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Bacterial Microcolonies in Gel Beads for High-throughput Screening

凝胶珠细菌微菌落的高通量筛选

Yolanda Schaerli Yolanda Schaerli

发布: 2018年07月05日第8卷第13期 DOI: 10.21769/BioProtoc.2911 浏览次数: 10881

评审: Elizabeth LibbyPierre-Yves ColinBeatrice Li

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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) search Microcolonies (微菌落) search Microdroplets (微滴) search Gel beads (凝胶珠) search Directed evolution (定向进化) search Combinatorial libraries (组合文库) search Synthetic biology (合成生物学) search

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

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

微生物学 > 异源表达系统 > 大肠杆菌

分子生物学 > DNA > 诱/突变

系统生物学 > 基因组学 > 筛选

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