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This protocol describes the methods used to engineer and deploy genetically encoded fluorescence activity reporters for nitrate and peptide transporter activity in yeast cells. Fusion of the dual-affinity nitrate transceptor CHL1/AtNRT1.1/AtNPF6.3 or four different peptide transporters (AtPTR1, 2, 4, and 5) from Arabidopsis to a pair of fluorescent proteins with different spectral properties, enabled us to engineer the NiTracs (nitrate transporter activity tracking sensors) and the PepTracs (peptide transporter activity tracking sensors), ratiometric fluorescence activity sensors that monitor the activity of the plasma membrane nitrate transceptor or the peptide transporters in vivo (Ho et al., 2014). The NiTrac1 sensor responds specifically and reversibly to the addition of nitrate, while the PepTracs respond to addition of dipeptides, either by a reduction in donor and acceptor emission, while acceptor-excited emission remains unaltered, or by a change in ratio of the fluorophore emission. All sensors are suitable for ratiometric imaging. The similarity of the biphasic kinetics of the NiTrac1 sensor response [from µM to mM (Liu and Tsay, 2003)] and the nitrate transport kinetics of the native nitrate transceptor, intimates that NiTrac1 provides information on conformational rearrangements during the transport cycle, thereby reporting transporter activity over a wide range of external nitrate concentrations. Several variants of NiTrac have been engineered, which differ with respect to their affinity for nitrate (NiTrac1: CHL1; NiTracT101A: CHL1T101A). NiTrac also recognizes chlorate. Here we describe a simple method for the design, implementation, and detection of nitrate transceptor activity in yeast cells using a spectrofluorimeter.

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Design and Functional Analysis of Fluorescent Nitrate and Peptide Transporter Activity Sensors in Yeast Cultures
酵母培养物中荧光硝酸盐和肽转运载体传感器的设计和功能分析

微生物学 > 异源表达系统 > 酿酒酵母
作者: Cheng-Hsun Ho
Cheng-Hsun HoAffiliation: Department of Plant Biology, Carnegie Science, Stanford, USA
Present address: Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan
For correspondence: zcybele3@sinica.edu.tw
Bio-protocol author page: a2900
 and Wolf B. Frommer
Wolf B. FrommerAffiliation: Department of Plant Biology, Carnegie Science, Stanford, USA
Bio-protocol author page: a1915
Vol 6, Iss 3, 2/5/2016, 1840 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1728

[Abstract] This protocol describes the methods used to engineer and deploy genetically encoded fluorescence activity reporters for nitrate and peptide transporter activity in yeast cells. Fusion of the dual-affinity nitrate transceptor CHL1/AtNRT1.1/AtNPF6.3 or four different peptide transporters (AtPTR1, 2, 4, and 5) from Arabidopsis to a pair of fluorescent proteins with different spectral properties, enabled us to engineer the NiTracs (nitrate transporter activity tracking sensors) and the PepTracs (peptide transporter activity tracking sensors), ratiometric fluorescence activity sensors that monitor the activity of the plasma membrane nitrate transceptor or the peptide transporters in vivo (Ho et al., 2014). The NiTrac1 sensor responds specifically and reversibly to the addition of nitrate, while the PepTracs respond to addition of dipeptides, either by a reduction in donor and acceptor emission, while acceptor-excited emission remains unaltered, or by a change in ratio of the fluorophore emission. All sensors are suitable for ratiometric imaging. The similarity of the biphasic kinetics of the NiTrac1 sensor response [from µM to mM (Liu and Tsay, 2003)] and the nitrate transport kinetics of the native nitrate transceptor, intimates that NiTrac1 provides information on conformational rearrangements during the transport cycle, thereby reporting transporter activity over a wide range of external nitrate concentrations. Several variants of NiTrac have been engineered, which differ with respect to their affinity for nitrate (NiTrac1: CHL1; NiTracT101A: CHL1T101A). NiTrac also recognizes chlorate. Here we describe a simple method for the design, implementation, and detection of nitrate transceptor activity in yeast cells using a spectrofluorimeter.
Keywords: Biosensor(生物传感器), Transporter(转运), Nitrogen(氮), FRET(烦恼), Genetically encoded sensor(基因编码的传感器)

[Abstract]

Materials and Reagents

  1. Monochromator-based spectrofluorimeter for 96 well plates [for instance: Safire or Infinite® M1000 (Tecan Trading AG)]
  2. 96 well microplates (flat bottom clear or black) (Greiner Bio-One GmbH, catalog numbers: 650101 and 650209 )
    Note: Black plates are more expensive, but have lower background.
  3. Multichannel (12) pipette (for 100 µl) (e.g. Sartorius AG, catalog number: 725240 )
  4. 50 ml sterile plastic tubes (for instance: Falcon®, or any other brand)
  5. Yeast Strain: protease-deficient yeast strain BJ5465 [MATa, ura3–52, trp1, leu2Δ1, his3Δ200, pep4::HIS3, prb1Δ1.6R, can1, GAL+] (ATCC, catalog number: 208289TM ), which was obtained from the Yeast Genetic Stock Center (University of California, Berkeley, CA)
  6. The nitrate transceptor CHL1/NRT1.1/NPF6.4 (Ho et al., 2009; Leran et al., 2013) or various PTR peptide transporters (Komarova et al., 2012; Tsay et al., 2007; Leran et al., 2013)
    Note: they were used as sensory domains for creating the nitrate (NiTracs) and peptide (PepTracs) sensor constructs. For this full length ORFs of CHL1, CHL1T101A, PTR1, PTR2, PTR4, and PTR5 from Arabidopsis (The Arabidopsis Information Resource) were cloned in the pTOPO Gateway Entry vector.
  7. Sensors: CHL1/PTRs
    Note: it was sandwiched between a yellow acceptor [Aphrodite t9: Aphrodite is a codon diversified Venus gene; t9 corresponds to a deletion of the C-terminus of 9 amino acids (Deuschle et al., 2006) and cyan donor fluorophore (mCerulean) (Rizzo et al., 2006)]. This was achieved by inserting the sensory domain in the Gateway yeast expression vector pDRFlip30.
    1. pDRFlip30-NiTrac1 (original dual-affinity sensor; Km ~ 75 μM and Km ~ 3.8 Mm) (Ho et al., 2014).
    2. pDRFlip30-NiTrac1T101A (Variant with low-affinity; Km ~ 3.5 mM) (Ho et al., 2014).
    3. pDRFlip30-PepTrac1 (PepTrac1 based on AtPTR1) (Ho et al., 2014)
    4. pDRFlip30-PepTrac2 (PepTrac2 based on AtPTR2) (Ho et al., 2014)
    5. pDRFlip30-PepTrac4 (PepTrac4 based on AtPTR4) (Ho et al., 2014)
    6. pDRFlip30-PepTrac5 (PepTrac5 based on AtPTR5) (Ho et al., 2014)
  8. Gly-Gly (Sigma-Aldrich, catalog number: G1002 ) or other di-/tri-peptides (Sigma-Aldrich)
  9. Potassium nitrate (Sigma-Aldrich, catalog number: P8394 )
  10. YNB, yeast nitrogen base w/o amino acids w/o ammonium sulfate (BD, Difco, catalog number: 233520 )
  11. D-(+)-Glucose monohydrate (Fluka Analytical, catalog number: 49159 )
  12. Agar (Sigma-Aldrich, catalog number: A1296 )
  13. Agarose (Sigma-Aldrich, catalog number: 05066 )
  14. MES hydrate (Sigma-Aldrich, catalog number: M2933 )
  15. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881 )
  16. MilliQ or distilled water
  17. 1, 4-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: DTT-RO )
  18. Carrier DNA [UltraPureTM Salmon Sperm DNA Solution (Thermo Fisher Scientific, InvitrogenTM, catalog number: 115632-011 )]
  19. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  20. KNO3
  21. KCl
  22. Stock
  23. 45% PEG4000
  24. Lithium acetate
  25. Tris-Cl (pH 7.5)
  26. 40x glucose solution
  27. -ura DropOut medium
(Takara Bio Company, Clontech, catalog number: 630416 ) (see Recipes)
  28. Wash buffer (see Recipes)
  29. Resuspension buffer (see Recipes)
  30. Substrate addition (see Recipes)
  31. PLATE mixture (see Recipes)

Equipment

  1. Tube rack (any brand)
  2. Orbital shaker, with temperature and velocity control (e.g. Eppendorf, New Brunswick Scientific, model: Innova 44 )
  3. Incubator for 28-30 °C incubation of yeast cells (any brand, e.g. VWR International)
  4. Centrifuge with swinging rotor for 50 ml tubes (Beckman Coulter, model: Allegra 25R )

Procedure

  1. Sensor design
    For yeast expression, CHL1/NRT1.1 or PTRs coding regions were inserted by Gateway LR reactions into the E.coli/yeast expression vectors pDRFlip30, a destination vector that sandwiches the sensory domain between Aphrodite t9 and mCerulean (Jones et al., 2014), following manufacturer’s instructions. The E. coli/yeast vector pDRFlip30 is used for expressing the NiTracs or PepTracs from a PMA1 (yeast proton ATPase) promoter fragment. pDRFlip30 contains the ADH (alcohol dehydrogenase) terminator, and the URA3 marker for auxotrophy selection in yeast (Figure 1). pDRFlip30 is a vector that allows us to sandwich the transporter of interest by translational fusion between an N-terminal Aphrodite t9 (AFPt9) variant (aphrodite is a codon-diversified gene producing Venus; Deuschle et al., 2006), lacking nine amino acids at its C-terminus and a C-terminal monomeric Cerulean (mCer; Rizzo et al., 2006).

  2. Yeast transformation
    The protease-deficient yeast strain BJ5465 [MATa ura3­52 trp1 leu2­Δ1 his3­Δ 200 pep4::HIS3 prb1­Δ1.6R can1 GAL] is transformed with the pDRFlip30 vector containing the desired NiTracs and PepTracs by using the modified Lithium Acetate method from Gietz et al., 1992. In brief:
    1. Inoculate cultures in YPD medium and grow at 30 °C overnight to OD600nm ~ 0.5.
    2. Spin down (2,000 x g) 1 ml of cells in microfuge tube (15 sec) for each transformation.
    3. Decant the supernatant and resuspend the cells in 100 μl of liquid medium by vortexing.
    4. Add 2 μl of 10 mg/ml carrier DNA, vortex.
    5. Add ~1 μg plasmid, vortex.
    6. Add 20 μl 1 M DTT, vortex.
    7. Add 0.5 ml of ‘PLATE mixture’ (100 ml stock containing 90 ml of 45% PEG4000, 10 ml of 1 M lithium acetate, 1 ml of 1 M Tris-Cl (pH 7.5), 0.2 ml of 0.5 M EDTA), vortex.
    8. Incubate at RT for 6-8 h or overnight.
    9. Heat-shock cells for 10 min at 42 °C.
    10. Place pipet tip directly into bottom of tube, withdraw 50-100 μl of cells and plate cells on solid -ura DropOut medium. Plates are wrapped with plastic cling wrap to prevent dehydration.
    11. Plates are incubated (lid down) at 30 °C for 2-3 days.

  3. Detection of NiTrac and PepTrac responses in yeast using a fluorimeter
    1. Single colonies are picked using sterile pipette tips and grown in a 50 ml tube containing 10 ml -ura DropOut liquid medium. Pick at least three independent colonies. Use fresh transformation; do not keep colonies for more than one week on plates to avoid mutations in yeast or plasmid.
    2. Place tubes in a rack and incubate in incubator for ~15 h under agitation (230 rpm) at 30 °C until the culture reaches OD600nm~0.5.
    3. Liquid cultures are subcultured after dilution to OD600nm 0.01 in the same liquid medium and grown at 30 °C until OD600nm reaches ~0.2.
    4. Collect the cells by centrifugation at 4,000 x g, RT for 7 min, to sediment the cells.
    5. Discard the supernatant and resuspend the sediment by vortexing in 10 ml ‘Wash buffer’, 15 sec, RT.
    6. Centrifuge as described above (step B4).
    7. Wash the sediment two more times as in step B4 to B6, to remove traces of growth medium.
    8. Resuspend the sediment to OD600nm ~0.5 in ‘Resuspension buffer’.
    9. Mix cells well and aliquot 100 µl of the culture into wells of a 96-well flat bottom plate.
    10. Fluorescence is measured in a fluorescence plate reader, in bottom reading mode using 7.5 nm bandwidth for both excitation and emission. Typically, emission spectra are recorded with the following instrument settings: λem 470-570 nm for donor (mCer), step size 5 nm, gain: 75; and λem 520-570 nm for AFPt9, step size 5 nm, gain: 75. Fluorescence from cultures harboring pDRFlip30 (donor: mCer) is measured with excitation at λexc 428 nm; AFPt9 is measured with excitation at λexc 500 nm.
    11. A single- or multichannel pipette is used to add 100 µl of the culture to wells (mix by pipetting up and down) and to add analyte solution to the cells. Set up at least three replicates per treatment. Try to add equal amounts of solutions to reduce variability and use well-calibrated pipettes since the assays are quantitative and sensitive to differences in volumes/ concentration of sensor and analyte.
    12. Record the fluorescence immediately (as fast as possible) after substrate or control solution addition. It takes about 10 min to read a full 96 well plate with the parameters mentioned above. For highly accurate analyses, measure only a few wells at a time to reduce differences in analysis time. It is also possible to use instruments with injectors that allow for immediate recording; use rapid switching between wells to record over time.

Data analysis

Subtract background fluorescence of yeast (using cells transformed with vector only) from all fluorescence values (for both spectra as well as single point measurements).

For NiTracs
NiTracs expressed in yeast respond to nitrate addition by decreasing fluorescence intensity of donor and acceptor emission (obtained with excitation at 428nm). Aphrodite-t9 emission was unaffected and served as a control or reference for normalization (obtained at 500nm excitation Figure 2A & inset). Nitrate addition (5 mM) induced a reduction in the emission spectrum, while emission of the acceptor after direct excitation of the acceptor did not change (Figure 2B). Since the Aphrodite-t9 emission is unaffected by nitrate when excited directly, Aphrodite-t9 emission can be used as a control and for normalization by using ratios instead of absolute values to compare between different cultures (e.g. mutants) (peak fluorescence intensity of Aphrodite-t9 excited at 500 nm over every point in the emission spectrum obtained with excitation at 428 nm).

For PepTracs
PepTracs respond to dipeptide by decreasing fluorescence intensity of donor and acceptor emissions (PepTrac1, PepTrac2, and PepTrac5) or by a ratio change (Aphrodite-t9 emission intensity/mCer emission intensity obtained with excitation at 428nm) in the case of PepTrac4 (Figure 3). For PepTrac1, PepTrac2, and PepTrac5, Aphrodite-t9 emission when excited at 500nm was unaffected by peptide addition in PepTracs (Figure 3A, B, & C, insect). The emission ratio change induced by addition of dipeptide for PepTrac4 is shown in Figure 2D).

Representative data


Figure 1. Map of pDRFlip30-CHL1/PTRs plasmids. Main components and their sizes (base pair, bp): PMA promoter fragment 452 bp, ADH terminator 333 bp, 2 micron replication origin 1165 bp, URA3 804 bp, Ampicillin 1863 bp, pUC origin 654 bp, CHL1 1770 bp, PTRs: PTR1 1710 bp, PTR2 1755 bp, PTR4 1635 bp, PTR5 1710 bp, Aphrodite t9 688 bp, and mCerulean 714 bp.


Figure 2. Decrease in emission intensity for NiTrac1 expressed in yeast cells. A. Excitation at 428 nm: addition of 5 mM potassium nitrate (red; control 5 mM KCl, blue), led to a reduction in fluorescence intensity of donor and acceptor emission. Inset: emission intensity of Aphrodite-t9 in NiTrac1 when excited at 500 nm. Inset: Aphrodite-t9 emission was unaffected. AU: arbitrary units); B. Nitrate triggers a decrease in the emission from the donor, and consequentially a reduced meission from the accepted when exciting only the donor. Nitrate-induced ratio change (peak fluorescence intensity of Aphrodite-t9 excited at 500 nm over emission spectrum at 485 nm obtained with excitation at 428 nm). Data are normalized to KCl-treated buffer (as negative control, C). The data are from same experiment as shown in (Ho et al., 2014), but are derived form a separate analysis of independent colonies.


Figure 3. Fluorescence response of PepTrac1 (A), PepTrac2 (B), PepTrac5 (C), and PepTrac4 (D) expressing yeast cells. Samples were excited at 428 nm: addition of 5 mM gly-gly (red; control: 5mM KCl, blue), led to a reduction of fluorescence intensity of donor and acceptor emission in the case of PepTrac1, 2, and 5. Inset: emission of Aphrodite-t9 in PepTracs when excited at 500 nm. Aphrodite t9 emission was unaffected in PepTrac1, 2, and 5. (D) Fluorescence ratio (excitation 428 nm; emission ratio 530nm/428 nm; corresponding to mCer and Aphrodite-t9 emission, respectively) for PepTrac4 before and after addition of 5mM gly-gly dipeptide. The data are from same experiment as shown in (Ho et al., 2014), but are derived form a separate analysis of independent colonies.

Recipes

  1. -ura DropOut medium

    0.23 g/L -ura DropOut
    1.7 g/L yeast nitrogen base w/o amino acids w/o ammonium sulfate
    40% sterile filtrated glucose 

    Autoclave, 121 °C, 15 psi, 15 min
    For liquid medium, when hand-warm, add glucose from 40% sterile filtrated stock to a final concentration of 2% under sterile hood (e.g. biosafety cabinet)
    For solid medium, add 20 g/L agar before autoclaving. Add sterile filtrated glucose from 40% stock to a final concentration of 2% when medium is hand-warm before pouring plates
    Adjust the pH of the -ura DropOut medium to pH 5.8 with NaOH before addition of agar and autoclaving
  2. Wash buffer
    50 mM MES buffer, adjust to pH 5.5 with NaOH
  3. Resuspension buffer

    Add agarose to final concentration 0.05% in wash buffer (Recipe 2), and then, microwave until the agarose dissolves completely and wait until medium cools to room temperature to delay sedimentation of the cells during the measurement.
  4. Substrate addition
    Add KNO3 or KCl from 1 M stock to resuspension buffer (Recipe 3) to generate analyte concentration for measurement
  5. PLATE mixture
    100 ml stock
    90 ml of 45% PEG4000
    10 ml of 1 M lithium acetate
    1 ml of 1 M Tris-Cl (pH 7.5)
    0.2 ml of 0.5 M EDTA

Acknowledgments

Methods were adapted from (Ho et al., 2014). Techniques were also adapted from other references as cited. This work has been supported by grants MCB-1021677 and MCB-1413254 from the National Science Foundation (to WBF).

References

  1. Deuschle, K., Chaudhuri, B., Okumoto, S., Lager, I., Lalonde, S. and Frommer, W. B. (2006). Rapid metabolism of glucose detected with FRET glucose nanosensors in epidermal cells and intact roots of Arabidopsis RNA-silencing mutants. Plant Cell 18(9): 2314-2325.
  2. 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.
  3. 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.
  4. Ho, C. H., Lin, S. H., Hu, H. C. and Tsay, Y. F. (2009). CHL1 functions as a nitrate sensor in plants. Cell 138(6): 1184-1194.
  5. Jones, A. M., Danielson, J. A., Manojkumar, S. N., Lanquar, V., Grossmann, G. and Frommer, W. B. (2014). Abscisic acid dynamics in roots detected with genetically encoded FRET sensors. Elife 3: e01741.
  6. Komarova, N. Y., Meier, S., Meier, A., Grotemeyer, M. S. and Rentsch, D. (2012). Determinants for Arabidopsis peptide transporter targeting to the tonoplast or plasma membrane. Traffic 13(8): 1090-1105.
  7. Leran, S., Varala, K., Boyer, J. C., Chiurazzi, M., Crawford, N., Daniel-Vedele, F., David, L., Dickstein, R., Fernandez, E., Forde, B., Gassmann, W., Geiger, D., Gojon, A., Gong, J. M., Halkier, B. A., Harris, J. M., Hedrich, R., Limami, A. M., Rentsch, D., Seo, M., Tsay, Y. F., Zhang, M., Coruzzi, G. and Lacombe, B. (2014). A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants. Trends Plant Sci 19(1): 5-9.
  8. Liu, K. H. and Tsay, Y. F. (2003). Switching between the two action modes of the dual-affinity nitrate transporter CHL1 by phosphorylation. EMBO J 22(5): 1005-1013.
  9. Rizzo, M. A., Springer, G., Segawa, K., Zipfel, W. R. and Piston, D. W. (2006). Optimization of pairings and detection conditions for measurement of FRET between cyan and yellow fluorescent proteins. Microsc Microanal 12(3): 238-254.
  10. Tsay, Y. F., Chiu, C. C., Tsai, C. B., Ho, C. H. and Hsu, P. K. (2007). Nitrate transporters and peptide transporters. FEBS Lett 581(12): 2290-2300.

材料和试剂

  1. 用于96孔板的基于单色仪的分光荧光计[例如:Safire或Infinite M1000(Tecan Trading AG)]
  2. 96孔微板(平底透明或黑色)(Greiner Bio-One GmbH,目录号:650101和650209)
    注意:黑板较贵,但背景较低。
  3. 多通道(12)移液管(100μl)(例如Sartorius AG,??目录号:725240)
  4. 50ml无菌塑料管(例如:Falcon 或任何其他品牌)
  5. 酵母菌株:蛋白酶缺陷型酵母菌株BJ5465 [MATa,ura3-52 ,trp1 ,leu2Δ1,his3Δ200 pep4 :: HIS3,prb1Δ1.6R,can1 ,GAL +](ATCC,目录号:208289 TM ) ,其从酵母遗传库中心(加利福尼亚大学,伯克利,加利福尼亚)获得
  6. 硝酸盐转导体CHL1/NRT1.1/NPF6.4(Ho等人,2009; Leran等人,2013)或各种PTR肽转运蛋白(Komarova >等人,2012; Tsay等人,2007; Leran等人,2013)
    注意:它们用作用于产生硝酸盐(NiTrac)和肽(PepTrac)传感器构建体的感觉结构域。对于来自拟南芥(拟南芥信息资源)的CHL1,CHL1T101A,PTR1,PTR2,PTR4和PTR5的全长ORF被克隆在pTOPO Gateway Entry载体中。
  7. 传感器:CHL1/PTRs
    注意:它夹在黄色受体[Aphrodite t9:Aphrodite是密码子多样化的金星基因; t9对应于9个氨基酸的C末端的缺失(Deuschle等人,2006)和青色供体荧光团(mCerulean)(Rizzo等人,2006)]。这通过在Gateway酵母表达载体pDRFlip30中插入感觉结构域来实现。
    1. pDRFlip30-NiTrac1(原始双亲和性传感器; m ?75μM和 K > m ?3.8 Mm)(Ho ,,2014)。
    2. pDRFlip30-NiTrac1T101A(具有低亲和力的变体; m <3.5mM)(Ho等人 2014)。
    3. pDRFlip30-PepTrac1(基于AtPTR1的PepTrac1)(Ho等人,2014)
    4. pDRFlip30-PepTrac2(基于AtPTR2的PepTrac2)(Ho等人,2014)
    5. pDRFlip30-PepTrac4(基于AtPTR4的PepTrac4)(Ho等人,2014)
    6. pDRFlip30-PepTrac5(基于AtPTR5的PepTrac5)(Ho等人,2014)
  8. Gly-Gly(Sigma-Aldrich,目录号:G1002)或其它二肽/三肽(Sigma-Aldrich)
  9. 硝酸钾(Sigma-Aldrich,目录号:P8394)
  10. YNB,酵母氮源w/o氨基酸w/o硫酸铵(BD,Difco,目录号:233520)
  11. D - (+) - 葡萄糖一水合物(Fluka Analytical,目录号:49159)
  12. 琼脂(Sigma-Aldrich,目录号:A1296)
  13. 琼脂糖(Sigma-Aldrich,目录号:05066)
  14. MES水合物(Sigma-Aldrich,目录号:M2933)
  15. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S5881)
  16. MilliQ或蒸馏水
  17. 1,4-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:DTT-RO)
  18. 载体DNA [UltraPure TM鲑鱼精子DNA溶液(Thermo Fisher Scientific,Invitrogen TM ,目录号:115632-011]]
  19. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758)
  20. KNO 3
  21. KCl
  22. 库存
  23. 45%PEG4000
  24. 乙酸锂
  25. Tris-Cl(pH7.5)
  26. 40x葡萄糖溶液
  27. DropOut培养基(Takara Bio Company,Clontech,目录号:630416)(参见配方)
  28. 洗涤缓冲液(见配方)
  29. 重悬缓冲液(见配方)
  30. 添加底物(参见配方)
  31. PLATE混合物(参见配方)

设备

  1. 管架(任何品牌)
  2. 轨道振动器,具有温度和速度控制(例如,Eppendorf,New Brunswick Scientific,型号:Innova 44)
  3. 用于酵母细胞(任何品牌,例如VWR International)的28-30℃孵育的孵育器
  4. 使用用于50ml管(Beckman Coulter,型号:Allegra 25R)的摆动转子离心,

程序

  1. 传感器设计
    对于酵母表达,通过Gateway LR反应将 CHL1/NRT1.1 或 PTRs 编码区插入大肠杆菌/酵母表达载体pDRFlip30 ,根据制造商的说明,将美洲狮t9和mCerulean之间的感觉结构域夹在中间的目的载体(Jones等人,2014)。 E。大肠杆菌/酵母载体pDRFlip30用于从PMA1(酵母质子ATP酶)启动子片段表达NiTrac或PepTrac。 pDRFlip30含有ADH(醇脱氢酶)终止子和用于酵母中营养缺陷型选择的URA3标记(图1)。 pDRFlip30是一种载体,其允许我们通过N末端阿芙罗狄蒂t9(AFPt9)变体(美洲先生是产生金星的密码子多样化基因; Deuschle等人)之间的翻译融合来夹心感兴趣的转运蛋白, 2006),其C末端缺少九个氨基酸和C末端单体Cerulean(mCer; Rizzo等人,2006)。

  2. 酵母转化
    通过使用来自Gietz等人的改进的乙酸锂方法,用含有所需的NiTrac和PepTrac的pDRFlip30载体转化蛋白酶缺陷型酵母菌株BJ5465 [MATa ura352 trp1leu2Δ1his3Δ200 pep4 :: HIS3prb1Δ1.6Rcan1 GAL] 。,1992。简而言之:
    1. 在YPD培养基中接种培养物并在30℃下生长过夜至OD 600nm <0.5。
    2. 对于每次转化,在微量离心管中旋转(2,000×g)1ml细胞(15秒)。
    3. 滗出上清液,通过涡旋重悬细胞在100μl液体培养基中。
    4. 加入2微升10毫克/毫升载体DNA,涡旋
    5. 加入?1μg质粒,涡旋
    6. 加入20μl1 M DTT,涡旋
    7. 加入0.5ml"板混合物"(100ml原料,含有90ml 45% PEG4000,10ml的1M乙酸锂,1ml的1M Tris-Cl(pH7.5),0.2 ?ml的0.5M EDTA),涡旋
    8. 在室温下孵育6-8小时或过夜
    9. 热休克细胞在42℃下10分钟。
    10. 将移液管尖端直接放入管底,取出50-100μl ?细胞和平板细胞在固体-ura DropOut培养基上。板被包裹 用塑料保鲜膜防止脱水。
    11. 将板在30℃温育(盖下)2-3天。

  3. 使用荧光计检测酵母中的NiTrac和PepTrac反应
    1. 使用无菌移液管吸头挑取单个菌落,并在50℃生长 ml管,其含有10ml-Drop Drop液体培养基。选择至少三个 ?独立殖民地。使用新鲜转换;不要保留殖民地 在平板上超过一周,以避免酵母或质粒中的突变
    2. 将管放在架子中,并在孵育器中孵育?15小时 在30℃下搅拌(230rpm)直至培养物达到OD 600nm <0.5
    3. 将液体培养物在稀释至OD 600nm 0.01后进行传代培养 ?相同的液体培养基中,并在30℃下生长直至OD 600nm达到?0.2。
    4. 通过在4,000xg下离心收集细胞,室温7分钟,沉淀细胞。
    5. 弃去上清液并通过在10ml'洗涤缓冲液'中,15秒,RT涡旋重悬沉淀物。
    6. 如上所述离心(步骤B4)
    7. 如步骤B4至B6,再洗涤沉淀两次,以除去微量的生长培养基
    8. 在"重悬缓冲液"中将沉淀物重悬于OD 600nm
    9. 将细胞充分混合,并将100μl培养物等分到96孔平底板的孔中。
    10. 在底部的荧光板读数器中测量荧光 读取模式,使用7.5nm带宽用于激发和发射。 通常,用以下仪器记录发射光谱 设置:供体(mCer)的λsub 470-570nm,步长为5nm,增益:75;和 ?对于AFPt9,λ 520-570nm,步长为5nm,增益:75。荧光来自 培养包含pDRFlip30(供体:mCer)的激活 在λsub 428nm处; AFPt9是在λexc500nm下激发测量的
    11. 单通道或多通道移液器用于添加100μl的 培养至孔(通过上下吹吸混合)并添加分析物 溶液。每次治疗至少设置三次重复。 尝试添加等量的解决方案,以减少变异性和使用经过良好校准的移液器,因为该测定是定量和敏感的 传感器和分析物的体积/浓度差异
    12. 后立即记录荧光(尽可能快) 底物或对照溶液添加。读大约需要10分钟 完全96孔板,具有上述参数。高度 准确分析,每次只测量几口井减少 分析时间的差异。也可以使用仪器 具有允许立即记录的注射器;使用快速切换 在井之间记录随着时间??的推移。

数据分析

从所有荧光值(对于两个光谱以及单点测量)减去酵母的背景荧光(使用仅用载体转化的细胞)。

适用于NiTrac
在酵母中表达的NiTrac通过降低供体和受体发射的荧光强度(在428nm激发获得)响应硝酸盐添加。 Aphrodite-t9发射不受影响,并且用作标准化的对照或参照(在500nm激发下获得,图2A&插图)。硝酸盐加入(5mM)诱导发射光谱的减少,而受体直接激发后受体的发射没有改变(图2B)。由于阿芙罗狄忒t9发射不受直接激发时硝酸盐的影响,因此可以通过使用比率而不是绝对值来比较不同培养物(例如突变体)的Aphrodite-t9发射作为对照和标准化(Aphrodite-在用在428nm激发下获得的发射光谱中的每个点在500nm激发的t9)。

对于PepTrac
PepTrac通过减少供体和受体发射(PepTrac1,PepTrac2和PepTrac5)的荧光强度或通过在PepTrac4的情况下的比率变化(Aphrodite-t9发射强度/在428nm激发下获得的mCer发射强度)响应二肽(图3 )。对于PepTrac1,PepTrac2和PepTrac5,当在500nm激发时的Aphrodite-t9发射不受PepTrac中肽添加的影响(图3A,B,& C,昆虫)。通过添加二肽用于PepTrac4诱导的发射比率变化显示在图2D)中。

代表数据


图1.pDRFlip30-CHL1/PTRs质粒的图谱。主要组分及其大小(碱基对,bp):PMA启动子片段452bp,ADH终止子333bp,2微米复制起点1165bp,URA3 804bp,氨苄青霉素1863bp,pUC起点654bp,CHL1 1770bp,PTRs:PTR1 1710bp,PTR2 1755bp,PTR4 1635bp,PTR51710bp,Aphrodite t9 688bp和mCerulean 714bp。

图2.在酵母细胞中表达的NiTrac1的发射强度的降低 A.在428nm处的激发:添加5mM硝酸钾(红色;对照5mM KCl,蓝色)导致减少供体的荧光强度和受体发射。插图:当在500nm激发时,Aphrodite-t9在NiTrac1中的发射强度。插图:Aphrodite-t9发射不受影响。 AU:任意单位);硝酸盐触发来自供体的发射的减少,因此,当仅激发供体时,接受的减少的发射。硝酸盐诱导的比率变化(在500nm激发的Aphrodite-t9的峰值荧光强度,在428nm处的发射光谱,在428nm处激发获得)。将数据归一化为KCl处理的缓冲液(作为阴性对照,C)。数据来自如(Ho等人,2014)中所示的相同实验,但是源自对独立菌落的单独分析。


图3.PrTrac1(A),PepTrac2(B),PepTrac5(C)和PepTrac4(D)表达酵母细胞的荧光反应。在428nm激发样品:加入5mM gly-在PepTrac1,2和5的情况下,gly(红色;对照:5mM KCl,蓝色)导致供体和受体发射的荧光强度降低。插图:当在500nm激发时,PepTrac中Aphrodite-t9的发射。 Aphrodite t9发射在PepTrac1,2和5中不受影响。(D)在添加5mM gly之前和之后的PepTrac4的荧光比(激发428nm;发射比530nm/428nm;分别对应于mCer和Aphrodite-t9发射)糖二肽。数据来自如(Ho等人,2014)中所示的相同实验,但是源自对独立菌落的单独分析。

食谱

  1. -ura DropOut介质
    0.23 g/L emura -ura DropOut
    1.7g/L酵母氮源w/o氨基酸w/o硫酸铵
    40%无菌过滤的葡萄糖 高压灭菌,121℃,15psi,15分钟
    对于液体介质,当手温时,在无菌罩(例如生物安全柜)下将葡萄糖从40%无菌过滤原料中添加至最终浓度为2%。
    对于固体培养基,在高压灭菌之前加入20g/L琼脂。当将培养基在倾倒平板前手温时,将无菌过滤的葡萄糖从40%储液添加至2%的终浓度 在加入琼脂和高压灭菌器之前,用NaOH将 -ura DropOut培养基的pH调节至pH 5.8
  2. 洗涤缓冲液
    50mM MES缓冲液,用NaOH调节pH至5.5
  3. 重悬缓冲液
    在洗涤缓冲液(配方2)中加入琼脂糖至终浓度为0.05%,然后,微波直到琼脂糖完全溶解,并等待介质冷却至室温,以延迟测量期间细胞的沉降。
  4. 底物加入
    将KNO 3或KCl从1M储备液加入重悬浮缓冲液(配方3),以产生用于测量的分析物浓度
  5. 平板混合物
    100 ml股票
    90ml的45%PEG4000
    加入10ml 1M乙酸锂 1ml 1M Tris-Cl(pH7.5) 0.2ml 0.5M EDTA

致谢

方法改编自(Ho等人,2014)。技术也适应于引用的其他参考文献。这项工作得到了来自国家科学基金会(到WBF)的MCB-1021677和MCB-1413254补助金的支持。

参考文献

  1. Deuschle,K.,Chaudhuri,B.,Okumoto,S.,Lager,I.,Lalonde,S.and Frommer,W??.B。(2006)。 使用FRET葡萄糖纳米传感器在表皮细胞和拟南芥的完整根中检测到的葡萄糖的快速代谢/em> RNA沉默突变体。 植物细胞 18(9):2314-2325。
  2. 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的方法核酸研究 20(6):1425.
  3. 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。
  4. Ho,C.H.,Lin,S.H.,Hu,H.C.and Tsay,Y.F。(2009)。 CHL1在植物中作为硝酸盐传感器。 138(6):1184-1194。
  5. Jones,A.M.,Danielson,J.A.,Manojkumar,S.N.,Lanquar,V.,Grossmann,G.and Frommer,W??.B。(2014)。 根据遗传编码FRET传感器检测到的根部脱落酸的动力学。 3:e01741。
  6. Komarova,N.Y.,Meier,S.,Meier,A.,Grotemeyer,M.S.and Rentsch,D。(2012)。 拟南芥 肽转运蛋白的决定簇,其靶向膜质或质膜。/a> 交通 13(8):1090-1105。
  7. Leran,S.,Varala,K.,Boyer,JC,Chiurazzi,M.,Crawford,N.,Daniel-Vedele,F.,David,L.,Dickstein,R.,Fernandez,E.,Forde, ,Gassmann,W.,Geiger,D.,Gojon,A.,Gong,JM,Halkier,BA,Harris,JM,Hedrich,R.,Limami,AM,Rentsch,D.,Seo,M.,Tsay,YF ,Zhang,M.,Coruzzi,G.and Lacombe,B。(2014)。 植物中的NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER家族成员的统一命名。 Trends Plant Sci 19(1):5-9。
  8. Liu,K.H。和Tsay,Y.F。(2003)。 通过磷酸化在双亲和性硝酸盐转运蛋白CHL1的两种作用模式之间切换。 EMBO J 22(5):1005-1013
  9. Rizzo,M.A.,Springer,G.,Segawa,K.,Zipfel,W.R.and Piston,D.W。(2006)。 优化配对和检测条件,用于测量青色和黄色荧光蛋白之间的FRET。 Microsc Microanal 12(3):238-254。
  10. Tsay,Y.F.,Chiu,C.C.,Tsai,C.B.,Ho,C.H。和Hsu,P.K。(2007)。 硝酸盐转运蛋白和肽转运蛋白。 FEBS Lett 581 12:2290-2300。
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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. Ho, C. and Frommer, W. B. (2016). Design and Functional Analysis of Fluorescent Nitrate and Peptide Transporter Activity Sensors in Yeast Cultures. Bio-protocol 6(3): e1728. DOI: 10.21769/BioProtoc.1728; Full Text
  2. 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.




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