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Detection of Protein Interactions in the Cytoplasm and Periplasm of Escherichia coli by Förster Resonance Energy Transfer
采用Förster共振能量转移法检测大肠埃希杆菌细胞质和周质中蛋白质的相互作用   

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本实验方案简略版
mBio
Oct 2013

 

Abstract

This protocol was developed to qualitatively and quantitatively detect protein-protein interactions in Escherichia coli by Förster Resonance Energy Transfer (FRET). The described assay allows for the previously impossible in vivo screening of periplasmic protein-protein interactions. In FRET, excitation of a donor fluorescent molecule results in the transfer of energy to an acceptor fluorescent molecule, which will then emit light if the distance between them is within the 1-10 nm range. Fluorescent proteins can be genetically encoded as fusions to proteins of interest and expressed in the cell and therefore FRET protein-protein interaction experiments can be performed in vivo. Donor and acceptor fluorescent protein fusions are constructed for bacterial proteins that are suspected to interact. These fusions are co-expressed in bacterial cells and the fluorescence emission spectra are measured by subsequently exciting the donor and the acceptor channel. A partial overlap between the emission spectrum of the donor and the excitation spectrum of the acceptor is a prerequisite for FRET. Donor excitation can cross-excite the acceptor for a known percentage even in the absence of FRET. By measuring reference spectra for the background, donor-only and acceptor-only samples, expected emission spectra can be calculated. Sensitized emission for the acceptor on top of the expected spectrum can be attributed to FRET and can be quantified by spectral unmixing.

Keywords: Bacteria (细菌), Cytoplasm (细胞质), Periplasm (周质), Protein interactions (蛋白质相互作用), FRET (FRET), mNeonGreen (mNeonGreen), SYFP2 (SYFP2), mKO (mKO), mCherry (mCherry)

Background

Determining how and which proteins interact to sustain life is at the core of molecular biology research. Many in vitro methods exist but may result in false positives as the interactions are taken out of their biological context. An in vivo method is crucial for the verification of in vitro obtained results. Moreover, in vivo methods allow for the observation of the often, dynamic processes that take place in the cell. Fluorescent proteins (FP)s are ideal for in vivo experiments as they can be fused to proteins of interest making it possible to microscopically track the localization and dynamics of the fused proteins in living cells. Energy can be transferred between FPs by Förster Resonance Energy Transfer (FRET) provided they have compatible physical properties. The emission spectrum of a donor FP must overlap with the excitation spectrum of an acceptor FP and the distance between them should be less than 10 nm. The larger the spectral overlap and the smaller the distance between the donor and acceptor FP the more FRET can occur. The stringent distance dependence for FRET is ideal to detect direct protein-protein interactions as they also occur in the nanometer range, whereas indirect protein interactions are usually on a larger distance scale and not detectable by FRET. For this reason, it gives very few false positives but due to the physical requirements of the involved fluorophores, negative results could be false negatives. The advantage of using FPs for FRET measurements is that the chromophore is enclosed in a protein barrel and therefore much better protected from the environment than chemical chromophores. The fluorescence of the latter is extremely sensitive to environmental changes such as pH, salt, and solvents. Consequently, they often cannot be used reliably to measure FRET in a complicated environment like the living cell. A drawback of the 3-4 nm in diameter protein-barrel that encloses the chromophore in FPs is that it reduces the detectable distance between proteins to 1-6 nm. Since 5-7 nm is the typical size of proteins, it usually does not pose a problem, but can again result in false negatives. We have used a spectroscopy based FRET method to analyze the interaction between proteins in the cytoplasm and in the periplasm of Escherichia coli (Alexeeva et al., 2010; Fraipont et al., 2011; van der Ploeg et al., 2013 and 2015; Meiresonne et al., 2017) and showed that the technique is suitable for mode of action studies of antibiotics (van der Ploeg et al., 2015) and medium throughput screening (Meiresonne et al., 2017) using various FP FRET pairs. This bio-protocol describes how to perform our FRET assay in bacteria to determine protein-protein interactions by spectral unmixing. Although this protocol is written for E. coli, the method of spectral unmixing is suitable for any organism, provided fusion proteins can be expressed in the species and fluorescence spectra can be collected. The protocol describes how to perform FRET experiments by 3 approaches: 1) Measurements on fixed cell using a fluorometer, the initial determination of protein interactions. 2) Measurements on fixed cells in the plate reader for the faster detection of already confirmed protein interactions that provide clear fluorescence signals. And 3) for established interactions, measurements on living cells grown in the plate reader. For instance, to monitor the real time inhibition of a particular interaction.

Materials and Reagents

  1. Sterile straight neck culturing flasks (DWK Life Sciences, DURAN, catalog number: 2177124 ) with aluminum anodized metal blue cap (DWK Life Sciences, DURAN, catalog number: 2901324 )
  2. 50 ml sterile Falcon tubes (SARSTEDT, catalog number: 62.657.254 )
  3. 2 ml sterile Eppendorf tubes (Greiner Bio One International, catalog number: 623201 )
  4. 1.5 ml sterile Eppendorf tubes (Greiner Bio One International, catalog number: 616201 )
  5. Disposable pipet tips 200 µl (Ultratip, Greiner Bio One International, catalog number: 739290 )
  6. Disposable pipet tips 1,000 µl (Ultratip, Greiner Bio One International, catalog number: 686290 )
  7. Wash bottles (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2401-0500 )
  8. Glass-bottomed black walled 96 wells plate (Porvair Sciences, catalog number: 324002 )
  9. Black walled clear bottomed 96 wells plastic plate (Greiner Bio One International, catalog number: 675096 )
  10. Stirred Quartz cuvettes (Hellma, catalog number: 119004F-10-40 )
  11. Lens cleaning tissue (Fischer Scientific, FisherbrandTM, catalog number: 10605955 )
    Note: This product has been discontinued.
  12. Filtropur S 0.2 µm filter (SARSTEDT, catalog number: 83.1826.001 )
  13. Ethanol 70% in lab wash bottle (VWR, Technisolv®, catalog number: 83801.360 )
  14. Ethanol 96% in lab wash bottle (VWR, Technisolv®, catalog number: 83804.360 )
  15. H2O in lab wash bottle
  16. 1 M β-D-1-thiogalactopyranoside (IPTG) (Duchefa Biochemie, catalog number: I1401 )
  17. Hellmanex III (Hellma, catalog number: 9-307-011-4-507 )
  18. Bacto tryptone (Duchefa Biochemie, catalog number: T1332 )
  19. Yeast extract (Duchefa Biochemie, catalog number: Y1333 )
  20. Sodium chloride (NaCl) (Merck, catalog number: 106404 )
  21. Glucose (Duchefa Biochemie, catalog number: G0802 )
  22. Thiamine hydrochloride (Sigma-Aldrich, catalog number: T4625-25G )
  23. L-Lysine monohydrochloride (Sigma-Aldrich, catalog number: L8662 )
  24. Ampicillin stock solution 100 mg ml-1 (Sigma-Aldrich, catalog number: A9518 ) in 50% ethanol
  25. Chloramphenicol stock solution 25 mg ml-1 (Duchefa Biochemie, catalog number: C0113 ) in 50% ethanol
  26. di-Potassium hydrogen phosphate (K2HPO4·3H2O) (VWR, catalog number: 26932.290 )
  27. Potassium dihydrogen phosphate (KH2PO4) (Merck, catalog number: 104873 )
  28. Ammonium sulfate, (NH4)2SO4 (Sigma-Aldrich, catalog number: A6387 )
    Note: This product has been discontinued.
  29. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Carl Roth, catalog number: T888.1 )
  30. Iron(II) sulfate heptahydrate (FeSO4·7H2O) (Sigma-Aldrich, catalog number: 215422 )
  31. Calcium nitrate tetrahydrate, Ca(NO3)2·4H2O (Sigma-Aldrich, catalog number: C1396 )
  32. L-Arginine (Sigma-Aldrich, catalog number: A8094 )
  33. L-Glutamine (Sigma-Aldrich, catalog number: G8540 )
  34. Thymidine (Sigma-Aldrich, catalog number: T1895 )
  35. Uracil (Sigma-Aldrich, catalog number: U1128 )
  36. Potassium chloride (KCl) (VWR, catalog number: 71003-522 )
  37. di-Sodium hydrogen phosphate dihydrate (Na2HPO4·2H2O) (Merck, catalog number: 106580 )
  38. Glutaraldehyde 25% (Merck, catalog number: 1042390250 )
  39. Formaldehyde ≥ 36.0% (Sigma-Aldrich, catalog number: 47608 )
  40. Lysine (Sigma-Aldrich, catalog number: L8662 )
  41. TY medium (see Recipes)
  42. 20% glucose solution (see Recipes)
  43. GB1 medium (see Recipes)
    1. MM1-10x
    2. MM2-10x
    3. MM3-100x
    4. MM4-100x
    5. Vitamin B1 stock solution (4 mg ml-1)
    6. Lysine stock solution (20 mg ml-1)
  44. Phosphate buffered saline (PBS 1x) (see Recipes)
  45. Formaldehyde and glutaraldehyde (FAGA) (see Recipes)

Equipment

  1. P20, P200 & P1000 Pipetman pipettes (Gilson, catalog number: F167300 )
  2. Gyrotory water bath shaker Model G76 (Eppendorf, New Brunswick Scientific, model: Model G76 or GFL-Gesellschaft für Labortechnik, catalog number: 1092 ) with holders for culturing flasks
  3. Photospectrometer (Biochrom, model: Libra S22 )
  4. Compressed air source
  5. Water purification system (Purelab flex with ELGA LC197-Biofilter from ELGA LabWater, Laner End, HP14 3BY, UK)
    Note: All H2O in the protocol indicates water of 11.5 MΩ from Water purification system.
  6. Centrifuge for 50 ml Falcon tubes (Eppendorf, model: 5804 R )
  7. Centrifuge for 1.5 and 2.0 ml Eppendorf tubes (Eppendorf, model: 5424 R )
  8. Spectrofluorometer Quantamaster 2000-4 (Photon Technology International, NJ) with a red-optimized set-up: R928P PMT tube (185-900 nm), 500 nm blaze (1,200 line/mm) both excitation and emission gratings
    Note: A PMT that will reliably detect fluorescence spectra up until 700 nm will be suitable for the described protocol.
  9. Optical filters:
    1. 500/10 nm BrightLine® single-band bandpass filter (Semrock, catalog number: FF01-500/10 )
    2. ET510LP (Chroma Technology, catalog number: ET510lp )
    3. 514/3 nm BrightLine® single-band bandpass filter (Semrock, catalog number: FF01-514/3 )
    4. 515 nm blocking edge BrightLine® long-pass filter (Semrock, catalog number: FF01-515/LP )
    5. HQ541/12 (Chroma Technology, catalog number: HQ541/12 )
      Note: This product has been discontinued. Filters with similar transmission properties can be used.
    6. E550LP (Chroma Technology, catalog number: E550lp )
    7. 587/11 nm BrightLine® single-band bandpass filter (Semrock, catalog number: FF01-587/11 )
    8. HQ600LP (Chroma Technology, catalog number: HQ600LP )
      Note: This product has been discontinued.
  10. Fluorescence scanning plate reader (BioTek Instruments, model: SynergyTM Mx , catalog number: SMATBC)
  11. Autoclave (SANYO, catalog number: MLS-3780 )

Software

  1. Fluorescence spectrophotometer software (PTI acquisition software FeliX32 version 1.2 Build 56)
  2. Microplate reader software Gen5 version 2.00 (Biotek)
  3. Excel (Microsoft)

Procedure

The FRET method from beginning to end encompasses several steps, which are discussed per subject. The procedure and sections are outlined in Figure 1.


Figure 1. Workflow of the bacterial spectral-based FRET method. The labeling of the panels corresponds to the section describing the specific protocols. A. Choosing the suitable FRET-pair and creating expression plasmids. B. Testing the constructed plasmids for functionality. C. Growing cultures steady state, inducing expression of the FRET constructs and harvesting cells. D. Sample preparation and data acquisition. E. Analysis of the data.

  1. Choosing a suitable FRET pair for your experiment and creating FP fusions
    1. Choosing a suitable FRET pair for your experiment
      Before starting FRET experiments, the most suitable FRET-pair should be selected, which will depend on the type of experiments that are planned. For instance, the compartment of the assayed protein-protein interaction or the desire to do in vivo measurements influence this choice (Table 1). In this bio-protocol mCherry (Shaner et al., 2004) is used as an acceptor FP for either mNeonGreen (Shaner et al., 2013) (mNG), super yellow fluorescent protein 2 (Kremers et al., 2006) (SYFP2) or monomeric Kusabira-Orange (Karasawa et al., 2004) (mKO) donor FPs. All combinations have their specific strengths and drawbacks. These proteins have been described as monomers and usually do not have a strong propensity to aggregate (Cranfill et al., 2016). Theoretically, the FRET pair with the largest Förster radius (R0) will result in the highest FRET values. The R0 value is a measure for the distance at which 50% energy transfer occurs from the donor to the acceptor fluorophore (Figure 2). This is determined by the overlap between the donor emission spectrum and the acceptor excitation spectrum, their respective quantum yield, extinction coefficient and orientation.


      Figure 2. Förster radii of the described FRET pairs. Plotted theoretical FRET efficiencies as a function of chromophore distance for the mNG-mCh, SYFP2-mCh and mKO-mCh FRET pairs using the spectral properties in Table 1. On the right, the zoomed-in graph shows the R0 or the distance between the fluorophores that will yield 50% energy transfer.

      Of the FRET pairs described here mKO-mCh has the highest R0 value with 5.9 nm. However, mKO has a long maturation time meaning that, although it is produced by the cells and its tertiary structure has folded, the amino acids that form the chromophore need time to rearrange their covalent bonds to be able to fluoresce, which is termed maturation. FRET experiments with not fully matured FPs can therefore lead to unbalanced signal acquisition and faulty subsequent calculations. If allowing for maturation time is no problem, then the mKO-mCh pair is preferred for cytoplasmic FRET experiments given its good dynamic range and least time consuming measuring time (Alexeeva et al., 2010; Fraipont et al., 2011; van der Ploeg et al., 2013). The SYFP2-mCh FRET pair has a slightly lower R0 value (5.7 nm) but will emit strong SYFP2 fluorescence, which would provide sufficient signal for interactions that occur between proteins of relatively low copy numbers per cell (van der Ploeg et al., 2015). With the good folding and maturation properties of SYFP2 live FRET should be possible using the SYFP2-mCh pair although only experiments with fixed cells were attempted yet. If the SYFP2-mCh FRET pair is used and the cells are fixed, then the SYFP2 signal needs to recover its fluorescence and the direct measuring of samples is not possible. Regardless, in our hands the SYFP2-mCherry FRET pair provided the broadest range of FRET detection. This is surprising as the mKO-mCh pair has a higher R0 but possibly SYFP2 performs somewhat better due to its high fluorescent intensity. The SYFP2-mCh pair could therefore be more suitable than the mKO-mCh pair if smaller differences in FRET values need to be measured for less abundant proteins. Of the three described FRET-pairs mNG-mCh has the lowest R0 value (5.5 nm) and consequently a modest detection range but it also has advantages. At the moment mNG is the only useful donor to mCherry that folds and matures in the periplasm. The mNG-mCh FRET pair could be used real time in living cell FRET experiments in both the cytoplasm and the periplasm (Meiresonne et al., 2017). Furthermore, the mNG-mCh FRET pair results obtained by spectral unmixing could be confirmed by a fluorescence life time based detection method (Meiresonne et al., 2017).

      Table 1. Information on the FRET pairs described in this bio-protocol

      *As described in the original papers (Alexeeva et al., 2010; van der Ploeg et al., 2013 and 2015; Meiresonne et al., 2017).
      Notes:
      1. For the R0 values presented in Table 1, the originally published extinction coefficient of 72,000 M-1 cm-1 of mCh was used (Shaner et al., 2004). Variation occurs in the description of FP extinction coefficients based on detection accuracy and sample conditions. The ever-improving field of FP research shows that the extinction coefficient of mCh is more in the range of 85,000 M-1 cm-1 (Cranfill et al., 2016; Bindels et al., 2017). Similarly, the quantum yield of mKO has been reported to be higher than originally thought (0.77) (Cranfill et al., 2016). This would lead to higher R0 values and different calculations for each of the described FRET pairs.
      2. Different FP pairs, especially CFP-YFP, have been used for bacterial FRET. For more information, see references (Sourjik and Berg, 2002; Detert Oude Weme et al., 2015; Sieger and Bramkamp, 2015).

    2. Creating FP fusions
      Bacterial genes encoding a protein of interest are fused to a gene encoding one of the described fluorescent proteins and expressed from an inducible expression vector. Any plasmid expression system can be used, provided that the plasmids are compatible (different origins of replication and different antibiotic resistance markers) and the expression level is low (typically 2,000 copies of the protein) to avoid nonspecific interactions. To give the FP and the protein of interest some freedom of movement and reduce steric hindrance, it can be useful to add a linker between the FP and the protein. GGS repeats are often used but we also have good results with ARNNNN and other amino acid sequences. Another consideration is the choice for an N- or C-terminal fusion. For instance, N-terminal fusions of proteins that encode signal sequences for downstream transport or processing should be avoided or circumvented. Additionally, the N- or C-terminus of a protein may be essential for its normal localization or functionality. Procedure B discusses testing the created fusions. The sequence of the plasmid should be confirmed before further experiments. A description of cloning and transforming E. coli with plasmids is beyond the scope of this protocol but other Bio-protocols like ‘[BIO101] Standard cloning’ and ‘[BIO101] The Inoue Method for Preparation and Transformation of Competent E. coli: “Ultra Competent” Cells’ (He, 2011; Im, 2011) may be useful.

  2. Testing the FP fusions
    Ideally the FP-fusions should be fully functional and expressed at levels close to their native levels while still resulting in measurable fluorescence intensities. Once the fusions are created on a plasmid, the expressed products should be tested for their effects on phenotype, fluorescence and correct cellular localization. If no fluorescence is observed, degradation of the FP may be assessed by Western blot analysis. Perhaps the fusion disrupts normal localization, functionality and interactions of the protein of interest. This may be amended by modifying the length of the linker or switching the position of the FP. Some proteins may neither be functional as N nor as C-terminal fusion but will work as a sandwich fusion (van der Ploeg et al., 2015). An FP-fusion should complement the loss of function from a deletion or depletion strain of that same protein. Complementation testing will depend on whether the loss of the gene results in a phenotype. If no complementation experiments are possible, FRET experiments can be done in either a wild-type or deletion background but it should be kept in mind that the fusion protein may not be functional and results could be misinterpreted.
    Note: Plasmid-based FP-fusions are a great way of quickly testing localization or the interaction of proteins of interest. However, plasmid-based complementation is not always possible due to stoichiometry imbalance. In such cases the FP-fusion could be inserted in the chromosome at the original locus. In fact, chromosomal insertions of well-established FRET partners would be preferred for easy assays. However, this would also require chromosomal references in an isogenic background because bacteria can respond differently to plasmid and chromosomal based protein expression with respect to growth differences in and possible autofluorescence due to plasmid maintenance.

    Preparation for the FRET experiment
    When the FP fusion proteins are ready for use, competent cells can be transformed with the donor and acceptor expressing plasmids. Any E. coli strain should be suitable. Because we are interested in proteins that affect morphology, we prefer to use wild-type strains like MC4100 or BW25113 that are morphogenetically homogenous. Note that, if deletion strains are used the references and controls should be in the wild-type variant. The FRET assay described, requires at least three reference samples that are expressed in cells of the same genetic background as the assessed interaction. Strongly advised is the use of positive and negative controls consisting of a plasmid expressing the donor FP fused to the acceptor FP and two plasmids expressing the donor or acceptor FPs fused to unrelated proteins that are known not to interact, respectively. The best controls are expressed in the same compartment as where the protein-protein interaction takes place to be able to assess molecular crowding effects. A list of samples for a minimal FRET experiment is shown in Table 2.

    Table 2. Samples and plasmids required for a minimal FRET experiment

    aTo detect the interaction between two proteins of interest at least three references are required. Positive and negative controls are advised. The described protocol is for two compatible plasmids that have a p15 origin of replication combined with chloramphenicol resistance and a ColE1 origin of replication combined with ampicillin resistance. Both plasmids have a weakened trc99A promoter and two terminators.

  3. Steady state growth, induction and harvesting cells
    Bacterial cells that are grown in rich medium often contain a high concentration of autofluorescing molecules that decrease the signal to noise ratio. For that reason, the cells are grown in minimal medium and FRET experiments performed in rich medium are discouraged. To allow comparison of the various bacterial cultures expressing the FP pairs the cells are grown to steady state in about 20 generations before the expression of the proteins is induced (Figure 3). This assures identical autofluorescent backgrounds in all samples. After induction of expression for at least 2 generations the cells are fixed and harvested.
    1. Day 0. Prepare starter cultures of all strains required to run your FRET experiment in 5 ml TY + ampicillin + chloramphenicol + 0.5% glucose medium at 37 °C while shaking until bacterial growth becomes visible.
      Note: Glucose is added to the medium to repress the expression of the lacIq controlled gene from the plasmids.
    2. Use the starter cultures to inoculate 25 ml (1:250) of Gb1 medium + ampicillin + chloramphenicol and continue overnight growth at 28 °C while shaking.
    3. Day 1. Dilute the overnight cultures 1:250 in fresh Gb1 medium + ampicillin + chloramphenicol and continue growth at 28 °C while shaking.
      Note: From now on periodically measure and note OD450 values of the cultures and do not let it reach above 0.2. If the cultures approach this value dilute with fresh pre-warmed medium.
    4. Calculate the doubling time (TD) for all grow cultures using the provided Excel sheet ‘Growth calculation.xlsx’ and use it to calculate the dilution factor to reach OD450 values of 0.1 the next morning when the experiment is continued.
    5. Day 2. Repeat the growing in fresh Gb1 medium + ampicillin + chloramphenicol while keeping the OD450 values below 0.2 and dilute for the next morning like in Step C4.
    6. Day 3. Repeat the growing in fresh Gb1 medium + ampicillin and chloramphenicol while keeping the OD450 values below 0.2 and dilute for the next morning like in Step C4 but this time aim for an OD450 value of 0.025 in 35 ml fresh Gb1 + ampicillin + chloramphenicol.
    7. Day 4. Measure OD450 values of the cultures and if they are around 0.03-0.04 induce expression from the plasmids by adding IPTG at an appropriate concentration (e.g., 10-20 µM) and continue growth.
    8. When the cultures approach OD450 values of ~0.2, prepare a sufficient amount of FAGA fixative (see Recipes).
      Note: The total volume of FAGA needed differs depending on the total volume of the to-be-fixed cultures, 1 ml of FAGA is required to fix 12.5 ml of culture.
    9. When the cells reach OD450 values of 0.2, fix the cells in the shaking water bath with FAGA, depending on how much of the culture’s volume is left for 10 min.
    10. Decant the fixed cultures in 50 ml Falcon tubes and pellet the cells by centrifugation for 10 min at 6,300 x g at room temperature (Eppendorf 5804 R).
    11. Discard the supernatant and wash 3 x with 1 ml PBS in a 1.5 ml Eppendorf tube centrifuging for 5 min at 4,600 x g at room temperature in a tabletop centrifuge (Eppendorf 5424 R).
    12. Resuspend the last pellet in 1 ml of PBS and depending on the FRET pair that is used incubate o/n at 37 °C or over weekend at 4 °C to allow chromophore maturation (mKO-mCh) or store the samples at 4 °C until ready for measuring (SYFP2-mCh & mNG-mCh).
      Note: SYFP2 and mNG fold fast and their chromophore (re)matures quickly so direct measurements could be considered, but it is more practical to do the spectral measurements the day after.


      Figure 3. Example of steady state growth. Flask culture growth curves of MC4100 based E. coli cultures were grown to steady state in Gb1 at 28 °C expressing periplasmic FP-fusions. The circles represent the time points at which overnight dilutions were made. At day 4 the cultures were induced with IPTG (arrow) to express periplasmic mNG- or mCh-fusions. The EV (Empty Vector) culture does not express any FP and serves as a control. The X-axis is given in ln OD450 to signify exponential growth. Cells were fixed and harvested after at least two mass doublings (asterisk). This figure was adapted from Meiresonne et al., 2017.

  4. Sample preparation and data acquisition
    1. Pellet the cells as described in Step C10 and resuspend in 1 ml PBS.
    2. Prepare at least 1,300 µl of samples of with OD450 values of 1.00 ± 0.005 by diluting the cells with fresh PBS and add 1,200 µl to the stirred quartz cuvette.
      Notes:
      1. The dilution can be made inside the photospectrometer; the more equal the OD450 values are; the more reliable will be the results.
      2. Do not forget to add the magnetic stirrer to the cuvette before measuring.
    3. Measure the acceptor emission spectrum using the settings and optical filters indicated in Table 3 and described in ‘Measuring spectra using a fluorometer–Fluorometer settings and practical setup’.
    4. Measure the donor emission spectrum using the settings and optical filters indicated in Table 3 and described in ‘Measuring spectra using a fluorometer–Fluorometer settings and practical setup’.
      Note: It is advisable to measure the acceptor and donor spectra of each sample in the same order to prevent possible differences in FP bleaching.
    5. Repeat Steps D2 to D4 for each sample and also measure a blank PBS only sample at the start of the measurement and the end of all measurements. This allows you to verify any change in the instrument response.
    6. Save the data in any convenient file format that can be exported to Microsoft Excel.
      Notes:
      1. Before and between receiving a sample, quartz cuvettes need to be thoroughly cleaned by performing steps outlined in the cleaning protocol below.
      2. By saving the OD450 = 1.00 samples they can directly be reused for spectral measurements with the plate reader described in ‘Measuring spectra with the plate reader’.

        Table 3. Filter setup for fluorometer spectral measurements

         aFilters with similar transmission properties can be used.

    Measuring spectra using a fluorometer–Fluorometer settings and practical setup
    Emission spectra are measured using a fluorometer. The fluorometer should excite a sample with a narrow band wavelength, which then emits fluorescence that is measured. A schematic overview of a fluorometer is shown in Figure 4. The light source emits white light, which passes a slit to the excitation monochromator where it is separated into its wavelength components. The light passes a slit before passing the excitation filter and hits the sample suspension in the quartz cuvette. The cells in the sample will be excited and emit light in every direction but excitation light also scatters from them. At an angle of 90°, the scattered light is filtered by an emission filter and the fluorescence emission passes a slit entering the emission monochromator. Here, the emission wavelengths are scanned and pass the last slit before entering a photomultiplier where quanta of photons are being transformed into a measurable electrical current. The resulting emission spectra are the raw data needed to calculate FRET. The measurement of different FPs requires different excitation, filter and measurement settings which are shown in Table 3. Slit widths are usually set to 6 nm and generally give a good result if the fluorescent proteins are expressed well. Smaller slit widths result in more specific measurements but will also let less light pass to be detected so slits should only be more closed when fluorescent signals are very high. If the fluorescent signals are low, the slits could be widened but this will result in less specific signals. When background fluorescence is an issue, this will increase in a proportional manner.
    Notes:
    1. The white light source has to heat up before the light output will be stable. Thirty minutes after turning it on check whether the power of the lamp is 75 W and if not adjust. Other fluorometers will have different requirements.
    2. The use of other white light sources or equipment may require different preparations.


      Figure 4. Schematic overview of measuring a sample in the fluorometer. The double black and gray circles at the monochromators represent adjustable slits.  

      Protocol cleaning quartz cuvettes
      1. Profusely rinse the inside and outside of the cuvette with H2O.
      2. Carefully shake off the excess H2O.
      3. Profusely rinse the inside and outside with 70% ethanol.
      4. Carefully shake off the excess 70% ethanol.
      5. Repeat Steps 1 to 4 (Protocol cleaning quartz cuvettes), three times.
      6. Profusely rinse the inside and outside with 96% ethanol.
      7. Carefully shake off the excess ethanol 96%.
      8. Blow dry the cuvette with compressed air.
      9. Polish the cuvette with lens tissue to get rid of accidental smudges like for instance fingerprints. Now the cuvettes are properly cleaned and can hold a sample for measurement.
      Notes:
      1. The stirrer beans are washed by the same rinsing protocol in the palm of your hand.
      2. After the last measurement wash all cuvettes and stirrer beans one more time and then submerge them in 2% Hellmanex III for storage. If cuvettes are not needed for longer time periods, store them clean and dry.
      3. Quartz cuvettes should be cared for meticulously as they are fragile and expensive. Only touch the cuvettes with your fingers close to the opening to prevent finger prints on the important part of the quartz. Carry the cuvette between your index finger and thumb while supporting the bottom with your pinky or ring finger as shown in Figure 5.


        Figure 5. Cartoon of how to hold quartz cuvettes properly i.e., between thumb and index finger supported by the little finger

    Measuring spectra with the plate reader
    A convenient way of measuring the emission spectra of your samples is by using a fluorescence plate reader. A word of caution is in order as the sensitivity of most fluorescence plate readers is lower than that of a dedicated fluorometer and provide weaker fluorescence signals. If fluorescence is low, the fluorometer should be used. This Bio-protocol describes the use of the BioTek synergy MX plate reader, which has excitation and emission monochromators without the possibility for additional optical filtering. In addition, this plate reader has slits with a minimal opening of 9 nm. The, on average, lower signal to noise and less specific signals from a plate reader may therefore lead to unmixing problems. It is thus recommended to first firmly establish FRET interactions using a fluorometer and then confirming them using the plate reader. If results are reproducible, experiments can be done with the plate reader only.


    Protocol plate reader FRET for fixed cells (mNG-mCh only)

    1. Prepare samples with OD450 values of 1.00 ± 0.005 like described in Steps D1 and D2.
    2. Dispense 200 µl replicates of the samples in a glass bottomed black walled 96 wells plate. If eight replicates are made, there is space for 11 prepared samples and 1 PBS measurement in a 96-well plate.
    3. Measure the donor and acceptor emission spectra of each well using the settings shown in Table 4.
    4. Export the emission data in an Excel compatible format and plot the replicates to check for obvious outlying data.
    5. Exclude the possible outliers based on large and obvious deviations from the other replicates and average the acceptor and donor emission spectra. These data will be used for spectral unmixing and FRET calculations.

      Table 4. Setting for spectral measurements using the plate reader

    6. *Optimal gain may differ between experiments. It is better to have slightly lower signals than to oversaturate the detector.
      Note: Measuring the OD450 values in the plate reader before the spectral measurements will give an indication how similar the samples are to each other.

      Protocol plate reader FRET for living bacteria experiment (mNG-mCh only)
      1. Grow your cells like described in Steps C1 and C2 in Procedure C for the growth of the samples.
      2. Dilute the Gb1 cultures to OD450 = 0.005 in at least 2,000 µl fresh Gb1 pre-warmed to 28 °C.
        Note: This is an important step and needs to be carefully done as the cultures now have to grow almost identically and corrections are virtually impossible. Cultures that have largely different growth rates upon expressing fusion proteins could give unreliable measurements compared to the other cultures and should be avoided.
      3. Dispense the diluted cells in 200 µl replicates in a black walled 96 wells plastic plate and continue growth in the plate reader at 28 °C while shaking at medium setting.
        Note: In our experience, it is better to use plastic bottomed black walled plates for these experiments as the glass bottomed ones give more variable OD450 readings after some growth time.
      4. As OD values approach OD450 = 0.1, dilute all wells 1:2 with fresh pre-warmed Gb1 medium containing IPTG to an end-concentration appropriate for regular experiments and continue growth.
      5. Pay close attention to the growth of the cells, as the cultures leave the exponential phase start spectral measurements as described in Steps c-e of the ‘Protocol plate reader FRET for fixed cells’ above in the current plate.
        Note: Measure OD450 values before and after the spectral measurements to observe possible outliers that should be excluded.
      6. An example of a full in vivo plate reader FRET experiment is shown at the end of Procedure E in Figure 8.

  5. Data analysis
    When the acceptor and donor emission spectra of all samples are measured either by the fluorometer or by the plate reader, they can be unmixed. Spectral unmixing dissects the emission data into its components based on the references that were provided. The donor channel is composed of background emission, donor-only emission, directly excited acceptor emission and possible FRET acceptor emission (Figure 6). The acceptor channel is composed of background emission and acceptor-only emission. Form this spectrum the amount of acceptor in the sample can be calculated using the background and reference spectra for the unmixing of the spectra. Knowing the amount of acceptor fluorescence, it can be calculated how much fluorescence is expected from the acceptor due to cross-excitation of the donor and the acceptor in the sample that contains both. Using the reference spectra and the knowledge on the amount of acceptor in the sample, the emission spectrum for a sample can be dissected into the background spectrum, the donor spectrum and the acceptor spectrum. Remaining is a spectrum of identical shape as the acceptor spectrum that cannot be accounted for if no sensitized emission (energy transfer) had occurred. This extra acceptor spectrum corresponds to the amount of energy transfer. The supplementary data of Alexeeva et al. (2010) gives a detailed description of the calculations based on biophysical work beyond the scope of a protocol (Clegg, 1992; Clegg et al., 1992; Wlodarczyk et al., 2008; Gadella, 2009). In the supplementary information, ready-to-use Excel sheets are provided to calculate FRET efficiencies for each of the described FRET pairs. ‘mNG-mCh.xlsx’ ‘mNG-mCh plate reader.xlsx’ ‘SYFP2-mCh.xlsx’ and ‘mKO-mCh.xlsx’.


    Figure 6. Principle of the periplasmic FRET assay. A. Excitation and emission spectra of mCh and mNG, indicating the wavelengths used to measure the acceptor channel (left) or the donor channel (right). The bold dotted line represents the used excitation wavelengths. The hatched blue area represents the spectral overlap between the mNG emission spectrum and the mCh excitation spectrum. The gray shaded area represents the wavelengths for which the emission was measured. B. Samples measured to calculate periplasmic FRET. References of background, mCh, and mNG are needed to calculate their contributions to the FRET within a sample. C. Unmixing of the periplasmic tandem FRET sample, showing the measured spectrum as black dots (Peri-tandem) and the calculated spectrum as a solid red line (Unmixed Calc.). The measured spectrum for the acceptor is composed of the background fluorescence (Unmixed Empty cells) and the amount of mCh (Unmixed Acceptor) present in the sample excited at 590 nm. The direct excitation of the acceptor is used to determine the amount of acceptor present in the sample. This amount is then used to calculate how much extra acceptor (sensitized emission) is detected in the donor excited sample. The measured spectrum for the donor contains the background (Unmixed Empty cells), mNG (Unmixed Donor), and mCh (Unmixed Acceptor) fluorescence and sensitized emission (Unmixed SE), which is the extra fluorescence that the unmixing algorithm cannot attribute to direct excitation of mCh. D. The low residual difference between the measured and calculated spectra is a measure of the quality of unmixing. This figure was originally published in Meiresonne et al., 2017.

    Protocol using the unmixing sheets
    An overview of how the unmixing process would appear on the computer screen is shown in Figure 7. High resolution screenshots are available in the supplementary data.
    Note: Be aware that the raw data may have different output formats depending on the equipment used and adjust accordingly.
    1. Copy the spectral data in the appropriate excel sheet 1 ‘Raw data’.
    2. Subtract the PBS measurement from all samples in sheet 2 ‘Minus PBS’.
    3. Copy the empty vector, acceptor reference and the donor reference in the indicated places in sheet 3 ‘Data’. This will automatically subtract the background signal from the acceptor and donor reference spectra.
    4. Copy sample data to the indicated place in sheet 3 ‘Data’ the sheet will unmix these spectra based on the given references.
      Note: The reference and sample spectra are plotted at the bottom of sheet 3 for inspection.
    5. Inspect the unmixing overview on sheet 4 ‘Result’ as described further below.
    6. Copy paste the results to sheet 5 ‘Summary’.
    7. Repeat Steps 4-6 for the remaining samples.


      Figure 7. The unmixing process for the calculation of FRET efficiencies as it would appear on the screen. Raw donor and acceptor unmixing calculations that will not be directly used are shown in the high-resolution screenshots in Supplementary file ‘Unmixing screens.pptx’.

      What determines a good measurement?
      Spectral unmixing is highly dependent on clear fluorescent signals that should be measured under identical conditions. The donor and acceptor reference spectra are obtained by subtracting the PBS and empty cells data from their respective emission measurements as described in ‘Protocol using the unmixing sheets’. This should yield a clean emission spectrum of the donor only or the acceptor only FP. Sometimes spectra that do not resemble the measured FP anymore or even negative spectra of especially the donor can be seen. This can be caused by strong levels of autofluorescence in the empty cells sample and low FP signals in the reference samples. If one of the references is not good, the unmixing of the spectra will not give trustworthy results. Alternatively, reference spectra of a previous measurement may be used to still get a rough indication of the FRET values of the samples. It is not advised to use these values as definite and the experiment needs to be repeated to yield trustworthy data. When the experiment results in good references, sample data can be unmixed. Emission signal that could not be attributed to the acceptor spectrum is called the unmixing difference. The unmixing difference is a good measure for the quality of the measured FRET spectra and in an ideal experiment it should be zero. More realistically, it should be a small percentage of the measured spectra as shown in Figures 6D and 8D.


      Figure 8. Full in vivo plate reader FRET experiment. A. Growth curves of cells grown in Gb1 at 28 °C show only small differences in doubling time for the periplasmic tandem mNG-mCh. OD450 values of all well-cultures were within ~10% of each other before measuring fluorescent spectra. The arrow indicates the time point at which induction of the constructs was started by 1:2 dilution. B. Average normalized fluorescence spectra of periplasmic references with standard deviation error bars showing only small intensity variation between replicates. C. Good quality unmixing of the acceptor channel on the left and of the donor channel on the right of the average periplasmic tandem sample with average references suggests reliable calculations of sensitized emission. D. Difference between the measured spectrum and the calculated spectrum showing only little unmixing differences for the acceptor and donor. This figure was originally published in Meiresonne et al., 2017.

Data analysis

This bio-protocol describes how to prepare, perform and analyze in vivo bacterial FRET experiments. At the relevant sections (Procedure E), information is provided on data analysis and references to in-depth technical information are provided. The significance of the obtained FRET values can be calculated using standard confidence interval statistical approaches. The FRET values should be significantly higher than the values obtained from non-interacting samples due to crowding. For this analysis, standard t-tests can be used.

Recipes

  1. TY medium
    For 1 L weigh:
    10 g Bacto tryptone
    5 g yeast extract
    5 g NaCl
    Add H2O up to 1 L and autoclave for 15 min at 121 °C
  2. 20% glucose solution
    For 200 ml 20% glucose solution weigh:
    40 g glucose
    Add H2O up to 200 ml and autoclave for 15 min at 121 °C
  3. Gb1 medium
    For 1 L combine (using aseptic technique):
    100 ml MM1-10x (Recipe 3a)
    100 ml MM2-10x (Recipe 3b)
    10 ml MM3-100x (Recipe 3c)
    10 ml MM4-100x (Recipe 3d)
    Note: For BW25113, TB28 and MG1655 based E. coli strains.
    20 ml glucose 20%
    1 ml thiamine hydrochloride 4 mg ml-1 (Recipe 3e)
    2.5 ml L-lysine monohydrochloride 20 mg ml-1 (Recipe 3f)
    1 ml ampicillin mg ml-1
    1 ml chloramphenicol 25 mg ml-1
    Add sterile H2O up to 1 L
    1. MM1-10x
      For 1 L MM1-10x weigh:
      63.3 g K2HPO4·3H2O
      29.5 g KH2PO4
      Add H2O up to 1 L and autoclave for 15 min at 121 °C
    2. MM2-10x
      For 1 L MM2-10x weigh:
      10.5 g (NH4)2SO4
      1 g MgSO4·7H2O
      Add H2O up to1 L and autoclave for 15 min at 121 °C
    3. MM3-100x
      For 1 L MM3-100x weigh:
      28 mg FeSO4·7H2O
      710 mg Ca(NO3)2·4H2O
      Add H2O up to 1 L and autoclave for 15 min at 121 °C
      Note: After sterilization, the MM3 will show a red precipitation that should be ignored.
    4. MM4-100x
      For 50 ml MM4-100x weigh:
      250 mg L-arginine
      250 mg L-glutamine
      10 mg thymidine
      100 mg uracil
      Add H2O up to 50 ml and filter sterilize with Filtropur S 0.2 µm filter
    5. Vitamin B1 stock solution (4 mg ml-1)
      For 50 ml vitamin B1 4 mg ml-1 stock solution weigh:
      200 mg thiamine hydrochloride
      Add H2O up to 50 ml and filter sterilize with Filtropur S 0.2 µm filter
    6. Lysine stock solution (20 mg ml-1)
      For 50 ml lysine 20 mg ml-1 stock solution weigh:
      1 g lysine
      Add H2O up to 50 ml and filter sterilize with Filtropur S 0.2 µm filter
  4. Phosphate buffered saline (PBS 1x)
    PBS buffer (pH 7.2)
    140 mM NaCl
    27 mM KCl
    10 mM Na2HPO4·2H2O
    2 mM KH2PO4
  5. Formaldehyde and glutaraldehyde (FAGA) fixative
    Per 1 ml of formaldehyde add 21 µl of glutaraldehyde

Acknowledgments

Adaptations of this bio-protocol have been used in the following publications (Alexeeva et al., 2010; Fraipont et al., 2011; van der Ploeg et al., 2013 and 2015; Meiresonne et al., 2017). The authors would like to thank prof. dr. Theodorus W. J. Gadella for developing the original Excel unmixing sheet (Alexeeva et al., 2010) and critically reading the manuscript and Laureen M. Y. Mertens for testing out the unmixing protocol. NYM was supported by the NWO, ALW open program (822.02.019), Svetlana Alexeeva by the NWO, ALW ‘Van Molecuul tot Cel’ program (805.47.200) and RvdP by the European Commission DIVINOCELL project (FP7-Health-2007-B-223431). The authors declare no conflict of interest.

References

  1. Alexeeva, S., Gadella, T. W., Jr., Verheul, J., Verhoeven, G. S. and den Blaauwen, T. (2010). Direct interactions of early and late assembling division proteins in Escherichia coli cells resolved by FRET. Mol Microbiol 77(2): 384-398.
  2. Bindels, D. S., Haarbosch, L., van Weeren, L., Postma, M., Wiese, K. E., Mastop, M., Aumonier, S., Gotthard, G., Royant, A., Hink, M. A. and Gadella, T. W., Jr. (2017). mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat Methods 14(1): 53-56.
  3. Clegg, R. M. (1992). Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol 211: 353-388.
  4. Clegg, R. M., Murchie, A. I., Zechel, A., Carlberg, C., Diekmann, S. and Lilley, D. M. (1992). Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction. Biochemistry 31(20): 4846-4856.
  5. Cranfill, P. J., Sell, B. R., Baird, M. A., Allen, J. R., Lavagnino, Z., de Gruiter, H. M., Kremers, G. J., Davidson, M. W., Ustione, A. and Piston, D. W. (2016). Quantitative assessment of fluorescent proteins. Nat Methods 13(7): 557-562.
  6. Detert Oude Weme, R. G., Kovacs, A. T., de Jong, S. J., Veening, J. W., Siebring, J. and Kuipers, O. P. (2015). Single cell FRET analysis for the identification of optimal FRET-pairs in Bacillus subtilis using a prototype MEM-FLIM system. PLoS One 10(4): e0123239.
  7. Fraipont, C., Alexeeva, S., Wolf, B., van der Ploeg, R., Schloesser, M., den Blaauwen, T. and Nguyen-Disteche, M. (2011). The integral membrane FtsW protein and peptidoglycan synthase PBP3 form a subcomplex in Escherichia coli. Microbiology 157(Pt 1): 251-259.
  8. Gadella, T. W. J. (2009). In: Theodorus, W. J. (Ed.). FRET and FLIM techniques. Elsevier.
  9. He, F. (2011). Standard DNA cloning. Bio-protocol 1(7):e52.
  10. Im, H. (2011). The inoue method for preparation and transformation of competent E. coli: "ultra competent" cells. Bio-protocol 1(20): e143.
  11. Karasawa, S., Araki, T., Nagai, T., Mizuno, H. and Miyawaki, A. (2004). Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem J 381(Pt 1): 307-312.
  12. Kremers, G. J., Goedhart, J., van Munster, E. B. and Gadella, T. W., Jr. (2006). Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Forster radius. Biochemistry 45(21): 6570-6580.
  13. Meiresonne, N. Y., van der Ploeg, R., Hink, M. A. and den Blaauwen, T. (2017). Activity-related conformational changes in d,d-carboxypeptidases revealed by in vivo periplasmic forster resonance energy transfer assay in Escherichia coli. MBio 8(5).
  14. Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N., Palmer, A. E. and Tsien, R. Y. (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22(12): 1567-1572.
  15. Shaner, N. C., Lambert, G. G., Chammas, A., Ni, Y., Cranfill, P. J., Baird, M. A., Sell, B. R., Allen, J. R., Day, R. N., Israelsson, M., Davidson, M. W. and Wang, J. (2013). A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods 10(5): 407-409.
  16. Sieger B. and Bramkamp M. (2015). Interaction sites of DivIVA and RodA from Corynebacterium glutamicum. Front Microbiol 5: 738.
  17. Sourjik, V. and Berg, H. C. (2002). Receptor sensitivity in bacterial chemotaxis. Proc Natl Acad Sci U S A 99(1): 123-127.
  18. van der Ploeg, R., Goudelis, S. T. and den Blaauwen, T. (2015). Validation of FRET assay for the screening of growth inhibitors of Escherichia coli reveals elongasome assembly dynamics. Int J Mol Sci 16(8): 17637-17654.
  19. van der Ploeg, R., Verheul, J., Vischer, N. O., Alexeeva, S., Hoogendoorn, E., Postma, M., Banzhaf, M., Vollmer, W. and den Blaauwen, T. (2013). Colocalization and interaction between elongasome and divisome during a preparative cell division phase in Escherichia coli. Mol Microbiol 87(5): 1074-1087.
  20. Wlodarczyk, J., Woehler, A., Kobe, F., Ponimaskin, E., Zeug, A. and Neher, E. (2008). Analysis of FRET signals in the presence of free donors and acceptors. Biophys J 94(3): 986-1000.

简介

该协议的开发是通过Förster共振能量转移(FRET)定性和定量检测大肠杆菌中的蛋白质 - 蛋白质相互作用。所描述的测定允许以前不可能的周质蛋白质 - 蛋白质相互作用的体内筛选。在FRET中,供体荧光分子的激发导致能量转移到受体荧光分子,如果它们之间的距离在1-10nm范围内,则受体荧光分子将发光。荧光蛋白质可以被遗传编码为与感兴趣的蛋白质的融合物并且在细胞中表达,因此FRET蛋白质 - 蛋白质相互作用实验可以在体内进行。供体和受体荧光蛋白融合体被构建用于被怀疑相互作用的细菌蛋白质。这些融合蛋白在细菌细胞中共表达,随后激发供体和受体通道测量荧光发射光谱。供体的发射光谱与受体的激发光谱之间的部分重叠是FRET的先决条件。即使在没有FRET的情况下,供体激发也可以使受体以已知百分比交叉激发。通过测量背景,仅供体和仅受体样品的参考光谱,可以计算预期的发射光谱。在预期光谱之上的受体的致敏发射可以归因于FRET,并且可以通过光谱解混来量化。

【背景】确定如何和哪些蛋白质相互作用维持生命是分子生物学研究的核心。存在许多体外方法,但可能导致误报,因为相互作用是从其生物学背景中取出的。 体内方法对体外验证结果至关重要。此外,“体内”方法允许观察在细胞中发生的经常发生的动态过程。荧光蛋白(FP)对于体内实验来说是理想的,因为它们可以融合到感兴趣的蛋白质上,从而可以在显微镜下追踪融合蛋白在活细胞中的定位和动态。通过Förster共振能量转移(FRET),能量可以在FP之间转移,前提是它们具有兼容的物理特性。供体FP的发射光谱必须与受体FP的激发光谱重叠,并且它们之间的距离应小于10nm。光谱重叠越大,供体和受体FP之间的距离越小,则FRET可能发生得越多。 FRET的严格距离依赖性是检测直接蛋白质 - 蛋白质相互作用的理想选择,因为它们也发生在纳米范围内,而间接蛋白质相互作用通常在更大的距离范围内且不被FRET检测到。由于这个原因,它提供了很少的假阳性,但是由于涉及荧光团的物理要求,阴性结果可能是假阴性。使用FP进行FRET测量的优点在于,发色团被包封在蛋白质桶中,因此比化学发色团更好地保护环境。后者的荧光对环境变化如pH,盐和溶剂极其敏感。因此,在活细胞这样的复杂环境中,它们通常不能可靠地用于测量FRET。在FPs中封装发色团的3-4nm直径蛋白质桶的缺点是它将蛋白质之间的可检测距离降低到1-6nm。由于5-7纳米是蛋白质的典型大小,所以通常不会造成问题,但是又会导致错误的结果。我们已经使用基于光谱的FRET方法来分析大肠杆菌细胞质和周质中蛋白质之间的相互作用(Alexeeva等人,2010; Fraipont等人, et al。,2011; van der Ploeg等人,2013和2015; Meiresonne等人,2017),并表明该技术适用于(van der Ploeg等人,2015)的作用模式研究和使用各种FP FRET对的中等通量筛选(Meiresonne et al。,2017)。这个生物协议介绍了如何执行我们的细菌FRET测定,以确定光谱解混蛋白质相互作用。虽然这个协议是为E写的。大肠杆菌,光谱解混的方法适用于任何生物体,只要在该物种中可以表达融合蛋白并且可以收集荧光光谱。协议描述了如何通过3种方法进行FRET实验:1)使用荧光计对固定细胞进行测量,即蛋白质相互作用的初始测定。 2)在读板仪上对固定细胞进行测量,以更快地检测已经证实的蛋白质相互作用,从而提供清晰的荧光信号。和3)建立的相互作用,在平板阅读器中生长的活细胞的测量。例如,要监视特定交互的实时抑制。

关键字:细菌, 细胞质, 周质, 蛋白质相互作用, FRET, mNeonGreen, SYFP2, mKO, mCherry

材料和试剂

  1. 无菌直颈培养瓶(DWK Life Sciences,DURAN,目录号:2177124)和铝阳极氧化金属蓝帽(DWK Life Sciences,DURAN,目录号:2901324)
  2. 50毫升无菌猎鹰管(SARSTEDT,目录号:62.657.254)
  3. 2毫升无菌Eppendorf管(Greiner Bio One International,目录号:623201)
  4. 1.5 ml无菌Eppendorf管(Greiner Bio One International,目录号:616201)
  5. 一次性移液吸头200μl(Ultratip,Greiner Bio One International,目录号:739290)
  6. 一次性移液吸头1,000μl(Ultratip,Greiner Bio One International,目录号:686290)
  7. 洗瓶(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:2401-0500)
  8. 玻璃底黑色96孔板(Porvair Sciences,目录号:324002)

  9. 黑壁清底96孔塑料板(Greiner Bio One International,目录号:675096)
  10. 搅拌石英比色杯(Hellma,目录号:119004F-10-40)
  11. 镜头清洁组织(Fischer Scientific,Fisherbrand TM,产品目录号:10605955)
    注:此产品已停产。
  12. Filtropur S 0.2μm过滤器(SARSTEDT,目录号:83.1826.001)
  13. 乙醇70%在实验室洗瓶(VWR,Technisolv®,目录号:83801.360)
  14. 乙醇96%在实验室洗瓶(VWR,Technisolv®,目录号:83804.360)
  15. H 2 O在实验室洗瓶中
  16. 1 Mβ-D-1-硫代吡喃半乳糖苷(IPTG)(Duchefa Biochemie,目录号:I1401)
  17. Hellmanex III(Hellma,目录号:9-307-011-4-507)
  18. 细菌用胰蛋白胨(Duchefa Biochemie,目录号:T1332)
  19. 酵母提取物(Duchefa Biochemie,目录编号:Y1333)
  20. 氯化钠(NaCl)(Merck,目录号:106404)
  21. 葡萄糖(Duchefa Biochemie,目录号:G0802)
  22. 盐酸硫胺素(Sigma-Aldrich,目录号:T4625-25G)
  23. L-赖氨酸一盐酸盐(Sigma-Aldrich,目录号:L8662)
  24. (西格玛奥德里奇,目录号:A9518)在50%乙醇中的氨苄青霉素储备溶液
  25. (50%乙醇)中的氯霉素储备溶液(Duchefa Biochemie,目录号:C0113)
  26. 二磷酸氢钾(K <子> 2 HPO <子> 4 •3H <子> 2 O)(VWR,目录号:26932.290)
  27. 磷酸二氢钾(KH 2 PO 4)(Merck,目录号:104873)
  28. 硫酸铵,(NH 4)2 SO 4(Sigma-Aldrich,目录号:A6387)
    注:此产品已停产。
  29. 硫酸镁七水合物(MgSO 4•7H 2 O)(Carl Roth,目录号:T888.1)
  30. 硫酸铁(II)七水合物(FeSO 4•7H 2 O)(Sigma-Aldrich,目录号:215422)
  31. 硝酸钙四水合物,Ca(NO3)2•4H2O(西格玛奥德里奇,目录号:C1396)
  32. L-精氨酸(Sigma-Aldrich,目录号:A8094)
  33. L-谷氨酰胺(Sigma-Aldrich,目录号:G8540)
  34. 胸苷(Sigma-Aldrich,目录号:T1895)
  35. 尿嘧啶(Sigma-Aldrich,目录号:U1128)
  36. 氯化钾(KCl)(VWR,目录号:71003-522)
  37. 二磷酸氢钠二水合物(娜<子> 2 HPO <子> 4 •2H <子> 2 O)(Merck公司,目录号:106580)
  38. 戊二醛25%(Merck,目录号:1042390250)
  39. 甲醛≥36.0%(Sigma-Aldrich,目录号:47608)
  40. 赖氨酸(Sigma-Aldrich,目录号:L8662)
  41. TY中等(见食谱)
  42. 20%的葡萄糖溶液(见食谱)
  43. GB1培养基(见食谱)
    1. MM1-10x
    2. MM2-10x
    3. MM3-100x
    4. MM4-100x
    5. 维生素B1储备液(4毫克ml-1)
    6. 赖氨酸原液(20毫克ml -1)
  44. 磷酸盐缓冲液(PBS 1x)(见食谱)
  45. 甲醛和戊二醛(FAGA)(见食谱)

设备

  1. P20,P200&amp; P1000 Pipetman移液器(Gilson,目录号:F167300)
  2. 旋转式水浴摇床G76型(Eppendorf,New Brunswick Scientific,型号:G76或GFL-GesellschaftfürLabortechnik,目录编号:1092),带有培养瓶的支架
  3. Photospectrometer(Biochrom,型号:天秤座S22)
  4. 压缩空气源
  5. 水净化系统(来自ELGA LabWater,Laner End,HP14 3BY,UK的ELGA LC197-Biofilter的Purelab flex)
    注意:协议中的所有H 2

  6. 离心50毫升Falcon管(Eppendorf,型号:5804 R)
  7. 用1.5和2.0ml Eppendorf离心管(Eppendorf,型号:5424R)离心
  8. 荧光分光光度计Quantamaster 2000-4(美国新泽西州Photon Technology International公司)采用红色优化设置:R928P PMT管(185-900 nm),500 nm闪光(1200线/ mm)激发和发射光栅。
    注意:能够可靠地检测直到700nm的荧光光谱的PMT将适用于所述的方案。
  9. 光学滤波器:
    1. 500 / 10nm BrightLine®单波段带通滤光片(Semrock,目录号:FF01-500 / 10)
    2. ET510LP(色度技术,目录号:ET510lp)
    3. 514/3 nm BrightLine®单波段带通滤光片(Semrock,目录号:FF01-514 / 3)
    4. 515纳米阻塞边BrightLine®长通滤波器(Semrock,产品目录号:FF01-515 / LP)
    5. HQ541 / 12(Chroma Technology,目录号:HQ541 / 12)
      注:本产品已停产。可以使用具有类似传输特性的滤波器。
    6. E550LP(Chroma Technology,目录号:E550lp)
    7. 587/11 nm BrightLine®单频带通滤波器(Semrock,产品目录号:FF01-587 / 11)
    8. HQ600LP(Chroma Technology,目录号:HQ600LP)
      注:此产品已停产。
  10. 荧光扫描板阅读器(BioTek Instruments,型号:Synergy TM Mx,目录号:SMATBC)

  11. 高压灭菌器(SANYO,目录号:MLS-3780)

软件

  1. 荧光分光光度计软件(PTI采集软件FeliX32版本1.2 Build 56)
  2. 酶标仪软件Gen5版本2.00(Biotek)
  3. Excel(微软)

程序

FRET方法从头到尾包含几个步骤,每个主题进行讨论。程序和部分在图1中概述。


图1.基于细菌谱的FRET方法的工作流程面板的标签对应于描述特定协议的部分。 A.选择合适的FRET对并创建表达质粒。 B.测试构建的功能性质粒。 C.培养稳定的培养物,诱导FRET构建体和收获细胞的表达。 D.样品制备和数据采集。 E.数据分析。

  1. 为您的实验选择合适的FRET对并创建FP融合
    1. 为您的实验选择合适的FRET对
      在开始FRET实验之前,应该选择最合适的FRET对,这取决于计划的实验类型。例如,所测定的蛋白质 - 蛋白质相互作用的间隔或进行体内测量的要求影响这种选择(表1)。在这个生物协议中,mCherry(Shaner等人,2004)被用作mNeonGreen(Shaner等人,2013)(mNG),super (SYFP2)或单体Kusabira-Orange(Karasawa等人,2004)(mKO)供体FPs。所有的组合都有其特定的优点和缺点。已经将这些蛋白质描述为单体,并且通常不具有强烈的聚集倾向(Cranfill等人,2016)。理论上,具有最大福斯特半径(R 0)的FRET对将导致最高的FRET值。 R 0值是从供体到受体荧光团发生50%能量转移的距离的量度(图2)。这由供体发射光谱和受体激发光谱之间的重叠,它们各自的量子产率,消光系数和取向来确定。


      图2.描述的FRET对的Forster半径。使用表1中的光谱性质绘制mNG-mCh,SYFP2-mCh和mKO-mCh FRET对的作为发色团距离的函数的理论FRET效率。在右侧,放大的图显示R <或者荧光团之间的距离将会产生50%的能量转移。

      在这里描述的FRET对中,mKO-mCh具有5.9nm的最高R 0值。然而,mKO具有较长的成熟时间,即虽然它是由细胞产生并且其三级结构已折叠,但形成发色团的氨基酸需要时间来重排它们的共价键以发荧光,这被称为成熟。没有完全成熟的FP的FRET实验因此可能导致不平衡的信号采集和错误的后续计算。如果考虑到成熟时间是没有问题的,那么mKO-mCh对优选用于细胞质FRET实验,因为其具有良好的动态范围和最少的耗时测量时间(Alexeeva等人,2010; Fraipont 等人,2011; van der Ploeg等人,2013)。 SYFP2-mCh FRET对具有略低的R0值(5.7nm),但会发射强的SYFP2荧光,这将提供足够的信号用于在每个细胞的相对较低拷贝数的蛋白质之间发生的相互作用van der Ploeg et al。 ,2015)。鉴于SYFP2具有良好的折叠和成熟性能,使用SYFP2-mCh对可以进行FRET,尽管只有固定细胞的实验尚未完成。如果使用SYFP2-mCh FRET配对并且细胞固定,则SYFP2信号需要恢复其荧光,并且不可能直接测量样品。无论如何,在我们手中,SYFP2-mCherry FRET对提供了最广泛的FRET检测范围。这是令人惊讶的,因为mKO-mCh对具有更高的R 0 0,但是由于其高荧光强度,可能SYFP 2表现出稍好的性能。因此,如果需要测量较少丰度蛋白质的FRET值的较小差异,则SYFP2-mCh对可能比mKO-mCh对更合适。在所描述的三个FRET对中,mNG-mCh具有最低的R 0值(5.5nm),因此具有适度的检测范围,但是其也具有优势。目前mNG是mCherry在周质中折叠成熟的唯一有用的捐献者。 mNG-mCh FRET对可以在细胞质和周质中的活细胞FRET实验中实时使用(Meiresonne等人,2017)。此外,通过光谱解混获得的mNG-mCh FRET对结果可以通过基于荧光寿命的检测方法来确认(Meiresonne等人,2017)。

      表1.本生物协议中描述的FRET对的信息

      *如原始文件(Alexeeva等人,2010; van der Ploeg等人,2013和2015; Meiresonne等人 >,2017)。
      注意:
      1. 对于表1中给出的R值,最初公布的消光系数为72,000M (Shaner -1 -1 等人,2004年)。基于检测精度和样品条件,在FP消光系数的描述中发生变化。不断改进的FP研究领域表明,mCh的消光系数更多地在85000M -1cm cm (Cranfill等,2016; Bindels等,2017)。同样,mKO的量子产率据报道高于最初的想象(0.77)(Cranfill等,2016)。这将导致每个描述的FRET对的更高的R值和不同的计算。
      2. 不同的FP对,尤其是CFP-YFP已经用于细菌FRET。欲了解更多信息,请参阅参考文献(Sourjik和Berg,2002; Detert Oude Weme等,2015; Sieger和Bramkamp,2015)。

    2. 创建FP融合
      编码感兴趣蛋白的细菌基因与编码所述荧光蛋白之一的基因融合,并由诱导型表达载体表达。可以使用任何质粒表达系统,条件是质粒是相容的(不同的复制起点和不同的抗生素抗性标记),并且表达水平低(通常为2,000个拷贝的蛋白质)以避免非特异性相互作用。为了使FP和感兴趣的蛋白质有一定的运动自由度并减少空间位阻,可以在FP和蛋白质之间加入接头。经常使用GGS重复序列,但是我们对ARNNNN和其他氨基酸序列也有很好的结果。另一个考虑是N-或C-末端融合的选择。例如,应避免或规避编码用于下游运输或加工的信号序列的蛋白质的N端融合物。另外,蛋白质的N-或C-末端对于其正常定位或功能可能是必需的。程序B讨论测试创建的融合。质粒的序列应在进一步实验前确认。用质粒克隆和转化大肠杆菌的描述超出了本方案的范围,但是其他生物学方案如“[BIO101]标准克隆”和“[BIO101] Inoue方法的制备和转化“大肠杆菌”:“超级感染细胞”(He,2011; Im,2011)可能是有用的。

  2. 测试FP融合
    理想情况下,FP融合蛋白应该是完全有功能的,并且表达水平接近其原生水平,同时仍然导致可测量的荧光强度。一旦在质粒上产生融合体,应该测试表达的产物对表型,荧光和正确的细胞定位的影响。如果没有观察到荧光,则可以通过Western印迹分析评估FP的降解。也许融合破坏了感兴趣的蛋白的正常定位,功能和相互作用。这可以通过修改链接器的长度或切换FP的位置来修改。一些蛋白质既不能作为N的功能也不能作为C端融合物,而是作为夹心融合物起作用(van der Ploeg等人,2015)。 FP-融合应补充相同蛋白质的缺失或缺失菌株的功能丧失。互补测试将取决于基因的丢失是否导致表型。如果不能进行互补实验,FRET实验可以在野生型或缺失背景下进行,但是应该记住融合蛋白可能不起作用,结果可能会被误解。
    注意:基于质粒的FP融合是快速测试定位或感兴趣的蛋白质相互作用的好方法。然而,由于化学计量不平衡,基于质粒的互补并不总是可行的。在这种情况下,可将FP融合体插入染色体的原始位置。实际上,已知的FRET配偶体的染色体插入对于简单的测定来说将是优选的。然而,这也将需要染色体参照在同基因背景中,因为细菌可以对质粒和基于染色体的蛋白质表达在生长差异和质粒维持可能的自发荧光方面有不同的响应。

    准备FRET实验
    当FP融合蛋白准备好使用时,可以用供体和受体表达质粒转化感受态细胞。任何 E。大肠杆菌菌株应该是合适的。因为我们对影响形态的蛋白质感兴趣,所以我们倾向于使用野生型菌株MC4100或BW25113,它们在形态上是均一的。请注意,如果使用缺失菌株,则参考和对照应该在野生型变体中。所描述的FRET测定需要至少三个参考样品,其在与所评估的相互作用相同的遗传背景的细胞中表达。强烈建议使用由表达与受体FP融合的供体FP的质粒和表达供体或受体FP的两种质粒分别与已知不相互作用的无关蛋白融合的阳性和阴性对照。最好的对照在与蛋白质 - 蛋白质相互作用发生的相同区室中表达,以便能够评估分子拥挤效应。表2列出了最小FRET实验的样品列表。

    表2.最小FRET实验所需的样品和质粒

    为了检测两个感兴趣的蛋白质之间的相互作用,至少需要三个参考文献。建议积极和消极的控制。所描述的方案针对两种相容性质粒,其具有与氯霉素抗性结合的p15复制起点和结合氨苄青霉素抗性的ColE1复制起点。两种质粒都具有弱化的99A启动子和两种终止子。

  3. 稳态增长,诱导和收获细胞
    生长在丰富培养基中的细菌细胞通常含有高浓度的自动荧光分子,降低了信噪比。出于这个原因,细胞在基本培养基中生长,不鼓励在富培养基中进行FRET实验。为了比较表达FP对的各种细菌培养物,在诱导蛋白质表达之前,细胞在约20代中生长至稳定状态(图3)。这确保了所有样品中相同的自发荧光背景。诱导表达至少2代后,固定和收集细胞。
    1. 制备在5ml TY +氨苄青霉素+氯霉素+ 0.5%葡萄糖培养基中在37℃下振荡直至细菌生长可见的所有菌株的起始培养物, > 注意:向培养基中加入葡萄糖以抑制来自质粒的lacI / qq受控基因的表达。
    2. 使用发酵剂培养物接种25ml(1:250)Gb1培养基+氨苄青霉素+氯霉素,并在摇动的同时在28℃继续过夜生长。
    3. 第1天。在新鲜的Gb1培养基+氨苄青霉素+氯霉素中稀释过夜培养物1:250,并在摇动的同时在28℃继续生长。
      注意:从现在开始,定期测量并注意培养物的OD值,不要让它达到0.2以上。如果培养物接近这个值,就用新鲜的预热培养基稀释。
    4. 使用提供的Excel工作表计算所有生长培养物的倍增时间(TD)“增长计算.xlsx “,并在继续实验时用它来计算第二天早上0.1的OD值450的稀释因子。
    5. 重复在新鲜的Gb1培养基+氨苄青霉素+氯霉素中生长,同时保持OD 450值低于0.2,并在第二天早晨稀释,如步骤C4所示。 >
    6. 重复在新鲜的Gb1培养基+氨苄青霉素和氯霉素中生长,同时保持OD 450值低于0.2,并在第二天早晨稀释,如在步骤C4中一样,但是这次的目标对于35ml新鲜的Gb1 +氨苄青霉素+氯霉素,其OD 450值为0.025。
    7. 测量培养物的OD 450值,如果它们在0.03-0.04左右,则通过加入适当浓度的IPTG诱导来自质粒的表达(例如<! - SIPO
    8. 当培养物接近〜0.2的OD 450值时,准备足够量的FAGA固定剂(见食谱)。
      注意:所需FAGA的总体积取决于待固定培养物的总体积,需要1 ml FAGA来固定12.5 ml培养物。
    9. 当细胞达到0.2的OD 450值时,用FAGA将细胞固定在振荡水浴中,这取决于培养物体积剩余10分钟。
    10. 将固定的培养物倒入50ml Falcon管中,并通过在室温(Eppendorf 5804R)在6,300×g下离心10分钟来沉淀细胞。
    11. 丢弃上清液,用1ml PBS在1.5ml Eppendorf管中在室温下在台式离心机(Eppendorf 5424R)中以4600×gg离心5分钟来洗涤3次。
    12. 在1ml PBS中重悬最后一个沉淀,取决于使用的FRET对,在37℃或在周末在4℃孵育o / n以允许发色团成熟(emo mKO-mCh 或将样品保存在4°C直到准备测量(SYFP2-mCh&amp; mNG-mCh)。
      注意:SYFP2和mNG的折叠速度很快,它们的发色团(快速)成熟很快,因此可以考虑直接测量,但是在第二天进行光谱测量更为实用。


      图3.稳态增长的例子基于MC4100的大肠杆菌培养物的培养物生长曲线在28℃下在Gb1中生长至稳态,表达周质FP-融合体。圆圈表示过夜稀释的时间点。在第4天,用IPTG诱导培养物(箭头)以表达周质性mNG-或mCh-融合物。 EV(空载体)文化不表示任何FP,并作为对照。在OD 450下给出X轴以表示指数增长。细胞在至少两次质量加倍(星号)后固定和收获。这个数字是根据Meiresonne et al。,2017年改编的。

  4. 样品制备和数据采集
    1. 如步骤C10所述将细胞沉淀,并重悬于1ml PBS中。
    2. 通过用新鲜的PBS稀释细胞,并将1,200μl加入到搅拌的石英比色皿中,制备至少1,300μl具有1.00±0.005的OD 450值的样品。
      注意:
      1. 可以在光谱分析仪内进行稀释。 OD值等于450就是等于450的值。结果就越可靠。
      2. 在测量之前,不要忘记将磁力搅拌器添加到比色杯中。
    3. 使用表3中所示的设置和光学过滤器测量受体发射光谱,并在'使用荧光计测量光谱 - 荧光计设置和实际设置'中描述。
    4. 使用表3中所示的设置和光学过滤器测量供体发射光谱,并在'<使用荧光计测量光谱< - 荧光计设置和实际设置>“中进行描述。
      注意:建议按照相同的顺序测量每个样品的受体和供体谱图,以防止FP漂白的可能差异。
    5. 对每个样品重复步骤D2到D4,并在测量开始和所有测量结束时测量空白PBS样品。这使您可以验证仪器响应中的任何变化。
    6. 将数据保存为可导出到Microsoft Excel的任何方便的文件格式。
      注意:
      1. 在接收样品之前和之间,需要通过执行下面清洁规程中列出的步骤彻底清洁石英比色皿。
      2. 450 使用读板仪测量光谱 '。

        表3.荧光光谱测量的过滤器设置

        a 可以使用具有类似传输属性的滤镜。

    使用荧光计测量光谱 - 荧光计设置和实际设置
    使用荧光计测量发射光谱。荧光计应激发窄带波长的样品,然后发射测量的荧光。荧光计的示意图如图4所示。光源发出白光,通过一个狭缝到达激发单色器,在那里被分离成它的波长成分。光通过一个狭缝,然后通过激发滤波器,并撞击石英比色皿中的样品悬浮液。样品中的细胞将被激发并在各个方向发光,但是激发光也会从中散射出去。在90°的角度,散射光被发射滤光器过滤,并且荧光发射通过进入发射单色器的狭缝。在这里,发射波长被扫描并通过最后一个狭缝,然后进入一个光子倍增器,在那里光子的量子被转换成一个可测量的电流。产生的发射光谱是计算FRET所需的原始数据。不同FPs的测量需要不同的激发,滤波和测量设置,如表3所示。狭缝宽度通常设置为6 nm,如果荧光蛋白表达良好,通常会得到很好的结果。较小的狭缝宽度导致更具体的测量,但是也将使较少的光通过,以便当荧光信号非常高时狭缝应当更加闭合。如果荧光信号较低,狭缝可能会变宽,但这会导致较少的特定信号。当背景荧光是一个问题,这将成比例地增加。
    注意:
    1. 在光输出稳定之前,白光源必须加热。开机30分钟后,检查灯泡功率是否为75W,如果不调整。其他荧光计将有不同的要求。
    2. 其他白光源或设备的使用可能需要不同的准备。


      图4.在荧光计中测量样品的示意图。单色仪上的双黑色和灰色圆圈表示可调整的狭缝。&nbsp;&nbsp;

      清洁石英比色皿

      1. 大量用H 2 O冲洗反应杯的内部和外部
      2. 仔细摆脱多余的H 2 O.

      3. 用70%的乙醇大量冲洗内外
      4. 小心摇掉多余的70%乙醇。
      5. 重复步骤1至4(协议清洁石英比色皿),三次。

      6. 用96%的乙醇大量冲洗内外

      7. 小心摇掉多余的乙醇96%

      8. 用压缩空气吹干比色杯
      9. 用镜头纸擦拭比色杯,去除指纹等意外污渍。现在比色杯已经被正确地清洗过,可以拿着样品进行测量。
      注意:
      1. 搅拌器豆在手掌上用同样的冲洗方法清洗。
      2. 最后一次测量后,再次将所有的比色杯和搅拌器豆冲洗干净,然后将其浸入2%Hellmanex III中保存。如果比色皿不需要更长的时间,请将其储存干净,干燥。
      3. 由于石英比色皿易碎且价格昂贵,因此应小心照料。只需用手指靠近开口处触摸小杯,以防止在石英的重要部分上印出指纹。在食指和拇指之间携带小杯,同时用小拇指或无名指支撑底部,如图5所示。


        图5如何正确握住石英比色皿 ,用拇指支撑拇指和食指

    使用读板器测量光谱
    测量样品发射光谱的便捷方法是使用荧光读板仪。因为大多数荧光读板器的灵敏度低于专用荧光计的灵敏度,并且提供较弱的荧光信号,所以要谨慎。如果荧光较低,则应使用荧光计。该Bio-protocol描述了BioTek协同MX读板机的使用,该读板机具有激发和发射单色器,而无需额外的光学过滤。此外,这款读板机具有最小9纳米开口的狭缝。平均而言,较低的信噪比和来自读板器的较少特定信号可能因此导致解混问题。因此,建议首先使用荧光计牢固地建立FRET相互作用,然后使用读板器进行确认。如果结果是可重复的,则只能使用读板仪进行实验。


    用于固定细胞的方案读板器FRET(仅限mNG-mCh)

    1. 如步骤D1和D2中所述,准备具有1.00±0.005的OD 450值的样品。
    2. 在玻璃底黑色96孔板中分配200μl样品复制品。如果进行8次重复,则可以在96孔板中留出11个准备样品和1个PBS测量的空间。
    3. 使用表4所示的设置测量每口井的供体和受体发射光谱。
    4. 以兼容Excel的格式导出排放数据,绘制重复数据以检查明显的偏离数据。
    5. 根据来自其他重复的大而明显的偏差排除可能的异常值,并平均受体和供体发射光谱。这些数据将用于光谱解混和FRET计算。

      表4.使用平板读取器进行光谱测量的设置

    6. *实验之间的最佳增益可能不同。
      信号略低于检测器过饱和度 注意:在光谱测量之前,在平板读数仪上测量OD值<450>
      用于活细菌实验的方案读板器FRET(仅限mNG-mCh)
      1. 如步骤C中的步骤C1和C2所述,培养细胞,以培养样本。
      2. 稀释Gb1文化至OD 450 = 0.005在至少2000升新鲜Gb1预热到28℃。
        注:这是一个重要的步骤,需要谨慎地做,因为文化现在必须几乎相同地增长,修正几乎是不可能的。与其他培养物相比,在表达融合蛋白时具有很大不同的生长速率的培养物可能会导致不可靠的测量,应该避免。
      3. 将稀释的细胞以200μl重复的样品分散在黑色的96孔塑料板中,并在28℃下在平板读数器上继续生长,同时在中等振荡的情况下摇动。
        注:根据我们的经验,最好使用塑料底黑色壁板进行这些实验,因为玻璃底板可以给出更多的可变的OD 450
      4. 当OD值接近OD 450 = 0.1时,用含有IPTG的新鲜预热的Gb1培养基将所有孔1:2稀释至适合定期实验并且持续生长的最终浓度。
      5. 密切关注细胞的生长,因为培养物离开指数阶段开始光谱测量,如上面在当前板中的'固定细胞的方案读板器FRET'的步骤ce中所述。 br /> 注意:在光谱测量之前和之后测量OD
      6. 完整的体内读板器FRET实验的一个例子显示在图8的程序E的末尾。

  5. 数据分析
    当所有样品的受体和供体发射光谱都由荧光计或平板读数器测量时,它们可以不混合。根据提供的参考,光谱分解将排放数据分解为其组成部分。供体通道由背景发射,仅供体发射,直接激发受体发射和可能的FRET受体发射(图6)组成。受体通道由背景发射和受体唯一发射组成。形成这个谱图,样品中受体的数量可以使用背景谱和参考谱来计算谱图的混合。知道受体荧光的量,可以计算由于供体和受体在包含两者的样品中的交叉激发而从受体预期有多少荧光。利用参考光谱和样品中受体数量的知识,可以将样品的发射光谱分解为背景光谱,供体光谱和受体光谱。剩下的是与受体光谱相同的光谱形状,如果没有发生敏化发射(能量转移),则不能说明这一点。这额外的受体谱对应于能量转移的量。 Alexeeva等人的补充数据(2010)给出了基于超出协议范围的生物物理学工作的详细描述(Clegg,1992; Clegg等人 1992; Wlodarczyk等人,2008; Gadella,2009)。在补充信息中,提供随时可用的Excel表格来计算每个描述的FRET对的FRET效率。 ' mNG-mCh.xlsx '' mNG-mCh plate reader.xlsx '' SYFP2-mCh.xlsx ”和“ mKO-mCh.xlsx ”。


    图6.周质FRET测定的原理A. mCh和mNG的激发和发射光谱,指示用于测量受体通道(左)或供体通道(右)的波长。粗虚线代表使用的激发波长。带阴影的蓝色区域表示mNG发射光谱与mCh激发光谱之间的光谱重叠。灰色阴影区域代表测量发射的波长。 B.测量样品以计算周质FRET。需要参考背景,mCh和mNG来计算样本中对FRET的贡献。 C.混合周质串联FRET样品,显示所测量的光谱为黑点(Peri-tandem)和计算的光谱为实红线(Unmixed Calc。)。测量的受体光谱由背景荧光(非混合空单元)和在590nm激发的样品中存在的mCh(未混合受体)的量组成。使用受体的直接激发来确定样品中存在的受体的量。然后使用该量来计算在供体激发样品中检测到多少额外的受体(致敏发射)。测量的供体谱包含背景(未混合的空单元格),mNG(未混合的供体)和mCh(未混合的受体)荧光和敏化发射(Unmixed SE),这是解混算法不能归因于直接激发的额外荧光mCh。 D.测量和计算光谱之间的低残差是解混的质量的量度。这个数字最初发表在Meiresonne 等,2017年。

    使用解混表单的协议
    计算机屏幕上的解混过程如何显示如图7所示。高分辨率屏幕截图可以在补充数据中找到。
    注意:根据使用的设备,原始数据可能有不同的输出格式,并进行相应的调整。
    1. 将光谱数据复制到相应的Excel表格1“原始数据”中。

    2. 减去第2页“减PBS”中所有样本的PBS测量结果
    3. 复制表3'数据'中指定位置的空矢量,受体参照和供体参照。这会自动从受体和供体参考光谱中减去背景信号。
    4. 将样品数据复制到表3'数据'中的指定位置,表格将根据给定的参考值将这些质谱图解混。
      注:参考和样品光谱绘制在表3的底部进行检查。
    5. 检查表4“结果”中的混合概述,如下所述。
    6. 将结果复制粘贴到表格5'摘要'。
    7. 对其余样本重复步骤4-6。


      图7.计算屏幕上显示的FRET效率的解混过程不会直接使用的原始捐助者和接受者解混合计算会显示在补充文件中的高分辨率屏幕截图中“取消混合screens.pptx ”。

      什么决定了一个好的测量?
      光谱分离高度依赖于应在相同条件下测量的清晰的荧光信号。供体和受体参考光谱是通过从其各自的发射测量结果中减去PBS和空单元格数据而获得的,如'使用解混表格的方案中所述。这应该只产生一个纯粹的发射光谱或只有受体FP。有时光谱不再类似测量的FP,甚至可以看到特别是供体的负光谱。这可能是由空白细胞样本中强烈的自体荧光水平和参考样本中低FP信号引起的。如果其中一个参考不好,光谱的混合将不会给出值得信赖的结果。或者,先前测量的参考光谱可以用于仍然获得样品的FRET值的粗略指示。建议不要将这些值作为确定的值,并且需要重复实验以获得可信的数据。当实验结果良好时,样本数据可以不混合。不能归因于受体谱的发射信号称为非混合差异。非混合差异是测量FRET光谱质量的一个很好的方法,在一个理想的实验中它应该是零。更实在的,它应该是测量光谱的一小部分,如图6D和8D所示。


      图8.完整的体内阅读板FRET实验。 :一种。在28℃下在Gb1中生长的细胞的生长曲线仅显示周质串联mNG-mCh的倍增时间的微小差异。在测量荧光光谱之前,所有良好培养物的OD 450值在彼此的〜10%之内。箭头指示通过1:2稀释开始诱导构建体的时间点。 B.具有标准偏差误差棒的周质参考的平均归一化荧光光谱显示重复之间只有小的强度变化。 C.左侧受体通道和右侧供体通道的高质量混合平均周质串联样品的平均参考值表明敏化发射的可靠计算。 D.测量的光谱和计算的光谱之间的差异仅显示受体和供体的很少的混合差异。这个数字最初发表在Meiresonne 等,2017年。

数据分析

这个生物协议描述了如何准备,执行和分析体内细菌FRET实验。在相关章节(程序E),提供有关数据分析的信息,并提供深入的技术信息。所获得的FRET值的重要性可以使用标准置信区间统计方法来计算。由于拥挤,FRET值应该显着高于从非相互作用样品获得的值。对于这个分析,可以使用标准的 t - 测试。

食谱

  1. TY中等
    1升重量:
    10克细菌胰蛋白胨
    5克酵母提取物
    5克NaCl
    加H 2 O至1L,121℃高压灭菌15分钟。
  2. 20%葡萄糖溶液
    对于200毫升20%的葡萄糖溶液重量:
    40克葡萄糖
    加入H 2 O至200ml,并在121℃高压灭菌15分钟。
  3. Gb1中等
    对于1升联合(使用无菌技术):
    100毫升MM1-10x(方案3a)
    100毫升MM2-10x(方案3b)
    10毫升MM3-100x(方案3c)
    10毫升MM4-100x(配方3d)
    注:对于基于BW25113,TB28和MG1655的大肠杆菌菌株
    20毫升葡萄糖20%
    1毫升盐酸硫胺素4毫克毫升(方案3e)
    2.5毫升L-赖氨酸一盐酸盐20毫克毫升(方案3f)
    1毫升氨苄青霉素毫克每毫升
    1毫升氯霉素25毫克ml -1
    加无菌H 2 O至1L
    1. MM1-10x
      对于1升MM1-10x重量:
      63.3克K 2 HPO 4•3H 2 O
      29.5克KH 2 PO 4 4 加H 2 O至1L,121℃高压灭菌15分钟。
    2. MM2-10x
      对于1 L MM2-10x重量:
      10.5克(NH 4)2 SO 4 1克MgSO 4•7H 2 O 0 加H 2 O至1L,121℃高压灭菌15分钟。
    3. MM3-100x
      对于1 L MM3-100x重量:
      28毫克FeSO 4•7H 2 O 710毫克Ca(NO3)2•4H2O 2
      加H 2 O至1L,121℃高压灭菌15分钟。
      注意:灭菌后,MM3会显示一个红色的降水,应该忽略。
    4. MM4-100x
      对于50毫升MM4-100x重量:
      250毫克L-精氨酸
      250毫克L-谷氨酰胺
      10毫克胸腺嘧啶
      100毫克尿嘧啶
      将H 2 O 2加至50ml,用Filtropur S0.2μm过滤器过滤灭菌。
    5. 维生素B1储备液(4毫克ml-1)
      对于50毫升维生素B1 4毫克毫升-1储备液称:
      200毫克盐酸硫胺素
      将H 2 O 2加至50ml,用Filtropur S0.2μm过滤器过滤灭菌。
    6. 赖氨酸原液(20毫克ml -1)
      对于50毫升赖氨酸20毫克毫升的储液重量:
      1克赖氨酸
      将H 2 O 2加至50ml,用Filtropur S0.2μm过滤器过滤灭菌。
  4. 磷酸盐缓冲液(PBS 1x)
    PBS缓冲液(pH 7.2)
    140 mM NaCl
    27 mM KCl
    10mM Na 2 HPO 4•2H 2 O 0/2 2 mM KH 2 PO 4 4/2
  5. 甲醛和戊二醛(FAGA)固定剂
    每1毫升甲醛添加21微升戊二醛

致谢

该生物方案的改变已经用于以下出版物(Alexeeva等人,2010; Fraipont等人,2011; van der Ploeg等人。,2013和2015; Meiresonne 等,2017)。作者想感谢教授。博士。 Theodorus W. J. Gadella开发了原始的Excel混合表单(Alexeeva et al。 ,2010),并严格阅读手稿和Laureen M. Y. Mertens测试了混合协议。 NYM由NWO,ALW开放计划(822.02.019),NWO的Svetlana Alexeeva,ALW的Van Molecuul tot Cel计划(805.47.200)以及RVDP由欧盟委员会DIVINOCELL项目(FP7-Health-2007- B-223431)。作者宣称没有利益冲突。

参考

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  2. Bindels,DS,Haarbosch,L.,van Weeren,L.,Postma,M.,Wiese,KE,Mastop,M.,Aumonier,S.,Gotthard,G.,Royant,A.,Hink,MA和Gadella, TW,Jr.(2017)。 mScarlet:用于细胞成像的明亮的单体红色荧光蛋白 Nat Methods 14(1):53-56。
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引用:Meiresonne, N. Y., Alexeeva, S., van der Ploeg, R. and den Blaauwen, T. (2018). Detection of Protein Interactions in the Cytoplasm and Periplasm of Escherichia coli by Förster Resonance Energy Transfer. Bio-protocol 8(2): e2697. DOI: 10.21769/BioProtoc.2697.
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