐转导体CHL1/NRT1.1和寡肽转运蛋白活性和调节的荧光传感器。 a> 3:e01917。
  5. Marini,A.M.,Soussi-Boudekou,S.,Vissers,S.and Andre,B。(1997)。 酿酒酵母中的铵转运蛋白家族。 Mol Cell Biol 17(8):4282-4293。   
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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. Ast, C., Frommer, W. B., Grossmann, G. and De Michele, R. (2015). Quantification of Extracellular Ammonium Concentrations and Transporter Activity in Yeast Using AmTrac Fluorescent Sensors. Bio-protocol 5(1): e1372. DOI: 10.21769/BioProtoc.1372; Full Text
  2. De Michele, R., Ast, C., Loque, D., Ho, C. H., Andrade, S. L., Lanquar, V., Grossmann, G., Gehne, S., Kumke, M. U. and Frommer, W. B. (2013). Fluorescent sensors reporting the activity of ammonium transceptors in live cells. Elife 2: e00800.




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