Bacterial Microcolonies in Gel Beads for High-throughput Screening
凝胶珠细菌微菌落的高通量筛选   

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Elizabeth Libby Elizabeth Libby
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Pierre-Yves Colin Pierre-Yves Colin
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Abstract

High-throughput screening of a DNA library expressed in a bacterial population for identifying potentially rare members displaying a property of interest is a crucial step for success in many experiments such as directed evolution of proteins and synthetic circuits and deep mutational scanning to identify gain- or loss-of-function mutants.

Here, I describe a protocol for high-throughput screening of bacterial (E. coli) microcolonies in gel beads. Single cells are encapsulated into monodisperse water-in-oil emulsion droplets produced with a microfluidic device. The aqueous solution also contains agarose that gelates upon cooling on ice, so that solid gel beads form inside the droplets. During incubation of the emulsion, the cells grow into monoclonal microcolonies inside the beads. After isolation of the gel beads from the emulsion and their sorting by fluorescence activated cell sorting (FACS), the bacteria are recovered from the gel beads and are then ready for a further round of sorting, mutagenesis or analysis. In order to sort by FACS, this protocol requires a fluorescent readout, such as the expression of a fluorescent reporter protein. Measuring the average fluorescent signals of microcolonies reduces the influence of high phenotypic cell-to-cell variability and increases the sensitivity compared to the sorting of single cells. We applied this method to sort a pBAD promoter library at ON and OFF states (Duarte et al., 2017).

Keywords: High-throughput screening (高通量筛选), Microcolonies (微菌落), Microdroplets (微滴), Gel beads (凝胶珠), Directed evolution (定向进化), Combinatorial libraries (组合文库), Synthetic biology (合成生物学)

Background

Fluorescence activated cell sorting (FACS) has an unmatched screening throughput of > 107 events/h (Davies, 2012). However, sorting of single cells according to their fluorescence by FACS to screen libraries of synthetic circuits (Schaerli and Isalan, 2013) is often hampered by high phenotypic cell-to-cell variability. Alternatively, it is possible to sort small cell colonies (microcolonies) contained in hydrogel beads (Weaver et al., 1991; Sahar et al., 1994; Zengler et al., 2002; Meyer et al., 2015). Beads with a diameter of approximately up to 50 μm can be sorted by FACS (Weaver et al., 1991; Sahar et al., 1994; Zengler et al., 2002; Fischlechner et al., 2014; Duarte et al., 2017). The microcolonies in the beads are monoclonal, if just a single cell per bead is initially encapsulated, and that cell then grows to a microcolony inside the bead. Highly monodisperse gel beads can be produced in water-in-oil emulsion droplets generated on a microfluidic device (Theberge et al., 2010).

This protocol describes the generation of 1% agarose gel beads (diameter ~50 µm) harboring bacterial microcolonies using microfluidics and their sorting by FACS to isolate the variants with the desired properties (Duarte et al., 2017). It is possible to sort for variants that take on different states (e.g., ON and OFF) under different conditions (e.g., different inducer concentrations) by performing multiple sequential rounds of this protocol. With this method, we sorted cells expressing a fluorescent reporter protein, while it could also be amended to screen for other readouts. If combined with a strategy to maintain fluorescent reaction products in the bead (Fischlechner et al., 2014), it can be used to screen for enzyme or pathway activities. Another option is to co-encapsulate sensor cells that fluoresce upon the production of a compound of interest (Meyer et al., 2015). It is also possible to assay cell growth by relying on the light scatter of the microcolonies or by staining them with a fluorescent biomass indicator dye (e.g., staining nucleic acids or proteins) (Weaver et al., 1991). When encapsulating multiple cells per bead, cell-cell interactions could also be screened for. Thus, the described protocol is broadly applicable in biology.

Materials and Reagents

  1. PTFE tubing with inner diameter of 0.8 mm and outer diameter of 1.6 mm (Cole-Parmer Instrument, catalog number: EW-06407-41 )
  2. Stainless steel catheter couplers, 20 ga x 15 mm, non-sterile (Instech laboratories, catalog number: SC20/15 )
  3. CellTrics filters, 50 µm yellow (Sysmex, catalog number: 04-0042-2317 )
  4. 1.5 ml micro tubes (for example SARSTEDT, catalog number: 72.706.400 )
  5. Falcon 5 ml round-bottom tubes, disposable, polystyrene (Corning, Falcon®, catalog number: 352054 )
  6. 1.4 ml Non coded Screw Cap tubes U-bottom Bulk (Micronic, catalog number: MP32062 )
  7. Adhesive tape
  8. Aluminium foil
  9. Kimwipes (KCWW, Kimberly-Clark, catalog number: 34120 )
  10. Optional: microscope slides for droplets analysis (for example Kova Glasstic Slide 10 With Counting Grids, Kova International, catalog number: 87144E )
  11. Gloves
  12. Small resealable plastic bag
  13. 2 SGE Gas Tight Syringes, Fixed Luer Lock, volume 100 µl (Trajan Scientific, SGE Analytical Science, catalog number: 005229 )
  14. SGE Gas Tight Syringe, Fixed Luer Lock, volume 5 ml (Trajan Scientific, SGE Analytical Science, catalog number: 008762 )
  15. Hamilton needles 20 gauge, Kel-F Hub NDL, 2 in, point style 3 (Hamilton, catalog number: 90520 )
  16. E. coli (or other bacterial) cells harboring the library to be screened
  17. Ice
  18. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  19. Mineral oil (Sigma-Aldrich, catalog number: M5904 )
  20. 3M Novec 7500 Engineered Fluid (known as HFE-7500 oil) (3M, catalog number: Novec 7500 )
  21. 5% (w/w) 008-FluoroSurfactant in HFE7500 (Ran Biotechnologies, catalog number: 008-FluoroSurfactant-5wtH-20G ) (Protect from light)
  22. Ultra-low Gelling Temperature agarose, type IX-A (Sigma-Aldrich, catalog number: A2576 )
  23. 1H,1H,2H,2H-Perfluoro-1-octanol (PFO), 97% (Sigma-Aldrich, catalog number: 370533 )
  24. Petri dishes (14 cm) (Thermo Fisher Scientific, NuncTM, catalog number: 249964 )
  25. Glass beads (2 mm) (Sigma-Aldrich, catalog number: Z273627 )
  26. Syringe Filters 0.22 µm pore size, 25 mm diameter (Corning, catalog number: 431219 )
  27. SYTO 9 Green Fluorescent Nucleic Acid Stain (Thermo Fisher scientific, catalog number: S34854 )
  28. Ammonium sulfate ((NH4)2SO4) (Sigma-Aldrich, catalog number: 09978 )
  29. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: 60356 )
  30. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5379 )
  31. Iron (II) sulfate heptahydrate (FeSO4·7H2O) (Sigma-Aldrich, catalog number: 215422 )
  32. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 221473 )
  33. Thiamine hydrochloride (Sigma-Aldrich, catalog number: T1270 )
  34. Casamino acids (BD, BactoTM, catalog number: 223050 )
  35. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M2643 )
  36. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  37. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  38. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
  39. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
  40. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
  41. Tryptone (BD, BactoTM, catalog number: 211705 )
  42. Yeast extract (BD, BactoTM, catalog number: 212750 )
  43. Kanamycin sulfate (Sigma-Aldrich, catalog number: K4000 )
  44. Medium for the bacteria (in our case M63 medium, see Recipes) containing the appropriate antibiotic and inducer concentrations
  45. 1x phosphate-buffered saline (PBS) (see Recipes)
  46. LB medium (see Recipes)
  47. LB-Agar plates containing the appropriate antibiotic (see Recipes)

Equipment

  1. Microfluidic devices to produce water-in-oil droplets (diameter 20-50 µm)
    Note: For example, PDMS devices purchased from Wunderlichips GmbH (As we do. The design of the device is chosen by the customer. If you would like to purchase devices with our design, please refer to this publication when contacting Wunderlichips.). Alternatively, the devices can be prepared as previously described in detail, including the surface modification required to render them hydrophobic (Devenish et al., 2013). The design file of the device used in this study (40 µm flow-focusing channel) is available from the author’s website: (Figure 1).


    Figure 1. Design of the microfluidic device used for droplet generation. The device contains an inlet for the oil phase, an inlet for the aqueous phase (bacteria, agarose, medium) and an exit outlet. The droplets are formed at the flow-focusing geometry (picture inset). For this protocol, the channel width at the flow focusing part is 40 µm and the height of the channels is also 40 µm. Using this device, droplets with a diameter of 40-50 µm can be produced. Scale bar = 40 µm.

  2. Syringe pumps (for example Aladdin infusion pump, World Precision Instruments, catalog number: AL300-220 )
  3. Inverted light microscope (for example Leica Microsystems, model: Leica DM IL LED ) (A conventional microscope is also possible, but the tubing of the microfluidic device might interfere with the optics.)
  4. Fluorescence activated cell sorter (for example BD, model: FACSAriaTM III )
  5. High-speed camera (for example Teledyne DALSA, model: Genie Nano M640 Mono, catalog number: G3-GM10-M0640 )
    Note: Standard cameras are not fast enough to observe/record continuous droplet formation. However, if the exposure time can be adjusted to ~50 µsec, droplet formation can be monitored with single pictures.
  6. Tubing cutter (Cole-Parmer, catalog number: EW-06438-10 )
  7. 2 Hot/cold compresses (from the pharmacy or grocery store)
  8. Lab jack (Bochem Instrumente, catalog number: 11020 )
    Note: Alternatively some box of the correct height to place the pump with the aqueous syringe at the height of the microscope stage.
  9. -80 °C freezer
  10. 4 °C fridge
  11. Set of pipettes covering 0.5-1,000 µl (for example from Gilson)
  12. Pliers
  13. Tweezers
  14. Scissors
  15. 37 °C incubator
  16. Autoclave
  17. Benchtop centrifuge for 1.5 ml tubes
  18. Spectrophotometer, nanodrop or plate reader to measure the absorbance of bacterial cultures
  19. Thermoblock for 1.5 ml tubes

Software

  1. Software to control the high-speed camera (for example Labview or Common vision blox, Stemmer imaging)
  2. Image processing program, such as ImageJ or Photoshop
  3. Software to control the FACS (for example BD FACSDIVA)
  4. Flow cytometry analysis software such as FlowJo (LLC)

Procedure

  1. Prepare droplet formation
    1. On the evening before the experiment, prepare pre-cultures of the cells to be sorted by inoculating them into ~5 ml LB containing the appropriate antibiotic (or another suitable medium) and incubate them at 37 °C for 12-18 h in a shaking incubator. Alternatively, the protocol can also be started from glycerol stocks (stored at -80 °C), for example, 15% (v/v) glycerol stocks prepared by adding 420 μl of the overnight culture to 180 μl of 50% (v/v) glycerol.
    2. Connect the high-speed camera to an inverted microscope (via C-mount).
    3. Adjust the settings of the high-speed camera. Provide enough light so that the exposure time can be adjusted to the minimum (~50 µsec) and the frame rate to the maximum. Reducing the picture dimension also allows increasing the frame rate.
      Note: To observe the droplet formation well, the frequency of the frames should be higher than the frequency of droplet formation. We are typically working at ≥ 600 frames per second.
    4. Place the syringe pumps close to the microscope. In order to keep the tubing short and avoid gelation of the agarose in the tubing, the pump for the syringe with the aqueous solution should be as close as possible to the microscope stage. We used a lab jack to place it at the right height (Figure 2).


      Figure 2. Equipment setup for producing microfluidic droplets. A. Overview of the whole setup. B. Close-up view on the microscope stage.

    5. Adjust the diameters in the pumps: 10.3 mm for the 5 ml syringe (oil) and 1.46 mm for the 100 µl syringe (aqueous phase).
    6. Bend three steel couplers with pliers to introduce a 90° bend in their center.
    7. Fix the device on the microscope stage. Inspect the device with a 10x objective and focus on the flow focusing part.
    8. Cut three pieces of tubing (best with a tubing cutter): used for the oil inlet, the aqueous solution inlet, and the outlet respectively. The tubes for the inlets must be long enough to connect the syringes with the device. The one for the oil can be quite long (typically 40 cm in our case). The one for the aqueous solution should be as short as possible to avoid gelation of the agarose in the tube. In our case, it is ≤ 5 cm. The exact length will depend on your microscope setup. The outlet tube is about 9 cm.
      Note: As the used plasmids carry an antibiotic resistance gene, we perform this protocol without sterile technique. It is, however, to autoclave the tubing and the steel couplers, and to disinfect the syringes and the device with 70% ethanol.
    9. Connect each tubing to a bent steel coupler by hand (wearing gloves).
    10. Connect the exit tube to the device by hand (wearing gloves).
    11. Use scissors to make a small hole in the lid of a 1.5 ml microcentrifuge tubing. Cut the lid off and put it onto another open microcentrifuge tube. Put the exit tube through the small hole into the microcentrifuge tube, which will serve to collect the droplets. The hole in the lid prevents the outlet tubing from falling out. Fix the tube with adhesive tape on the microscope stage and cool it with a small resealable plastic bag full of ice.
    12. Prepare 5-10 ml solution of fluorinated oil (HFE-7500) with 0.5% 008-FluoroSurfactant by pipetting 0.5-1 ml of 5% 008-FluoroSurfactant in HFE-7500 to 4.5-9 ml HFE-7500. It can be kept for several months at room temperature. Protect the tube from light with aluminum foil.
    13. Fill the 5 ml syringe with the oil/surfactant. Remove air bubbles.
    14. Connect the tubing for the oil to the needle of the syringe and fill it completely with oil. Confirm that the oil comes out of the steel coupler (into a Kimwipes tissue) and no air bubbles are left in the tubing.
    15. Fix the 5 ml syringe with the oil on the pump.
    16. Connect the tubing with the oil to the device by inserting the steel coupler into the oil inlet of the device.
    17. Prepare 1-2 ml 2% (w/v) agarose in your medium of choice (M63 in our case), including the appropriate antibiotic. The easiest way to do this is to weigh ~20 mg agarose into a microcentrifuge tube and add the correct amount of medium (1 ml in the case of 20 mg). Melt the agarose by incubating it for at least 15 min in a thermoblock at 60 °C.
      Note: I do not use temperatures higher than 60 °C for melting the agarose to prevent potential cell death by elevated temperature in Step A22.
    18. Get bacterial cells from a fresh pre-culture or a glycerol stock. Spin them down (~3,000 x g, 5 min) and resuspend in the medium you are going to use in the experiment.
    19. Measure the absorbance at 600 nm of the cells with a spectrophotometer, nanodrop or plate reader.
    20. Calculate the number of cells to be added to get the desired loading of your beads with cells. This depends on the droplet size and follows a Poisson distribution (see Notes for a step by step example of the calculations).
    21. Prepare ~500 µl of cells at the calculated density in the medium.
    22. Add 100 µl of the melted agarose (2% (w/v)) to 100 µl of the cells resulting in an aqueous solution containing the cells and 1% (w/v) agarose.
    23. From now on, you should take care of keeping the agarose solution warm. Work fast to avoid gelation of the agarose and keep it warm with the pre-warmed (e.g., in a 60 °C incubator) hot/cold compress.
    24. Fill a 100 µl syringe with the aqueous agarose solution. Try to avoid air bubbles. It might be necessary to expel and taking the liquid up several times until there are no air bubbles left.
    25. Connect the tubing for the aqueous solution to the syringe. Fill the tubing completely with the aqueous solution. Check that the solution comes out of the steel coupler (into a Kimwipes tissue) and no air bubbles are left in the tubing.
    26. Fix the syringe on the pump. Put a pre-warmed (e.g., in a 60 °C incubator) hot/cold compress on to the syringe and the tubing to avoid gelation of the agarose.
    27. Connect the tubing with the aqueous solution to the device (via the steel coupler).

  2. Droplet formation and microcolony growth
    1. Start the pump for the oil. Use approximately 2,000 μl/h until you see oil entering the device (< 5 min).
    2. Start the pump for the aqueous solution. Use approximately 150 μl/h until you see droplets forming. Then slow down the flow rate to 80 μl/h. Wait until droplet formation is stable (< 5 min).
      Notes:
      1. These are flow rates that we are typically using. Device design and the composition of the aqueous phase influence the optimal flow rates. The chosen flow rates affect droplet size and frequency of droplet formation.
      2. Typical reasons for unstable droplet formations are: “jetting” – the aqueous phase goes beyond the flow focusing part of the device and forms irregular droplets (reduce the aqueous flow rate), the agarose has started solidifying into a gel (keep the syringe warmer), the device is blocked by some dust or the PDMS device delaminated from the glass slides around the channels or inlets (change device).
    3. Take some pictures of the droplets in the device, so that you can determine their size (see Notes 1-2).
    4. Let the device run until you have produced as many droplets as you want. From time to time replace the hot/cold compress with a warm one. Ensure that the syringes do not run out of liquid.
      Note: With these flow rates we typically have a frequency of around 340 Hz, i.e., we produce 1.2 x 106 droplets/h. Seventy-four percent of them will not contain any cell. Some beads will also be lost during the process.
    5. If you want multiple aliquots of the same sample (e.g., to test different incubation times), it is easier and more accurate to change the collection tube after a specific amount of time (e.g., 15 min for each aliquot) than later having to pipette aliquots of the emulsion. I suggest keeping one aliquot as a negative control without incubation.
    6. If you want to make a second sample, let the device run while preparing the second sample in a second 100 μl syringe (+ a fresh aqueous inlet and outlet tubing).
    7. Remove the exit tubing and the collection tube. The emulsion remaining in the tubing can be recovered by pushing air through it with a syringe.
    8. Turn off the pump for the aqueous solution and reduce the oil flow rate to about 200 μl/h. Replace the syringe and tubing for the aqueous phase. Increase the oil rate again to 2,000 μl/h and start the aqueous solution again at 150 μl/h until you have droplets and lower it to 80 μl/h. Keeping the oil running reduces the risk of blocking the device.
      Note: Occasionally, blocking can happen since some dust enters the device and blocks the flow-focusing part of the device. You can try to remove it by pushing the oil syringe by hand to flush it out of the device.
    9. Keep the emulsion samples on ice until all samples are ready.
    10. Remove as much fluorinated oil (bottom layer) as you can without losing emulsion. Collect the fluorinated oil in a separated tube for recycling.
      Note: The fluorinated oil with the surfactant can be recycled and re-used multiple times. Simply filter it through a 0.22 µm syringe filter.
    11. To prevent evaporation, I recommend overlaying the emulsion samples with a thin layer of mineral oil.
    12. Incubate the samples at 37 °C (or at the temperature of choice) in a thermoblock or incubator for the chosen amount of time without shaking. Keep the negative control sample on ice in a 4 °C fridge.
      Notes:
      1. The incubation time depends on the cells and medium. For the M63 medium, we incubate overnight, for LB medium 4-8 h is enough. The first time it might be worth making a time course by incubating aliquots for different amounts of time and analyzing them on the flow cytometer (see Figure 2 in Duarte et al., 2017).
      2. Growth in the emulsion is limited by the amount of medium provided in the droplet. A way to increase cell growth is to break the emulsion before incubation. The beads can then be incubated in ample growth medium and oxygen leading to higher cell density than when incubated in the emulsion. The disadvantage of this strategy is that all beads share the same medium. It is, for example, not possible to screen for cells that excrete a quorum sensing molecule that then activates a network in the cells from the same bead. The chemical would also enter neighboring beads, where the molecule is not produced. In this form of incubation, it is also more likely that some cells escape the beads and then grow outside the bead in the growth medium. These free bacteria can then decrease sorting purity and a filtering step where the beads are separated from the free bacteria might become necessary.
    13. After incubation, cool the emulsion on ice for at least 15 min. This is to solidify the gel beads.

  3. Recover gel beads
    1. If you would like to look at your droplets using (fluorescence) microscopy, you can pipette them onto a microscope slide.
    2. Pipette 500 µl phosphate buffered saline (PBS) to the emulsion layer.
    3. Add 20-50 μl 1H,1H,2H,2H-perfluorooctanol (PFO) to the emulsion.
    4. Shortly vortex (5 sec) the tube and briefly centrifuge (10 sec, 2,500 x g).
    5. The emulsion (white) should be gone now. If there is still some emulsion left, repeat the last two steps (i.e., add more PFO, vortex and centrifuge).
    6. You have now three phases in the tube. From bottom to top: fluorinated oil and PFO, PBS containing the beads and mineral oil (if added) (Figure 3). Transfer the PBS phase to a fresh 1.5 ml microcentrifuge tube by pipetting. Try to get as much of it as possible without taking any oil. If you have also transferred some oil, transfer the PBS again into a fresh 1.5 ml microcentrifuge tube. The oil tends to stick to the tube walls.
      Note: Dispose of the mineral oil and the fluorinated oil containing the PFO separately as chemical waste.
    7. If you need to stain the cells (e.g., with Syto 9), you can do that now. If they are already fluorescent, you can proceed to the next step.


      Figure 3. Three phases after breaking the emulsion

  4. Sort gel beads
    1. To prevent clogging of the FACS, filter the samples through a 50 μm CellTrics mesh into the 1.4 ml screw cap tubes.
    2. Using tweezers, place the 1.4 ml tubes with the samples into the 5 ml round-bottom tubes which will fit to the flow cytometer. Like this, you have a minimal sample volume that cannot be sorted at the bottom of the tube.
    3. Analyse/sort the beads on the flow cytometer. Many universities will have a facility to help you with this.
    4. Use a 100 μm nozzle and the laser and filters suitable for detecting the fluorescence of your cells (488 nm laser, 530/30 nm filter for GFP).
    5. The width of the sideward scatter (SSC-W) versus the height of the forward scatter (FSC-H) is the combination of scatter parameters that separates the beads best from other particles (free bacteria, agarose debris, dust) or electronic noise of the machine. Therefore, first gate all beads (with and without microcolonies) on the SSC-W – FSC-H dot-plot (Figure 4A). For separating beads with and without microcolonies, we use the SSC-H – FSC-H dot plot (Figure 4B).
    6. The thresholds on the FSC-H and SSC-H can be set so that particles smaller than the beads are not recorded.
    7. Measure your controls (beads not incubated, beads with WT cells…).
    8. On the fluorescence histogram (Figure 4C) or the FSC-H–fluorescence dot plot set the gate for the beads with the fluorescence of interest, for example the 5% of beads with the highest fluorescence.


      Figure 4. Gating strategy. A. Width of the sideward scatter (SSC-W) versus the height of the forward scatter (FSC-H) is the combination of scatter parameters that separates the beads best from other particles (free bacteria, agarose debris, dust) or electronic noise of the machine. Therefore, we first gate all beads (with and without microcolonies) on the SSC-W – FSC-H dot-plot. B. We then plot the gated beads in the SSC-H – FSC-H dot plot for gating the beads with microcolonies. The negative control should only contain the beads without microcolonies. C. We then plot the fluorescence of the beads containing microcolonies as histogram and set the gate for sorting, for example the 5% brightest beads.

    9. Sort them into a 1.5 ml tube containing 200 µl of LB medium.
      Note: The 50 µm agarose beads actually break open when sorted with the 100 µm nozzle. This is convenient for recovering the bacteria from the beads, as no additional agarose digest and/or melting has to be performed. If it is important for your experiments to keep the beads intact, they can be strengthened before the sorting, for example with a layer of polyacrylamide (Spencer et al., 2016).
    10. Plate the beads out on pre-warmed LB-agar plate (supplemented with the appropriate antibiotic) and incubate overnight at 37 °C. We typically plate out each sorted sample on two big LB-agar plates (14 cm Ø) and typically recover > 10x more colonies than the number of beads collected.
    11. The colonies from the plates can be individually picked or resuspended all together in medium using glass beads. The resuspended libraries can be stored as glycerol stocks (15% v/v) at -80 °C. Samples can be analyzed, e.g., by sequencing, (single cell) flow cytometry, plate reader experiments etc. The glycerol stocks can be used to start another round of this protocol.

Data analysis

To analyze and plot the flow cytometry data recorded during the sorting, we use the program FlowJo and apply the gating strategy as described in Figure 4.
Analysis of the libraries and/or individual clones, e.g., by sequencing, (single cell) flow cytometry and plate reader experiments will indicate if you enriched for the desired properties and whether another round of sorting is required. See Duarte et al. (2017) for examples to analyze the libraries.

Notes

Calculation of the cell numbers:

  1. Determine the diameter of droplets
    1. Take a picture of the microscope ruler with a microscope camera.
    2. Use the measuring tool of Photoshop (or a similar program) to determine how many pixels correspond to 1 µm.
    3. Take pictures of the droplets (as a monolayer). Do not use coverslips as this might distort the diameter measurement.
    4. Measure their diameters (d) in pixels.
    5. Calculate the diameter in µm.
  2. Calculate the volume (V) of your droplets

    r: radius
    1 m3= 1,000 L
    Example: d = 50 µm → r = 25 x 10-6 m → V = 6.5 x 10-14 m3 = 6.5 x 10-11 L
  3. Choose how many cells/droplet you want
    Cells are distributed into droplets according the Poisson distribution

    e=2.71828 (Euler’s number)
    λ: average number of cells/droplet
    k: number of cells in a droplet (0, 1, 2, 3, 4…)
    Poisson distribution calculators can be found online, for example here: http://stattrek.com/online-calculator/poisson.aspx.
    An example: λ = 0.3 → P(0, 0.3) = 0.74; P(1, 0.3) = 0.22; P(2, 0.3) = 0.033; P(3, 0.3) = 0.003.
    This means, if on average we have 0.3 cells/droplet we will have 74% empty droplets, 22% droplets with 1 cell, 3.3% droplets with 2 cells and 0.3% droplets with 3 cells.
    The average of 0.3 cells/droplet is a good balance between empty droplets and droplets with more than one cells.
  4. Convert optical density at 600 nm (OD600) to cell density
    1. To correlate the measured OD600 with your cell density, you need to calibrate your instrument for your cell type.
    2. Here is an example of a detailed protocol how to do this:
      http://delrio.dcccd.edu/jreynolds/microbiology/2421/lab_manual/counts.pdf.
    3. For E. coli, a rough estimate is that OD600 of 1 corresponds to 5 x 108 -1 x 109 cells/ml.
  5. Calculate your cell dilutions
    Example:
    1. We have droplets with a 50 µm diameter.
    2. We want an average of 0.3 cell/droplet.
    3. We calibrated that the OD600 of 1 corresponds to 5 x 108 cells/ml.
    4. We measured the OD600 of our culture to be 0.2.
    5. Using equation (1) we calculate the volume of our droplets to be 6.5 x 10-11 L, this means, we have 1.5 x 1010 droplets/L and 1.5 x 107 droplets/ml of aqueous solution.
    6. For an average of 0.3 cell we thus need 0.3 x 1.5 x 107 cells/ml = 4.5 x 106 cells/ml.
    7. With an OD600 of 1 corresponding to 5 x 108 cells/ml, 4.5 x 106 cells/ml correspond to an OD600 of 0.009. This means the final OD600 of our sample should be 0.009. As we measured an OD600 of 0.2 we will need to dilute the cells overall 22-fold. A 2-fold dilution is achieved by mixing the cells with the agarose, hence we prepare a 11-fold dilution.
    8. These calculations will give you a first indication for your cell density. If the flow cytometry analysis (Figure 4) indicates that too many or too few beads contain cells, the cell density should be accordingly adjusted in the next experiment.
    9. Your cells might settle down in the syringe and the tubing, causing a time-dependent decrease in the number of cells entering the device and therefore lead to more empty droplets than expected by Poisson distribution. The extend of this effect is dependent on cell type, solution viscosity, tubing length and duration of your experiment. To prevent it, you can use a density matching agent such as Percoll or alginate. See Devenish et al. (2013) for details.

Recipes

  1. M63 medium (supplemented with 0.2% (w/v) glycerol, 0.025% (w/v) casamino acids, 0.00005% (w/v) thiamine)
    Prepare 5x stock:
    1. Add the following to 800 ml dH2O:
      10 g (NH4)2SO4
      68 g KH2PO4
      2.5 g FeSO4·7H2O
    2. Adjust pH to 7.0 using KOH
    3. Adjust volume to 1 L with dH2O
    4. Sterilize by autoclaving
    Prepare the 1x Working Solution:
    1. Add the following:
      200 ml 5x stock
      0.1 ml 0.5% thiamine hydrochloride
      1.25 ml 20% Casamino Acids
      10 ml of sterile 20% glycerol
      1 ml of sterile 1 M MgSO4
      Antibiotic (e.g., 50 µg/ml kanamycin)
    2. Adjust volume to 1 L with dH2O
  2. 1x PBS
    1. Add the following to 800 ml dH2O:
      8 g of NaCl
      0.2 g of KCl
      1.44 g of Na2HPO4
      0.24 g of KH2PO4
    2. Adjust the pH to 7.4 with HCl
    3. Add distilled water to a total volume of 1 L
    4. Sterilize by autoclaving
  3. LB medium
    1. Add the following to 800 ml dH2O
      10 g Tryptone
      5 g yeast extract
      10 g NaCl
    2. Adjust pH to 7.0 using NaOH
    3. Adjust volume to 1 L with dH2O
    4. Sterilize by autoclaving
  4. LB-Agar
    1. Add the following to 800 ml dH2O
      10 g Tryptone
      5 g yeast extract
      10 g NaCl
      15 g agar
    2. Adjust pH to 7.0 using NaOH
    3. Adjust volume to 1 L with dH2O
    4. Sterilize by autoclaving
    5. After autoclaving, cool to 50 °C, add antibiotic (if needed, e.g., 50 µg/ml kanamycin), and pour into Petri dishes.
    6. Let harden, then invert and store at 4 °C

Acknowledgments

I thank Içvara Barbier for critical reading. I acknowledge funding from the Swiss National Science Foundation (project 31003A_175608). This protocol was adapted from procedures published in (Duarte et al., 2017). The author declares no competing interests.

References

  1. Davies, D. (2012). Cell separations by flow cytometry. Methods Mol Biol 878: 185-199.
  2. Devenish, S. R., Kaltenbach, M., Fischlechner, M. and Hollfelder, F. (2013). Droplets as reaction compartments for protein nanotechnology. Methods Mol Biol 996: 269-286.
  3. Duarte, J. M., Barbier, I. and Schaerli, Y. (2017). Bacterial microcolonies in gel beads for high-throughput screening of libraries in synthetic biology. ACS Synth Biol 6(11): 1988-1995.
  4. Fischlechner, M., Schaerli, Y., Mohamed, M. F., Patil, S., Abell, C. and Hollfelder, F. (2014). Evolution of enzyme catalysts caged in biomimetic gel-shell beads. Nat Chem 6(9): 791-796.
  5. Meyer, A., Pellaux, R., Potot, S., Becker, K., Hohmann, H. P., Panke, S. and Held, M. (2015). Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors. Nat Chem 7(8): 673-678.
  6. Sahar, E., Nir, R. and Lamed, R. (1994). Flow cytometric analysis of entire microbial colonies. Cytometry 15(3): 213-221.
  7. Schaerli, Y. and Isalan, M. (2013). Building synthetic gene circuits from combinatorial libraries: screening and selection strategies. Mol Biosyst 9(7): 1559-1567.
  8. Spencer, S. J., Tamminen, M. V., Preheim, S. P., Guo, M. T., Briggs, A. W., Brito, I. L., D, A. W., Pitkanen, L. K., Vigneault, F., Juhani Virta, M. P. and Alm, E. J. (2016). Massively parallel sequencing of single cells by epicPCR links functional genes with phylogenetic markers. ISME J 10(2): 427-436.
  9. Theberge, A. B., Courtois, F., Schaerli, Y., Fischlechner, M., Abell, C., Hollfelder, F. and Huck, W. T. (2010). Microdroplets in microfluidics: an evolving platform for discoveries in chemistry and biology. Angew Chem Int Ed Engl 49(34): 5846-5868.
  10. Weaver, J. C., Bliss, J. G., Powell, K. T., Harrison, G. I. and Williams, G. B. (1991). Rapid clonal growth measurements at the single-cell level: gel microdroplets and flow cytometry. Biotechnology (N Y) 9(9): 873-877.
  11. Zengler, K., Toledo, G., Rappe, M., Elkins, J., Mathur, E. J., Short, J. M. and Keller, M. (2002). Cultivating the uncultured. Proc Natl Acad Sci U S A 99(24): 15681-15686.

简介

在细菌群体中表达的DNA文库的高通量筛选用于鉴定显示感兴趣性质的潜在稀有成员是在许多实验中成功的关键步骤,例如蛋白质和合成回路的定向进化以及用于鉴定增益的深度突变扫描 - 或功能丧失的突变体。

在这里,我描述了一种用于高通量筛选凝胶珠中细菌(大肠杆菌)微菌落的方案。将单细胞包封成用微流体装置产生的单分散油包水乳液液滴。水溶液还含有琼脂糖,其在冰上冷却时凝胶化,从而在液滴内部形成固体凝胶珠。在乳液温育期间,细胞在珠内生长成单克隆微菌落。在从乳液中分离凝胶珠并通过荧光激活细胞分选(FACS)分选后,从凝胶珠中回收细菌,然后准备进行进一步的分选,诱变或分析。为了通过FACS分类,该方案需要荧光读数,例如荧光报告蛋白的表达。测量微小菌落的平均荧光信号降低了高表型细胞间变异性的影响,并且与单细胞分选相比提高了灵敏度。我们应用这种方法在ON和OFF状态下对pBAD启动子文库进行分类(Duarte et al。,2017)。

【背景】荧光激活细胞分选(FACS)具有> 10 7 事件/ h的无与伦比的筛选通量(Davies,2012)。然而,通过FACS根据其荧光分选单个细胞以筛选合成回路的文库(Schaerli和Isalan,2013)经常受到高表型细胞间变异性的阻碍。或者,可以对水凝胶珠中所含的小细胞集落(微集落)进行分类(Weaver et al。,1991; Sahar et al。,1994; Zengler et al。,2002; Meyer et al。,2015)。直径约50μm的珠子可以通过FACS分类(Weaver et al。,1991; Sahar et al。,1994; Zengler et al 。,2002; Fischlechner et al。,2014; Duarte et al。,2017)。珠子中的微菌落是单克隆的,如果每个珠子最初仅包封一个细胞,那么该细胞就会生长到珠子内部的微菌落中。高度单分散的凝胶珠可以在微流体装置上产生的油包水乳液液滴中产生(Theberge 等人,,2010)。

该协议描述了使用微流体生成含有细菌微菌落的1%琼脂糖凝胶珠(直径~50μm),并通过FACS分选以分离具有所需特性的变体(Duarte et al。,2017) 。通过执行多个连续循环,可以在不同条件下(例如,不同的诱导浓度)对不同状态(例如,ON和OFF)进行排序的变体进行排序协议。使用这种方法,我们对表达荧光报告蛋白的细胞进行分选,同时也可以对其进行修正以筛选其他读数。如果结合维持珠子中荧光反应产物的策略(Fischlechner et al。,2014),它可用于筛选酶或途径活性。另一种选择是共同包封在感兴趣的化合物产生时发荧光的传感器细胞(Meyer 等人,,2015)。也可以通过依赖微小菌落的光散射或通过用荧光生物质指示染料(例如,染色核酸或蛋白质)染色来测定细胞生长(Weaver et al 。,1991)。当每个珠子包封多个细胞时,也可以筛选细胞 - 细胞相互作用。因此,所描述的方案广泛适用于生物学。

关键字:高通量筛选, 微菌落, 微滴, 凝胶珠, 定向进化, 组合文库, 合成生物学

材料和试剂

  1. PTFE管,内径0.8 mm,外径1.6 mm(Cole-Parmer仪器,产品目录号:EW-06407-41)
  2. 不锈钢导管连接器,20 ga x 15 mm,非无菌(Instech实验室,目录号:SC20 / 15)
  3. CellTrics过滤器,50μm黄色(Sysmex,目录号:04-0042-2317)
  4. 1.5毫升微管(例如SARSTEDT,目录号:72.706.400)
  5. Falcon 5毫升圆底管,一次性聚苯乙烯(Corning,Falcon ®,目录号:352054)
  6. 1.4毫升非编码螺旋盖管U型底部散装(Micronic,目录号:MP32062)
  7. 胶带
  8. 铝箔
  9. Kimwipes(KCWW,Kimberly-Clark,目录号:34120)
  10. 可选:用于液滴分析的显微镜载玻片(例如Kova Glasstic Slide 10 With Counting Grids,Kova International,目录号:87144E)
  11. 手套
  12. 小型可重复密封的塑料袋
  13. 2个SGE气体紧密注射器,固定鲁尔锁,体积100μl(Trajan Scientific,SGE Analytical Science,目录号:005229)
  14. SGE Gas紧密注射器,固定鲁尔锁,容积5 ml(Trajan Scientific,SGE Analytical Science,目录号:008762)
  15. Hamilton针20规格,Kel-F Hub NDL,2英寸,点式3(Hamilton,目录号:90520)
  16. 电子。含有待筛选文库的大肠杆菌(或其他细菌)细胞

  17. 甘油(Sigma-Aldrich,目录号:G5516)
  18. 矿物油(Sigma-Aldrich,目录号:M5904)
  19. 3M Novec 7500工程液(称为HFE-7500油)(3M,目录号:Novec 7500)
  20. HFE7500中的5%(w / w)008-氟表面活性剂(Ran Biotechnologies,目录号:008-FluoroSurfactant-5wtH-20G)(避光)
  21. 超低凝胶温度琼脂糖,IX-A型(Sigma-Aldrich,目录号:A2576)
  22. 1H,1H,2H,2H-全氟-1-辛醇(PFO),97%(Sigma-Aldrich,目录号:370533)
  23. 培养皿(14厘米)(Thermo Fisher Scientific,Nunc TM ,目录号:249964)
  24. 玻璃珠(2毫米)(Sigma-Aldrich,目录号:Z273627)
  25. 注射器过滤器孔径0.22μm,直径25 mm(Corning,目录号:431219)
  26. SYTO 9绿色荧光核酸染色(Thermo Fisher scientific,目录号:S34854)
  27. 硫酸铵((NH 4 )2SO 4 )(Sigma-Aldrich,目录号:09978)
  28. 磷酸氢二钾(K 2 HPO 4 )(Sigma-Aldrich,目录号:60356)
  29. 磷酸二氢钾(KH 2 PO 4 )(Sigma-Aldrich,目录号:P5379)
  30. 硫酸铁(II)七水合物(FeSO 4 ·7H 2 O)(Sigma-Aldrich,目录号:215422)
  31. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:221473)
  32. 盐酸硫胺素(Sigma-Aldrich,目录号:T1270)
  33. 酪蛋白氨基酸(BD,Bacto TM ,目录号:223050)
  34. 硫酸镁(MgSO 4 )(Sigma-Aldrich,目录号:M2643)
  35. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  36. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  37. 磷酸氢二钠(Na 2 HPO 4 )(Sigma-Aldrich,目录号:S3264)
  38. 盐酸(HCl)(Sigma-Aldrich,目录号:258148)
  39. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S8045)
  40. 胰蛋白胨(BD,Bacto TM ,目录号:211705)
  41. 酵母提取物(BD,Bacto TM ,目录号:212750)
  42. 硫酸卡那霉素(Sigma-Aldrich,目录号:K4000)
  43. 含有适当抗生素和诱导剂浓度的细菌培养基(在我们的例子中是M63培养基,参见食谱)
  44. 1x磷酸盐缓冲盐水(PBS)(见食谱)
  45. LB培养基(见食谱)
  46. LB-Agar平板含有适当的抗生素(见食谱)

设备

  1. 微流体装置产生油包水滴(直径20-50μm)
    注意:例如,从Wunderlichips GmbH购买的PDMS设备(正如我们所做的那样。设备的设计由客户选择。如果您想购买我们设计的设备,请在联系Wunderlichips时参考本出版物。 )。或者,可以如先前详细描述的那样制备装置,包括使它们疏水所需的表面改性(Devenish等,2013)。本研究中使用的设备的设计文件(40μm流聚焦通道)可从作者的 网站 :(图1)。


    图1.用于产生液滴的微流体装置的设计该装置包含油相入口,水相入口(细菌,琼脂糖,培养基)和出口。液滴在流动聚焦几何形状处形成(图片插图)。对于该协议,流聚焦部分处的沟道宽度为40μm,沟道的高度也为40μm。使用该装置,可以生产直径为40-50μm的液滴。比例尺=40μm。

  2. 注射泵(例如阿拉丁输液泵,World Precision Instruments,目录号:AL300-220)
  3. 倒置光学显微镜(例如Leica Microsystems,型号:Leica DM IL LED)(传统显微镜也是可能的,但微流体装置的管道可能会干扰光学器件。)
  4. 荧光激活细胞分选仪(例如BD,型号:FACSAria TM III)
  5. 高速摄像机(例如Teledyne DALSA,型号:Genie Nano M640 Mono,目录号:G3-GM10-M0640)
    注意:标准相机的速度不足以观察/记录连续的液滴形成。但是,如果曝光时间可以调整到约50微秒,可以用单张照片监控液滴形成。
  6. 油管切割机(Cole-Parmer,目录号:EW-06438-10)
  7. 2热/冷压缩(来自药房或杂货店)
  8. Lab jack(Bochem Instrumente,目录号:11020)
    注意:或者使用正确高度的一些盒子将泵与水性注射器放在显微镜载物台的高度。
  9. -80°C冰柜
  10. 4°C冰箱
  11. 一套覆盖0.5-1,000μl的移液器(例如来自Gilson)
  12. 钳子
  13. 镊子
  14. 剪刀
  15. 37°C培养箱
  16. 高压灭菌器
  17. 台式离心机用于1.5 ml管
  18. 分光光度计,nanodrop或读板仪测量细菌培养物的吸光度
  19. 用于1.5 ml管的热块

软件

  1. 用于控制高速摄像头的软件(例如Labview或Common vision blox,Stemmer成像)
  2. 图像处理程序,如ImageJ或Photoshop
  3. 用于控制FACS的软件(例如BD FACSDIVA)
  4. 流式细胞仪分析软件如FlowJo(LLC)

程序

  1. 准备液滴形成
    1. 在实验前的晚上,通过将它们接种到含有适当抗生素(或另一种合适的培养基)的约5ml LB中,并在摇动中将它们在37℃下孵育12-18小时,制备待分选细胞的预培养物。孵化器。或者,该方案也可以从甘油储备(储存在-80℃)开始,例如,通过将420μl过夜培养物加入180μl50%制备的15%(v / v)甘油储液(v / v)甘油。
    2. 将高速摄像头连接到倒置显微镜(通过C接口)。
    3. 调整高速摄像机的设置。提供足够的光线,以便将曝光时间调整到最小(~50μsec)并将帧速率调到最大。缩小图像尺寸还可以提高帧速率。
      注意:为了很好地观察液滴形成,帧的频率应高于液滴形成的频率。我们通常以每秒≥600帧的速度工作。
    4. 将注射泵放在靠近显微镜的位置。为了保持管道短路并避免管道中琼脂糖凝胶化,注射器与水溶液的泵应尽可能靠近显微镜载物台。我们使用实验室千斤顶将其放置在正确的高度(图2)。


      图2.用于产生微流体液滴的设备设置。 A.整个设置的概述。 B.显微镜载物台上的特写视图。

    5. 调整泵的直径:5毫升注射器(油)为10.3毫米,100微升注射器(水相)为1.46毫米。
    6. 用钳子弯曲三个钢制连接器,在其中心引入90°弯曲。
    7. 将设备固定在显微镜载物台上。用10倍物镜检查设备,并专注于流量聚焦部分。
    8. 切割三段管(最好用管道切割器):分别用于进油口,水溶液入口和出口。入口管必须足够长,以便将注射器与设备连接起来。油的那个可能很长(在我们的情况下通常为40厘米)。用于水溶液的溶液应尽可能短,以避免管中琼脂糖的凝胶化。在我们的例子中,它是≤5厘米。确切的长度取决于您的显微镜设置。出口管约9厘米。
      注意:由于使用的质粒带有抗生素抗性基因,我们在没有无菌技术的情况下执行该方案。然而,高压灭菌管道和钢制耦合器,并用70%乙醇对注射器和设备进行消毒。
    9. 用手将每根管子连接到弯曲的钢制连接器上(戴手套)。
    10. 用手将出口管连接到设备上(戴手套)。
    11. 用剪刀在1.5毫升微量离心管的盖子上做一个小孔。盖上盖子,将其放在另一个打开的微量离心管上。将出口管穿过小孔进入微量离心管,用于收集液滴。盖子上的孔可以防止出口管子掉出来。用胶带将管子固定在显微镜载物台上,然后用一个装满冰块的可重新密封的塑料袋冷却。
    12. 通过将0.5-1ml的5%008-氟表面活性剂在HFE-7500中移液至4.5-9ml HFE-7500,制备5-10ml含0.5%008-氟表面活性剂的氟化油(HFE-7500)溶液。它可以在室温下保存几个月。用铝箔保护灯管不受光照。
    13. 在5毫升注射器中注入油/表面活性剂。去除气泡。
    14. 将油管连接到注射器的针头,并用油完全填充。确认油从钢制连接器中流出(进入Kimwipes纸巾)并且管道中没有留下气泡。
    15. 用泵上的油固定5毫升注射器。
    16. 将钢制连接器插入设备的进油口,将油管与油管连接。
    17. 在您选择的培养基(我们的病例为M63)中制备1-2 ml 2%(w / v)琼脂糖,包括适当的抗生素。最简单的方法是将约20毫克琼脂糖称入微量离心管中,并加入适量的培养基(在20毫克的情况下为1毫升)。将琼脂糖在60°C的热块中孵育至少15分钟,使琼脂糖融化。
      注意:我没有使用高于60°C的温度来融化琼脂糖,以防止步骤A22中因温度升高导致细胞死亡。
    18. 从新鲜的预培养物或甘油原液中获取细菌细胞。旋转它们(~3,000 x g ,5分钟)并重新悬浮在您将要在实验中使用的培养基中。
    19. 用分光光度计,nanodrop或读板仪测量细胞在600nm处的吸光度。
    20. 计算要添加的细胞数量,以获得所需的细胞负载量。这取决于液滴尺寸并遵循泊松分布(有关计算的逐步示例,请参阅注释)。
    21. 在培养基中以计算的密度制备~500μl细胞。
    22. 将100μl熔化的琼脂糖(2%(w / v))加入100μl细胞中,得到含有细胞和1%(w / v)琼脂糖的水溶液。
    23. 从现在开始,您应该注意保持琼脂糖溶液温暖。快速工作以避免琼脂糖凝胶化,并使用预热(例如,在60°C培养箱中)热/冷压缩保持温度。
    24. 用琼脂糖水溶液填充100μl注射器。尽量避免气泡。可能需要将液体排出并取出几次,直到没有气泡留下。
    25. 将水溶液的管道连接到注射器。用水溶液完全填充管道。检查解决方案是否来自钢制连接器(进入Kimwipes纸巾)并且管道中没有留下气泡。
    26. 将注射器固定在泵上。将预热(例如,在60°C培养箱中)热/冷压缩到注射器和管道上以避免琼脂糖凝胶化。
    27. 将管道与水溶液连接到设备(通过钢制连接器)。

  2. 液滴形成和微菌落生长
    1. 启动泵用于润滑油。使用大约2,000μl/ h直到您看到油进入设备(<5分钟)。
    2. 启动泵用于水溶液。使用约150μl/ h直至看到液滴形成。然后将流速减慢到80μl/ h。等到液滴形成稳定(<5分钟)。
      注意:
      1. 这些是我们通常使用的流量。装置设计和水相的组成影响最佳流速。选择的流速会影响液滴尺寸和液滴形成的频率。
      2. 不稳定液滴形成的典型原因是:“喷射” - 水相超出装置的流动聚焦部分并形成不规则液滴(降低水流速),琼脂糖开始凝固成凝胶(保持注射器加热器),设备被一些灰尘堵塞,或者PDMS设备从通道或入口周围的玻璃滑块上脱落(更换设备)。
    3. 拍摄设备中液滴的一些照片,以便确定它们的大小(参见注释1-2)。
    4. 让设备运行,直到您生成任意数量的液滴。不时用温暖的热敷替换热/冷敷。确保注射器没有液体用完。
      注意:对于这些流速,我们通常具有大约340 Hz的频率,即,我们产生1.2 x 10 6 液滴/ H。其中百分之七十四不会包含任何细胞。在此过程中也会丢失一些珠子。
    5. 如果您想要同一样品的多个等分试样(例如,以测试不同的孵育时间),在特定的时间后更换收集管更容易,更准确(例如
    6. 如果您想制作第二个样品,请在第二个100μl注射器(+新鲜的水性进样口和出口管)中准备第二个样品时运行设备。
    7. 取下出口管和收集管。留在管中的乳液可以通过用注射器推动空气通过它来回收。
    8. 关闭泵用于水溶液并将油流速降低至约200μl/ h。更换水相的注射器和管道。再次将油速提高至2,000μl/ h,并以150μl/ h的速度再次启动水溶液,直至出现液滴并降至80μl/ h。保持机油运行可降低阻塞设备的风险。
      注意:偶尔会发生阻塞,因为有些灰尘会进入设备并阻挡设备的流聚焦部分。您可以尝试用手推动注油器将其从设备中冲出来将其取出。
    9. 将乳液样品保存在冰上,直到所有样品都准备好。
    10. 尽可能多地去除氟化油(底层)而不会损失乳液。将氟化油收集在分开的管中进行回收。
      注意:含有表面活性剂的氟化油可以回收再利用多次。只需通过0.22μm注射器过滤器过滤即可。
    11. 为防止蒸发,我建议用一层薄薄的矿物油覆盖乳液样品。
    12. 将样品在37℃(或选择的温度)下在热块或培养箱中孵育选定的时间而不摇动。将阴性对照样品放在冰上4°C冰箱中。
      注意:
      1. 孵育时间取决于细胞和培养基。对于M63培养基,我们孵育过夜,对于LB培养基4-8小时就足够了。第一次通过将等分试样孵育不同的时间并在流式细胞仪上进行分析来进行时间过程是值得的(参见Duarte等人,2017年的图2)。
      2. 乳液中的生长受到液滴中提供的介质量的限制。增加细胞生长的方法是在孵育前破坏乳液。然后可将珠子在充足的生长培养基中培养,并且氧气导致比在乳液中孵育时更高的细胞密度。该策略的缺点是所有珠子共享相同的介质。例如,不可能筛选排泄群体感应分子的细胞,然后该细胞从同一珠子激活细胞中的网络。该化学物质也会进入相邻的珠子,而不会产生分子。在这种孵育形式中,一些细胞更可能逃离珠子,然后在生长培养基中的珠子外生长。然后,这些游离细菌可以降低分选纯度,并且可能需要将珠子与游离细菌分离的过滤步骤。
    13. 孵育后,将乳液在冰上冷却至少15分钟。这是为了凝固凝胶珠。

  3. 恢复凝胶珠
    1. 如果您想使用(荧光)显微镜观察液滴,可以将它们移液到显微镜载玻片上。
    2. 移取500μl磷酸盐缓冲盐水(PBS)至乳剂层。
    3. 在乳液中加入20-50μl1H,1H,2H,2H-全氟辛醇(PFO)。
    4. 将管短暂涡旋(5秒)并短暂离心(10秒,2,500 x g )。
    5. 乳液(白色)现在应该消失了。如果还剩下一些乳液,重复最后两步(即,添加更多PFO,涡旋和离心)。
    6. 你现在管中有三个阶段。从下到上:氟化油和PFO,含有珠子和矿物油的PBS(如果添加)(图3)。通过移液将PBS相转移至新鲜的1.5ml微量离心管中。尝试在不加油的情况下尽可能多地获得它。如果您还转移了一些油,将PBS再次转移到新的1.5 ml微量离心管中。油往往会粘在管壁上。
      注意:将矿物油和含有PFO的氟化油分别作为化学废物处理。
    7. 如果您需要染色细胞(例如,使用Syto 9),您现在可以这样做。如果它们已经发荧光,您可以继续下一步。


      图3.破乳后的三个阶段

  4. 排序凝胶珠
    1. 为了防止FACS堵塞,将样品通过50μmCellTrics筛网过滤到1.4 ml螺旋盖管中。
    2. 使用镊子,将带有样品的1.4 ml试管放入5 ml圆底管中,该管适合流式细胞仪。像这样,您的样品体积很小,无法在管底部进行分类。
    3. 在流式细胞仪上分析/分选珠子。许多大学将有一个设施来帮助你解决这个问题。
    4. 使用100μm喷嘴和适用于检测细胞荧光的激光和滤光片(488 nm激光,用于GFP的530/30 nm滤光片)。
    5. 侧向散射(SSC-W)的宽度与前向散射(FSC-H)的高度是散射参数的组合,其将珠子与其他粒子(游离细菌,琼脂糖碎片,灰尘)或电子噪声分离得最好。机器。因此,首先在SSC-W-FSC-H点图上对所有珠子(有和没有小菌落)进行门控(图4A)。为了分离有和没有微集落的珠子,我们使用SSC-H-FSC-H点图(图4B)。
    6. 可以设置FSC-H和SSC-H的阈值,以便不记录小于珠子的粒子。
    7. 测量您的对照(未孵育的珠子,与WT细胞的珠子......)。
    8. 在荧光直方图(图4C)或FSC-H-荧光点图上设置具有感兴趣荧光的珠子的门,例如5%具有最高荧光的珠子。


      图4.门控策略。 A.侧向散射(SSC-W)与前向散射(FSC-H)高度的宽度是散射参数的组合,可将珠子与其他散珠最佳地分开颗粒(游离细菌,琼脂糖碎片,灰尘)或机器的电子噪音。因此,我们首先在SSC-W-FSC-H点图上对所有珠子(有和没有小菌落)进行门控。 B.然后,我们在SSC-H-FSC-H点图中绘制门控珠,用于对具有微菌落的珠进行门控。阴性对照应仅包含没有微菌落的珠子。 C.然后我们绘制含有微菌落的珠子的荧光作为直方图,并设置分选的门,例如5%最亮的珠子。

    9. 将它们分成含有200μlLB培养基的1.5ml管。
      注意:当使用100μm喷嘴分选时,50μm琼脂糖珠实际上会破裂。这对于从珠子中回收细菌是方便的,因为不必进行额外的琼脂糖消化和/或融化。如果对于实验来说保持珠子完整是很重要的,可以在分选前加强它们,例如用一层聚丙烯酰胺(Spencer et al。,2016)。
    10. 将珠子在预热的LB-琼脂平板(补充有适当的抗生素)上铺板并在37℃下孵育过夜。我们通常将每个分选的样品放在两个大的LB琼脂平板(14cmØ)上,并且通常恢复&gt;比收集的珠子数多10倍的菌落。
    11. 来自平板的菌落可以使用玻璃珠单独挑选或重新悬浮在培养基中。重悬的文库可以在-80℃下作为甘油储液(15%v / v)储存。可以通过测序,(单细胞)流式细胞术,平板阅读器实验等分析样品例如。甘油储备可用于开始该方案的另一轮。

数据分析

为了分析和绘制分选过程中记录的流式细胞仪数据,我们使用程序FlowJo并应用如图4所示的门控策略。
通过测序,(单细胞)流式细胞术和平板读数器实验分析文库和/或单个克隆例如将指示您是否富含所需特性以及是否需要另一轮分选。有关分析库的示例,请参见Duarte et al。(2017)。

笔记

计算细胞数量:

  1. 确定液滴的直径
    1. 用显微镜照相机拍摄显微镜尺。
    2. 使用Photoshop(或类似程序)的测量工具确定有多少像素对应1μm。
    3. 拍摄水滴(作为单层)。不要使用盖玻片,因为这可能会扭曲直径测量值。
    4. 以像素为单位测量它们的直径(d)。
    5. 以μm为单位计算直径。
  2. 计算液滴的体积(V)

    r:半径
    1 m 3 = 1,000 L
    示例:d =50μm→r = 25 x 10 -6 m→V = 6.5 x 10 -14 m 3 = 6.5 x 10 -11 L
  3. 选择您想要的细胞/液滴数量
    根据泊松分布将细胞分布成液滴
    e = 2.71828(欧拉数)
    λ:平均细胞数/液滴
    k:液滴中的细胞数(0,1,2,3,4 ......)
    泊松分布计算器可以在网上找到,例如: http://stattrek.com/online-calculator /poisson.aspx
    例如:λ= 0.3→P(0,0.3)= 0.74; P(1,0.3)= 0.22; P(2,0.3)= 0.033; P(3,0.3)= 0.003。
    这意味着,如果平均我们有0.3个细胞/液滴,我们将有74%的空液滴,22%的液滴与1个细胞,3.3%的液滴与2个细胞和0.3%的液滴与3个细胞。
    0.3个细胞/液滴的平均值是空液滴和具有多个细胞的液滴之间的良好平衡。
  4. 将600nm处的光密度(OD 600 )转换为细胞密度
    1. 要将测得的OD 600 与细胞密度相关联,您需要根据细胞类型校准仪器。
    2. 以下是如何执行此操作的详细协议示例:
      http://delrio.dcccd.edu/jreynolds/microbiology/2421/lab_manual /counts.pdf。
    3. 对于 E。大肠杆菌,粗略估计OD 600 1对应于5 x 10 8 -1 x 10 9 细胞/ ml 。
  5. 计算细胞稀释度
    例:
    1. 我们的液滴直径为50微米。
    2. 我们想要平均0.3个细胞/液滴。
    3. 我们校准OD 600 为1对应于5 x 10 8 细胞/ ml。
    4. 我们测量了我们培养物的OD 600 为0.2。
    5. 使用等式(1)我们计算出液滴的体积为6.5 x 10 -11 L,这意味着,我们有1.5 x 10 10 液滴/ L和1.5 x 10 7 液滴/ ml水溶液。
    6. 因此,对于平均0.3个细胞,我们需要0.3×1.5×10 5个细胞/ ml = 4.5×10 6个细胞/ ml。
    7. OD 600 为1,相当于5×10 8 细胞/ ml,4.5×10 6 细胞/ ml对应于OD 600 的0.009。这意味着我们样品的最终OD 600 应为0.009。当我们测量OD 600 为0.2时,我们需要将细胞总体稀释22倍。通过将细胞与琼脂糖混合来实现2倍稀释,因此我们制备11倍稀释液。
    8. 这些计算将为您提供细胞密度的第一个指示。如果流式细胞术分析(图4)表明过多或过少的珠子含有细胞,则应在下一个实验中相应地调整细胞密度。
    9. 您的细胞可能会沉淀在注射器和管道中,导致进入设备的细胞数量随时间减少,从而导致比泊松分布预期更多的空液滴。这种效应的延伸取决于细胞类型,溶液粘度,管道长度和实验持续时间。为了防止它,您可以使用密度匹配代理,如Percoll或alginate。有关详细信息,请参阅Devenish et al。(2013)。

食谱

  1. M63培养基(补充0.2%(w / v)甘油,0.025%(w / v)酪蛋白氨基酸,0.00005%(w / v)硫胺素)
    准备5x库存
    1. 将以下物质添加到800 ml dH 2 O:
      10 g(NH 4 ) 2 SO 4
      68 g KH 2 PO 4
      2.5g FeSO 4 ·7H 2 O
    2. 使用KOH将pH调节至7.0
    3. 用dH 2 O将音量调节到1 L
    4. 通过高压灭菌消毒
    准备1x工作解决方案
    1. 添加以下内容:
      200毫升5倍库存
      0.1毫升0.5%盐酸硫胺素
      1.25毫升20%酪蛋白氨基酸
      10毫升无菌20%甘油
      1毫升无菌1M MgSO 4
      抗生素(例如,50μg/ ml卡那霉素)
    2. 使用dH 2 O将音量调节至1 L.
  2. 1x PBS
    1. 将以下物质添加到800 ml dH 2 O:
      8克NaCl
      0.2克KCl
      1.44克Na 2 HPO 4
      0.24克KH 2 PO 4
    2. 用HCl调节pH至7.4
    3. 加入蒸馏水至总体积1L
    4. 通过高压灭菌消毒
  3. LB媒体
    1. 将以下物质添加到800 ml dH 2 O
      10克胰蛋白胨
      5克酵母提取物
      10克NaCl
    2. 使用NaOH将pH调节至7.0
    3. 用dH 2 O将音量调节到1 L
    4. 通过高压灭菌消毒
  4. LB琼脂
    1. 将以下物质添加到800 ml dH 2 O
      10克胰蛋白胨
      5克酵母提取物
      10克NaCl
      15克琼脂
    2. 使用NaOH将pH调节至7.0
    3. 用dH 2 O将音量调节到1 L
    4. 通过高压灭菌灭菌
    5. 高压灭菌后,冷却至50°C,加入抗生素(如果需要,例如,50μg/ ml卡那霉素),倒入培养皿中。
    6. 让它变硬,然后倒转并在4°C下储存

致谢

我感谢IçvaraBarbier的批判性阅读。我感谢瑞士国家科学基金会(项目31003A_175608)的资助。该方案改编自(Duarte et al。,2017)中公布的程序。作者声明没有竞争利益。

参考

  1. 戴维斯,D。(2012年)。 通过流式细胞仪进行细胞分离。 方法Mol Biol 878 :185-199。
  2. Devenish,S.R.,Kaltenbach,M.,Fischlechner,M。和Hollfelder,F。(2013)。 液滴作为蛋白质纳米技术的反应区。 方法Mol Biol 996:269-286。
  3. Duarte,J.M。,Barbier,I。和Schaerli,Y。(2017)。 凝胶珠中的细菌菌落用于高通量筛选合成生物学中的文库。 ACS Synth Biol 6(11):1988-1995。
  4. Fischlechner,M.,Schaerli,Y.,Mohamed,M.F。,Patil,S.,Abell,C。和Hollfelder,F。(2014)。 嵌入仿生凝胶壳珠中的酶催化剂的演变。 Nat Chem 6(9):791-796。
  5. Meyer,A.,Pellaux,R.,Potot,S.,Becker,K.,Hohmann,H.P.,Panke,S。和Held,M。(2015)。 通过在纳升反应器内部使用遗传编码的产品传感器优化全细胞生物催化剂。 Nat Chem 7(8):673-678。
  6. Sahar,E.,Nir,R。和Lamed,R。(1994)。 整个微生物菌落的流式细胞术分析。 Cytometry 15 (3):213-221。
  7. Schaerli,Y。和Isalan,M。(2013)。 从组合库构建合成基因回路:筛选和选择策略。 Mol Biosyst 9(7):1559-1567。
  8. Spencer,S.J.,Tamminen,M.V.,Preheim,S.P.,Guo,M.T.,Briggs,A.W.,Brito,I.L.,D,A.W.,Pitkanen,L.K.,Vigneault,F.,Juhani Virta,M.P。和Alm,E.J。(2016)。 epicPCR对单个细胞进行大规模平行测序,将功能基因与系统发育标记联系起来。 ISME J 10(2):427-436。
  9. Theberge,A.B.,Courtois,F.,Schaerli,Y.,Fischlechner,M.,Abell,C.,Hollfelder,F。和Huck,W。T.(2010)。 微流体中的微滴:化学和生物学发现的不断发展的平台。 Angew Chem Int Ed Engl 49(34):5846-5868。
  10. Weaver,J.C.,Bliss,J.G.,Powell,K.T.,Harrison,G.I。和Williams,G.B。(1991)。 单细胞水平的快速克隆生长测量:凝胶微滴和流式细胞术。 生物技术(N Y)9(9):873-877。
  11. Zengler,K.,Toledo,G.,Rappe,M.,Elkins,J.,Mathur,E.J.,Short,J.M。and Keller,M。(2002)。 培养没有文化的人。 Proc Natl Acad Sci USA 99 (24):15681-15686。
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引用:Schaerli, Y. (2018). Bacterial Microcolonies in Gel Beads for High-throughput Screening. Bio-protocol 8(13): e2911. DOI: 10.21769/BioProtoc.2911.
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