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Phototaxis Assays of Synechocystis sp. PCC 6803 at Macroscopic and Microscopic Scales
宏观和微观尺度上集胞藻PCC 6803的趋光性分析   

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Abstract

Phototaxis is a mechanism that allows cyanobacteria to respond to fluctuations in the quality and quantity of illumination by moving either towards or away from a light source. Phototactic movement on low concentration agar or agarose plates can be analyzed at macroscopic and microscopic scales representing group behavior and single cell motility, respectively. Here, we describe a detailed procedure for phototaxis assays on both scales using the unicellular cyanobacterium Synechocystis sp. PCC 6803.

Keywords: Cyanobacteria(蓝藻细菌), Synechocystis(集胞藻属), Motility(动力性), Phototaxis(趋光性), Photoreceptors(光受体)

Background

The model organism Synechocystis sp. PCC 6803 uses retractile type IV pili (T4P) to move across moist surfaces in a jerky motion referred to as twitching motility. Two secretion ATPases (PilB and PilT) are responsible for the extension and retraction of the pilus apparatus, thus pulling the cells forward. Synechocystis sp. PCC 6803 harbors a variety of photoreceptors covering the entire visible spectrum. Absorption of light can stimulate either positive or negative phototaxis depending on wavelength and intensity. Recently, it was demonstrated that single cells of Synechocystis sp. PCC 6803 are able to directly detect unidirectional illumination by focusing the light in a sharp focal point on the distal side (Schuergers et al., 2016). Moreover, it was shown that the direction of twitching motility correlates with a specific proximal localization of the motor ATPase PilB (Schuergers et al., 2015). A model was proposed that the focusing leads to a local inhibition of the motility apparatus, thus determining the direction of movement of single cells as a photophobic response away from the focal light spot (Schuergers et al., 2016).

Materials and Reagents

  1. Sterile square (120 x 120 x 17 mm) Petri dishes (Greiner Bio One International, catalog number: 688161 ; supplied by VWR)
  2. Sterile 1 µl inoculation loops (SARSTEDT, catalog number: 86.1567.010 )
  3. Sterile 1.5 ml Eppendorf tubes 3810X (Eppendorf, catalog number: 0030125150 )
  4. Sterile µ-dishes 35 mm high glass bottom (ibidi, catalog number: 81158 )
  5. Microscope coverslips 20 x 20 mm (Carl Roth, catalog number: H873 )
  6. Sterile syringes 50 ml (SARSTEDT, catalog number: 94.6077.137 )
  7. Sterile syringe filters Filtropur S 0.2 (SARSTEDT, catalog number: 83.1826.001 )
  8. Sterile 50 ml Greiner centrifuge tubes (Greiner Bio One International, catalog number: 227261 )
  9. Serological pipettes 25 ml (SARSTEDT, catalog number: 86.1685.020 )
  10. Serological pipettes 5 ml (SARSTEDT, catalog number: 86.1253.025 )
  11. Synechocystis sp. PCC 6803 strain (motile wild type obtained from S. Shestakov, Moscow State University, Russia), resequenced by Trautmann et al. (2012)
  12. Liquid paraffin, viscous (Carl Roth, catalog number: 8904 )
  13. Ultrapure water
  14. Ethylenediamine tetraacetic acid disodium salt dehydrate (Na2EDTA·2H2O) (Carl Roth, catalog number: 8043 )
  15. Sodium hydroxide (NaOH) (Carl Roth, catalog number: 6771 )
  16. di-Potassium hydrogen phosphate trihydrate (K2HPO4·3H2O) (EMD Millipore, catalog number: 105099 )
  17. Sodium carbonate (Na2CO3) (Carl Roth, catalog number: P028 )
  18. Boric acid (H3BO3) (Carl Roth, catalog number: 6943 )
  19. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Carl Roth, catalog number: T881 )
  20. Zinc sulphate heptahydrate (ZnSO4·7H2O) (Carl Roth, catalog number: T884 )
  21. Sodium molybdate dehydrate (Na2MoO4·2H2O) (Carl Roth, catalog number: 0274 )
  22. Copper(II) sulphate pentahydrate (CuSO4·5H2O) (Carl Roth, catalog number: P024 )
  23. Cobalt(II) nitrate hexahydrate, Co(NO3)2·6H2O (Carl Roth, catalog number: HN16 )
  24. Ammonium ferric citrate (Carl Roth, catalog number: CN77 )
  25. 2-[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES) (Carl Roth, catalog number: 9137 )
  26. Sodium thiosulphate (Na2S2O3) (Carl Roth, catalog number: HN25 )
  27. D(+)-glucose (Carl Roth, catalog number: X997 )
  28. Sodium nitrate (NaNO3) (Carl Roth, catalog number: A136 )
  29. Magnesium sulphate heptahydrate (MgSO4·7H2O) (Carl Roth, catalog number: P027 )
  30. Calcium chloride dihydrate (CaCl2·2H2O) (Carl Roth, catalog number: 5239 )
  31. Citric acid (Carl Roth, catalog number: X863 )
  32. Agar-agar, Kobe I (Carl Roth, catalog number: 5210 )
  33. UltraPureTM agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500500 )
  34. 0.5 M Na2EDTA pH 8.0 (see Recipes)
  35. 3% w/v K2HPO4·3H2O solution (see Recipes)
  36. 2% w/v Na2CO3 solution (see Recipes)
  37. Trace metal mix solution (see Recipes)
  38. 0.6% w/v ammonium ferric citrate solution (see Recipes)
  39. 1 M TES buffer pH 8.0 (see Recipes)
  40. 30% w/v Na2S2O3 solution (see Recipes)
  41. 40% w/v D(+)-glucose solution (see Recipes)
  42. 100x BG11 medium (see Recipes)
  43. 2x BG11 medium (see Recipes)
  44. 1x BG11 medium (see Recipes)
  45. 1% w/v agar solution (see Recipes)
  46. 0.6% w/v agarose solution (see Recipes)
  47. Macroscopic phototaxis plate medium (see Recipes)
  48. Microscopic phototaxis plate medium (see Recipes)

Equipment

  1. Laboratory bottles 1,000 ml (Duran Group, catalog number: 21 801 54 5 )
  2. Laboratory bottles 500 ml (Duran Group, catalog number: 21 801 44 5 )
  3. Laboratory bottles 250 ml (Duran Group, catalog number: 21 801 36 5 )
  4. Laboratory bottles 100 ml (Duran Group, catalog number: 21 801 24 5 )
  5. Non-transparent square box (127 x 127 x 19 mm) with a one-sided opening (custom-made from polyvinyl chloride)
  6. Silicon ring (øo: 30 mm, øi: 16 mm) custom-made
  7. Non-transparent hollow cylinder (ø: 40 mm, h: 30 mm) with 4 holes (ø: 5 mm, h: 7 mm) positioned in increments of 90° for the insertion of LEDs (custom-made from polyvinyl chloride)
  8. Balance (Denver Instrument)
  9. pH meter (Mettler-Toledo)
  10. UV/Vis spectrophotometer (Shimadzu, model: UV-2401PC )
  11. Light source (Philips Lighting Holding, model: MASTER TL-D Super 80 18W/840 1SL/25 )
  12. Pipetting aid PIPETBOY acu 2 (INTEGRA Biosciences, model: PIPETBOY acu 2, catalog number: 155 017 )
  13. Quantum Sensor LI-190R (LI-COR, model: LI-190R )
  14. Transmitted light scanner (Epson, model: Epson Perfection V700 Photo , with adjustable cover)
  15. Fluorescence microscope (Nikon Instruments, model: Eclipse Ni-U , with CFI Plan Fluor 40X/0.75)
  16. Digital CCD camera (Hamamatsu Photonics, model: ORCA®-05G )
  17. Microcontroller board (Arduino, model: Arduino UNO R3 )
  18. RGB-LEDs (625 nm, 525 nm, 470 nm) 5 mm (World Trading Net)
  19. Biological safety cabinet (NuAire, model: Class II Type A2 )

Software

  1. NIS-Elements Basic Research 4.20.01
  2. Arduino 1.0.6
  3. MATLAB Runtime 8.3
  4. BacteriaMobilityQuant (https://web.fe.up.pt/~dee11017/software/BacterialMobilityQuant.zip)
  5. R

Procedure

  1. Macroscopic phototaxis assay (Figure 1)


    Figure 1. Macroscopic phototaxis assay. Consecutive steps as described in steps A1-A8.

    1. Prepare 80 ml of the macroscopic phototaxis plate medium (see Recipes).
    2. Mix very gently to avoid any air bubbles.
    3. Pour the medium carefully into a square Petri dish and close the lid to avoid over-drying of the phototaxis plate. Let it solidify on an even surface. Plates should always be freshly prepared.
    4. Scrape cyanobacterial cells with a sterile 1 µl inoculation loop from freshly grown agar plates or from a macroscopic phototaxis plate (see Note 5) and resuspend in 50 µl 1x BG11 medium (see Recipes) in a sterile 1.5 ml Eppendorf tube by vigorously twisting the loop (approximately OD750 40 or very dark green color).
    5. Place the phototaxis plate on a template with three equally spaced parallel rows drawn over the surface area of the plate. Spot 5 µl of the cell suspension in triplicates in each row. Close the lid and allow the droplets to soak into the plate. This step may take up to an hour depending on the surface of the plate and the density of the cells.
    6. Place the phototaxis plate upside down in a non-transparent square box with a one-sided opening. Align the rows of the phototaxis plate parallel to the opening.
    7. Incubate the phototaxis plate at 30 °C under a unidirectional white light source with an angle of about 45° (e.g., Philips MASTER TL-D Super 80 18W/840 1SL/25) at approximately 35 µmol photons m-2 sec-1 measured with a quantum sensor at the front side of the phototaxis box.
    8. After 1-2 days the finger-like projections of the motile cells can be recorded by scanning the plate or taking a picture (Figure 2). For reproducibility, all results should be recorded after the same time.


      Figure 2. Macroscopic phototaxis plates. Finger-like projections of motile wild-type cells after A) one day of incubation on agar-agar, Kobe I and B) one week of incubation on standard BactoTM agar under unidirectional white light (35 µmol photons m-2 sec-1).

  2. Microscopic phototaxis assay (Figure 3)


    Figure 3. Microscopic phototaxis assay. Consecutive steps as described in steps B1-B12.

    1. Prepare 50 ml of the microscopic phototaxis plate medium (see Recipes).
    2. Mix very gently using a pipetting aid to avoid any air bubbles.
    3. Pour 5 ml of the medium carefully into each sterile glass bottom µ-dish. Use a pipetting aid and close the lids to avoid over-drying of the phototaxis plates. Let the medium solidify on a level surface to prevent unevenness. As above plates should always be freshly prepared.
    4. Scrape motile cyanobacterial cells with a sterile 1 µl inoculation loop from a fresh macroscopic phototaxis plate and resuspend in 110 µl 1x BG11 medium (see Recipes) in a sterile 1.5 ml Eppendorf tube by vigorously twisting the loop (approximately OD750 1.0 or very light green color). Be careful not to scrape any agar from the surface. The density of the cells is crucial for the outcome of the experiment. If the density is too low, the cells will not move properly. If the density is too high, the cells will obstruct each other and tracking will be negatively affected.
    5. Spot 5 x 2 µl droplets of the cell suspension in the center of each plate and let dry for 10-15 min. Avoid over-drying and exposure to intense light (e.g., sunlight on the bench).
    6. Carefully place a coverslip on top of the cells immediately after all the liquid has been soaked in. Avoid air bubbles but do not press down with force. If the coverslip is applied too early or pressed down with force, the cells will float and Brownian movement will be observed, predominantly. If the coverslip is applied too late, over-drying of the surface will hinder cell movement.
    7. Lubricate one side of a silicon ring with liquid paraffin and place the ring on the plate. The ring should cover all of the exposed agarose surface to minimize evaporation and prevent jittering of the sample while imaging. Be careful not to contaminate the surface of the coverslip with the liquid paraffin.
    8. Incubate the samples in a dark environment for 2-3 h.
    9. Place the phototaxis plate on the stage of an upright microscope equipped with a 40x objective and cover it with a non-transparent hollow cylinder with 4 holes positioned in increments of 90°. Insert the LEDs into the holes and align the pins horizontally to the stage. Make sure that the LEDs touch the center of the plate and focus on the cells. The LEDs are positioned at the same height as the agarose surface.
    10. Start the microscope software NIS-Elements Basic Research 4.20.01. Dim the condenser light of the microscope so that it is barely visible. Typically, the histogram displayed in the microscope software should be in the lower range for acquisition times > 100 msec. If the intensity of the condenser light is too high, the cells will show impaired directional motility. If the intensity of the condenser light is too low, tracking will be negatively affected.
    11. Switch on the RGB-LEDs as desired using a microcontroller board (e.g., Arduino UNO R3 with the software Arduino 1.0.6). For a standard phototaxis assay directional white light illumination from RGB-LEDs (470/525/625 nm at equal intensities) with a total intensity of 10 µmol photons m-2 sec-1, measured with a quantum sensor, is used.
    12. Acquire images as necessary. For a standard 5 min time lapse video a frame rate of 1 frame every 3 sec at an acquisition time of 200 msec is used (Video 1).

      Video 1. Time lapse video of wild-type cells illuminated with 10 µmol photons m-2 sec-1 from the right side

Data analysis

  1. Import your data into the BacteriaMobilityQuant software. Adjust settings such as acquisition time and scale in the parameters → update tab. Run the program. The outlines of the cells are detected separately for each frame (Figure 4A) before the actual tracking of the cells (Figure 4B).
    Note: BacteriaMobilityQuant requires MATLAB Runtime 8.3 to be installed on your PC.
  2. The program returns two .txt files containing the tracking information of the cells. Trajectories are displayed in a .png file (Figure 4C) and a movie of the tracked cells is saved in .avi format (Video 2).

    Video 2. BacteriaMobilityQuant cell tracking of wild-type cells illuminated with 10 µmol photons m-2 sec-1 from the right side

  3. Data analysis of the raw tracks can be performed using the software R. Some form of data cleansing is recommended to discard irrelevant tracks from the dataset (Figure 4D). To avoid artifacts due to possible mismatching of cells between frames by the tracking algorithm we only consider cells that can be tracked for at least 25 consecutive frames with an average velocity below 0.4 µm sec-1 and a maximum displacement of less than 8 µm between two frames (3 sec). Moreover, for tracking we discard the subgroup of immotile cells with an average velocity below 0.05 µm sec-1. The best values depend on overall cell speed and frame rate and should be evaluated carefully. If desired, directional statistics of the data like a Rayleigh test of uniformity can be performed using the CIRCULAR package (Agostinelli and Lund, 2013) implemented in R.


    Figure 4. BacteriaMobilityQuant analysis of wild-type cells illuminated with 10 µmol photons m-2 sec-1 from the right side. A. BacteriaMobilityQuant cell detection; B. BacteriaMobilityQuant cell tracking; C. BacteriaMobilityQuant cell trajectories; D. Data cleansing with R to remove unreliable tracks and immotile cells.

Notes

  1. All solutions are prepared using ultrapure water (resistivity > 18.18 MΩ cm at 25 °C) and analytical grade reagents.
  2. 1% w/v agar solution and 0.6% w/v agarose solution should be prepared freshly prior to each phototaxis assay.
  3. For reproducibility, it is highly recommended to use agar-agar, Kobe I for the macroscopic phototaxis plates and UltraPureTM agarose for the microscopic phototaxis plates. In our group, a significant increase in motility was observed when changing from standard BactoTM agar to agar-agar, Kobe I (Figure 2).
  4. For reproducibility, Na2S2O3 should be used in the plates (Thiel et al., 1989) (see Recipes) and all results should be recorded after the same time.
  5. For the macroscopic phototaxis assays, we achieve the best results when we take cells from a freshly grown agar plate and incubate them on a phototaxis plate under unidirectional white light for three days. The front line of the cells can now be used for the actual phototaxis assay.
  6. Microscopic phototaxis assays are conducted approximately 2-3 h after spotting the cells on the agarose plates and letting them adjust to the surface in a dark environment. In this phase of motility cells show a strongly biased movement towards a directional light source.
  7. For the microscopic phototaxis assays, it is also possible to use an inverted microscope if the protocol is adjusted accordingly.
  8. During the microscopic phototaxis assay pay attention to the orientation of possible air bubbles between plate and coverslip. Never place air bubbles between light source and bacteria to prevent scattering.
  9. Microscopic phototaxis assays are carried out at room temperature (approximately 22 °C).
  10. Every experimental step involving the handling of cyanobacterial cell cultures must be performed in a biological safety cabinet to prevent contamination.

Recipes

  1. 0.5 M Na2EDTA, pH 8.0 (1,000 ml)
    186.12 g Na2EDTA·2H2O
    Add 800 ml ddH2O
    Adjust to pH 8.0 with NaOH
    Note: Use highly concentrated NaOH, Na2EDTA dissolves only when the pH of the solution is adjusted to 8.0.
    Add ddH2O to 1,000 ml
    Autoclave and store at room temperature
  2. 3% w/v K2HPO4·3H2O solution (50 ml)
    1.5 g K2HPO4·3H2O
    Add ddH2O to 50 ml
    Filter sterilize using a syringe and a filter
    Store at 4 °C
  3. 2% w/v Na2CO3 solution (50 ml)
    1.0 g Na2CO3
    Add ddH2O to 50 ml
    Filter sterilize using a syringe and a filter
    Store at 4 °C
  4. Trace metal mix solution (500 ml)
    400 ml ddH2O
    1.43 g H3BO3
    900 mg MnCl2·4H2O
    110 mg ZnSO4·7H2O
    195 mg Na2MoO4·2H2O
    39.5 mg CuSO4·5H2O
    24.7 mg Co(NO3)2·6H2O
    Add ddH2O to 500 ml
    Filter sterilize using a syringe and a filter
    Store at 4 °C
  5. 0.6% w/v ammonium ferric citrate solution (50 ml)
    300 mg ammonium ferric citrate
    Add ddH2O to 50 ml
    Filter sterilize using a syringe and a filter
    Store at 4 °C
  6. 1 M TES buffer pH 8.0 (50 ml)
    30 ml ddH2O
    11.45 g 2-[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES)
    Adjust to pH 8.0 with NaOH
    Add ddH2O to 50 ml
    Filter sterilize using a syringe and a filter
    Store at 4 °C
  7. 30% w/v Na2S2O3 solution (50 ml)
    15 g Na2S2O3
    Add ddH2O to 50 ml
    Filter sterilize using a syringe and a filter
    Store at 4 °C
  8. 40% w/v D(+)-glucose solution (50 ml)
    20 g D(+)-glucose
    Add ddH2O to 50 ml
    Filter sterilize using a syringe and a filter
    Aliquot in 1.5 ml Eppendorf tubes and store at 4 °C
  9. 100x BG11 medium (500 ml)
    74.79 g NaNO3
    3.75 g MgSO4·7H2O
    1.80 g CaCl2·2H2O
    0.30 g citric acid
    0.28 ml 0.5 M Na2EDTA pH 8.0
    Add ddH2O to 500 ml
    Autoclave and store at 4 °C
  10. 2x BG11 medium (1,000 ml)
    900 ml ddH2O
    20 ml 100x BG11 medium
    2 ml 3% w/v K2HPO4·3H2O solution
    2 ml 2% w/v Na2CO3 solution
    2 ml trace metal mix solution
    Add ddH2O to 998 ml
    Autoclave
    2 ml 0.6% w/v ammonium ferric citrate solution
    Store at room temperature
  11. 1x BG11 medium (1,000 ml)
    900 ml ddH2O
    10 ml 100x BG11 medium
    10 ml 1 M TES buffer pH 8.0
    1 ml 3% w/v K2HPO4·3H2O solution
    1 ml 2% w/v Na2CO3 solution
    1 ml trace metal mix solution
    Add ddH2O to 999 ml
    Autoclave
    1 ml 0.6% w/v ammonium ferric citrate solution
    Store at room temperature
  12. 1% w/v agar solution (200 ml)
    2 g agar-agar, Kobe I
    Add ddH2O to 200 ml
    Autoclave and use freshly
  13. 0.6% w/v agarose solution (100 ml)
    0.6 g UltraPureTM agarose
    Add ddH2O to 100 ml
    Autoclave and use freshly
  14. Macroscopic phototaxis plate medium (80 ml)
    0.8 ml 1 M TES buffer pH 8.0
    0.8 ml 30% w/v Na2S2O3 solution
    0.4 ml 40% w/v D(+)-glucose solution
    39 ml 2x BG11 medium
    39 ml molten 1% w/v agar solution
    Mix and pour very gently to avoid any air bubbles and unevenness of the surface
    Avoid drying of the plate
  15. Microscopic phototaxis plate medium (50 ml)
    0.5 ml 1 M TES buffer pH 8.0
    0.5 ml 30% w/v Na2S2O3 solution
    0.25 ml 40% w/v D(+)-glucose solution
    24.375 ml 2x BG11 medium
    24.375 ml molten 0.6% w/v agarose solution
    Mix and pour very gently using a pipetting aid to avoid any air bubbles and unevenness of the surface
    Avoid drying of the plates

Acknowledgments

This protocol is based on the methods described in Schuergers et al. (2016) and Schuergers et al. (2015). BacteriaMobilityQuant software for cell tracking was developed by Tiago Esteves and Maja Temerinac-Ott (Schuergers et al., 2016). BG11 medium is prepared according to Rippka et al. (1979). This work was funded by the Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School).

References

  1. Agostinelli, C. and Lund, U. (2013). R package “circular”: circular statistics (version 0.4-7).
  2. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. and Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol (111): 1-61.
  3. Schuergers, N., Lenn, T., Kampmann, R., Meissner, M. V., Esteves, T., Temerinac-Ott, M., Korvink, J. G., Lowe, A. R., Mullineaux, C. W. and Wilde, A. (2016). Cyanobacteria use micro-optics to sense light direction. eLIFE (5): e12620.
  4. Schuergers, N., Nurnberg, D. J., Wallner, T., Mullineaux, C. W. and Wilde, A. (2015). PilB localization correlates with the direction of twitching motility in the cyanobacterium Synechocystis sp. PCC 6803. Microbiology 161(Pt 5): 960-966.
  5. Thiel, T., Bramble, J. and Rogers, S. (1989). Optimum conditions for growth of cyanobacteria on solid media. FEMS Microbiol Lett 52(1-2): 27-31.
  6. Trautmann, D., Voss, B., Wilde, A., Al-Babili, S. and Hess, W. R. (2012). Microevolution in cyanobacteria: re-sequencing a motile substrain of Synechocystis sp. PCC 6803. DNA Res 19(6): 435-448.

简介

Phototaxis是允许蓝细菌通过朝向或远离光源移动来响应照明质量和数量的波动的机制。分别在低浓度琼脂或琼脂糖平板上的光镜移动可以分别表示组织行为和单细胞运动性的宏观和微观尺度。在这里,我们描述了使用单细胞蓝细菌集胞藻的两种鳞片上的光趋化测定的详细程序。 PCC 6803。

背景 模型生物 Synechocystis sp。 PCC 6803使用伸缩型IV皮脂(T4P)以潮汐表面移动,称为抽动动力。两个分泌的ATP酶(PilB和PilT)负责扩增和缩回菌毛装置,从而将细胞向前拉。集胞藻 sp。 PCC 6803拥有覆盖整个可见光谱的各种光感受器。光的吸收可以根据波长和强度刺激正或负光趋向性。最近,已经证明,单细胞集胞藻 PCC 6803能够通过将光聚焦在远侧的尖锐焦点上来直接检测单向照明(Schuergers等人,2016)。此外,显示抽动运动的方向与运动ATPase PilB的特定近端定位相关(Schuergers等人,2015)。提出了一种模型,即聚焦导致对运动装置的局部抑制,从而确定单个细胞作为远离焦点的光电响应的移动方向(Schuergers等人,2016) )。

关键字:蓝藻细菌, 集胞藻属, 动力性, 趋光性, 光受体

材料和试剂

  1. 无菌方形(120×120×17mm)培养皿(Greiner Bio One International,目录号:688161;由VWR提供)
  2. 无菌1μl接种环(SARSTEDT,目录号:86.1567.010)
  3. 无菌1.5 ml Eppendorf管3810X(Eppendorf,目录号:0030125150)
  4. 无菌μ盘35毫米高玻璃底(ibidi,目录号:81158)
  5. 显微镜盖玻片20 x 20毫米(Carl Roth,目录号:H873)
  6. 无菌注射器50ml(SARSTEDT,目录号:94.6077.137)
  7. 无菌注射器过滤器Filtropur S 0.2(SARSTEDT,目录号:83.1826.001)
  8. 无菌50ml格林纳离心管(Greiner Bio One International,目录号:227261)
  9. 血清移液管25毫升(SARSTEDT,目录号:86.1685.020)
  10. 血清移液管5ml(SARSTEDT,目录号:86.1253.025)
  11. 集胞藻 sp。 PCC 6803菌株(由S.Shestakov,Moscow State University,Russia获得的活性野生型),由Trautmann等人(2012)
    重新排序。
  12. 液体石蜡,粘稠(Carl Roth,目录号:8904)
  13. 超纯水
  14. 乙二胺四乙酸二钠盐脱水(Na 2 EDTA·2H 2 O)(Carl Roth,目录号:8043)
  15. 氢氧化钠(NaOH)(Carl Roth,目录号:6771)
  16. 磷酸氢二氢钾三水合物(K 2 HPO 4·3H 2 O)(EMDMicipore,目录号:105099) >
  17. 碳酸钠(Na 2 CO 3)(Carl Roth,目录号:P028)
  18. 硼酸(H 3 3 BO 3)(Carl Roth,目录号:6943)
  19. 氯化锰(II)四水合物(MnCl 2·4H 2 O)(Carl Roth,目录号:T881)
  20. 硫酸锌七水合物(ZnSO 4·7H 2 O)(Carl Roth,目录号:T884)
  21. 钼酸钠脱水(Na 2 MoO 4·2H 2 O)(Carl Roth,目录号:0274)
  22. 硫酸铜(II)五水合物(CuSO 4·5H 2 O)(Carl Roth,目录号:P024)
  23. 硝酸钴(II)六水合物Co(NO 3 3)2·6H 2 O(Carl Roth,目录号:HN16) >
  24. 柠檬酸铵(Carl Roth,目录号:CN77)
  25. 2 - [(2-羟基-1,1-双(羟甲基)乙基)氨基]乙磺酸(TES)(Carl Roth,目录号:9137)
  26. 硫代硫酸钠(Na 2 S 2 O 3 O 3)(Carl Roth,目录号:HN25)
  27. D(+) - 葡萄糖(Carl Roth,目录号:X997)
  28. 硝酸钠(NaNO 3)(Carl Roth,目录号:A136)
  29. 硫酸镁七水合物(MgSO 4·7H 2 O)(Carl Roth,目录号:P027)
  30. 氯化钙脱水(CaCl 2·2H 2 O)(Carl Roth,目录号:5239)
  31. 柠檬酸(Carl Roth,目录号:X863)
  32. 琼脂,神户一(Carl Roth,目录号:5210)
  33. UltraPure TM琼脂糖(Thermo Fisher Scientific,Invitrogen TM,目录号:16500500)
  34. 0.5 M Na 2 EDTA pH 8.0(参见食谱)
  35. 3%w / v K 2 HPO 4·3H 2 O溶液(参见食谱)
  36. 2%w / v Na 2 CO 3溶液(参见食谱)
  37. 痕量金属混合溶液(见配方)
  38. 0.6%w / v柠檬酸铁铵溶液(参见食谱)
  39. 1 M TES缓冲液pH 8.0(见配方)
  40. 30%w / v Na 2 S 2 O 3 O 3溶液(参见食谱)
  41. 40%w / v D(+) - 葡萄糖溶液(参见食谱)
  42. 100x BG11培养基(参见食谱)
  43. 2x BG11培养基(见食谱)
  44. 1x BG11培养基(见食谱)
  45. 1%w / v琼脂溶液(见配方)
  46. 0.6%w / v琼脂糖溶液(参见食谱)
  47. 宏观光趋向板培养基(见食谱)
  48. 微观光轴平板培养基(见食谱)

    设备

    1. 实验室瓶1000毫升(杜兰集团,目录号:21 801 54 5)
    2. 实验室瓶500毫升(杜兰集团,目录号:21 801 44 5)
    3. 实验室瓶250毫升(杜兰集团,目录号:21 801 36 5)
    4. 实验室瓶100毫升(杜兰集团,目录号:21 801 24 5)
    5. 不透明方箱(127 x 127 x 19毫米),单面开口(由聚氯乙烯定制)
    6. 硅环(øø>::øøø::::::::::::::::::::::::::::))))))))))))
    7. 不透明中空圆柱体(ø:40 mm,h:30 mm),带有4个孔(ø:5 mm,h:7 mm),以90°为增量,用于插入LED(由聚氯乙烯定制) br />
    8. 平衡(丹佛仪器)
    9. pH计(Mettler-Toledo)
    10. UV / Vis分光光度计(Shimadzu,型号:UV-2401PC)
    11. 光源(Philips Lighting Holding,型号:MASTER TL-D Super 80 18W / 840 1SL / 25)
    12. 移液辅助剂PIPETBOY acu 2(INTEGRA Biosciences,型号:PIPETBOY acu 2,目录号:155 017)
    13. 量子传感器LI-190R(LI-COR,型号:LI-190R)
    14. 传输光扫描仪(爱普生,型号:Epson Perfection V700照片,带可调盖)
    15. 荧光显微镜(Nikon Instruments,型号:Eclipse Ni-U,CFI Plan Fluor 40X / 0.75)
    16. 数码CCD相机(Hamamatsu Photonics,型号:ORCA ® -05G)
    17. 微控制器板(Arduino,型号:Arduino UNO R3)
    18. RGB-LED(625nm,525nm,470nm)5mm(世界贸易网)
    19. 生物安全柜(NuAire,型号:Class II Type A2)

      软件

      1. NIS-Elements基础研究4.20.01
      2. Arduino 1.0.6
      3. MATLAB Runtime 8.3
      4. BacteriaMobilityQuant( https://web.fe.up .pt /〜dee11017 / software / BacterialMobilityQuant.zip
      5. R

        程序

        1. 宏观光滴测定(图1)


          图1.宏观光滴测定。 如步骤A1-A8中所述的连续步骤。

          1. 准备80毫升的宏观光轴平台培养基(见食谱)
          2. 混合非常轻轻地避免任何气泡。
          3. 将培养基小心地倒入正方形陪替氏培养皿中并关闭盖子,以避免过度干燥光感受板。让它在平滑的表面凝固。板材应该总是准备好。
          4. 用新鲜生长的琼脂平板或宏观光感受态板(见附注5)从无菌的1μl接种环中刮掉蓝细菌细胞,并将其在50μl1x BG11培养基(参见食谱)中悬浮于无菌的1.5ml Eppendorf管中,通过剧烈扭转环(大约OD 750或非常深绿色)。
          5. 将光趋势板放置在模板上,三个等距间隔的平行排在板的表面区域上。将5μl细胞悬浮液每一列重复一次。关闭盖子,让液滴浸入板中。该步骤可能需要长达一小时,具体取决于板的表面和细胞的密度。
          6. 将光倾斜板倒置在带有单面开口的不透明的方形盒中。将光照板的行平行于开口对齐。
          7. 在约35μmol光子下以30°C的单向白光源以约45°(例如,Philips MASTER TL-D Super 80 18W / 840 1SL / 25)的角度孵育光趋向板使用量子传感器在光趋向盒的前侧测量的m -2 sec -1
          8. 1-2天后,可以通过扫描板或拍摄照片来记录运动细胞的手指状突起(图2)。对于重复性,所有结果应在同一时间后记录。


            图2.宏观光趋向板在A)在琼脂,神户I和B上孵育一天后,运动性野生型细胞的手指状突起在标准Bacto TM 琼脂在单向白光(35μmol光子m -2 sec -1 )。

          9. 显微光度测定(图3)


            图3.微观光趋化测定。 如步骤B1-B12所述的连续步骤。

            1. 准备50毫升微观光趋向板培养基(见食谱)。
            2. 使用吸液辅助剂轻轻混合,以避免任何气泡。
            3. 将5 ml的培养基小心倒入每个无菌玻璃底部μ碟中。使用移液辅助剂并关闭盖子,以避免过度干燥光致变色板。让介质在水平面上凝固,以防止凹凸不平。如上所述,应始终做好新鲜准备
            4. 用无菌的1μl接种环路从新鲜的宏观光滑板上刮下运动性蓝细菌细胞,并通过大力扭转环路(大约OD 750)重新悬浮于无菌的1.5ml Eppendorf管中的110μl1x BG11培养基(参见食谱) sub> 1.0或非常浅绿色)。小心不要从表面刮掉任何琼脂。细胞的密度对于实验的结果是至关重要的。如果密度太低,细胞将不能正常移动。如果密度太高,细胞会相互阻碍,跟踪将受到不利影响
            5. 将5×2微升的细胞悬浮液滴在每个板的中心,并干燥10-15分钟。避免过度干燥和暴露于强光(例如,,台灯上的阳光)。
            6. 所有液体浸入后,请小心地将盖玻片放在细胞顶部。避免气泡,但不要用力按压。如果盖玻片施加得太早或受力压下,细胞将浮起来,主要会观察到布朗运动。如果盖玻片应用太晚,表面的过度干燥将阻碍细胞运动。
            7. 用液体石蜡润滑硅环的一面,并将环放在板上。该环应覆盖所有暴露的琼脂糖表面,以最小化蒸发,并防止样品在成像时发生抖动。注意不要用液体石蜡污染盖玻片的表面。
            8. 在黑暗环境中孵育样品2-3小时。
            9. 将光趋向板放在装有40x物镜的立式显微镜的平台上,并用不透明的中空圆柱盖住,四个孔以90°的增量放置。将LED插入孔中,并将引脚水平对准舞台。确保LED触摸板的中心并聚焦在单元格上。 LED位于与琼脂糖表面相同的高度。
            10. 启动显微镜软件NIS-Elements Basic Research 4.20.01。减去显微镜的聚光灯,使其几乎看不到。通常,显微镜软件中显示的直方图应在采集时间的下限范围内> 100毫秒如果冷凝器光的强度太高,则细胞将显示有限的方向运动。如果聚光灯的强度太低,跟踪将受到不利影响
            11. 使用微控制器板(例如,Arduino UNO R3,使用软件Arduino 1.0.6)根据需要打开RGB-LED。对于来自RGB-LED(470/525/625nm,等强度)的定向白光照射,总强度为10μmol光子m sec -1 < sup>,用量子传感器测量。
            12. 根据需要获取图像。对于标准的5分钟时间流逝视频,使用在200msec的采集时间每3秒1帧的帧速率(视频1)。

          数据分析

          1. 将您的数据导入BacteriaMobilityQuant软件。在参数→更新选项卡中调整采集时间和比例等设置。运行程序。在实际跟踪单元格之前,为每个帧(图4A)单独检测单元格的轮廓(图4B)。
            注意:BacteriaMobilityQuant需要在PC上安装MATLAB Runtime 8.3。
          2. 该程序返回两个包含单元格跟踪信息的.txt文件。轨迹显示在.png文件中(图4C),跟踪的单元格的影片将以.avi格式保存(视频2)。

          3. 可以使用软件R执行原始轨迹的数据分析。建议使用某种形式的数据清理来从数据集中丢弃不相关的轨迹(图4D)。为了避免由于跟踪算法在帧之间的单元可能的不匹配造成的伪像,我们只考虑可以跟踪至少25个连续帧的单元,平均速度低于0.4μmsec -1,并且最大位移在两帧之间小于8μm(3秒)。此外,对于跟踪,我们以平均速度低于0.05μmsec -1的方式丢弃不动细胞的亚组。最佳值取决于整体单元速度和帧速率,应仔细评估。如果需要,可以使用R中实现的CIRCULAR软件包(Agostinelli和Lund,2013)执行诸如均匀性瑞利测试之类的数据的方向统计。


            图4.来自右侧的10μmol光子m -2 sec -1 的野生型细胞的细菌迁移量定量分析A.细菌迁移量细胞检测; B.细菌迁移量细菌迁移量细胞轨迹; D.用R清除数据,清除不可靠的轨迹和不动细胞。

          笔记

          1. 所有溶液都是使用超纯水(25℃下的电阻率>18.18MΩcm)和分析级试剂制备的。
          2. 1%w / v琼脂溶液和0.6%w / v琼脂糖溶液应在每次光滴测定之前新鲜制备。
          3. 为了重现性,强烈建议将琼脂,神户I用于宏观光轴平板和UltraPure TM 琼脂糖用于显微光轴向平板。在我们组中,当从标准的Bacto TM 琼脂转变为琼脂,神户I(图2)时,观察到运动性显着增加。
          4. 为了重现性,应在板中使用Na 2 O 3 O 3 O 3(Thiel等人,1989) )(见配方),所有结果应在同一时间后记录。
          5. 对于宏观光趋化测定,当我们从新鲜生长的琼脂平板上取出细胞并在单向白光下将它们在光感受板上孵育三天时,我们获得最佳结果。细胞的前线现在可以用于实际的光趋化测定。
          6. 在琼脂糖板上点样细胞约2-3小时后进行微观光照测定,并使其在黑暗环境中调整至表面。在这个运动阶段,细胞表现出朝向定向光源的强烈的偏向移动
          7. 对于微观光趋化测定,如果协议相应调整,也可以使用倒置显微镜
          8. 在微观光趋化测定期间,注意板和盖玻片之间可能的气泡的取向。不要在光源和细菌之间放置气泡,以防止散射
          9. 微观光照测定在室温(约22℃)下进行
          10. 必须在生物安全柜中进行涉及处理蓝细菌细胞培养物的每个实验步骤,以防止污染

          食谱

          1. 0.5M Na 2 EDTA,pH 8.0(1,000ml) 186.12g Na 2 EDTA·2H 2 O 加入800毫升ddH 2 O - / - 用NaOH调节至pH 8.0 注意:只有当溶液的pH调节至8.0时,才能使用高浓度的NaOH,钠溶解。 >
            将ddH 2 O添加到1,000 ml
            高压灭菌并在室温下储存
          2. 3%w / v K 2 HPO 4·3H 2 O溶液(50ml)
            1.5g K 2 HPO 4 / 3H 2 O
            将ddH 2 O添加到50 ml
            使用注射器和过滤器过滤灭菌
            储存于4°C
          3. 2%w / v Na 2 CO 3溶液(50ml)
            1.0g Na 2 CO 3
            将ddH 2 O添加到50 ml
            使用注射器和过滤器过滤灭菌
            储存于4°C
          4. 痕量金属混合溶液(500 ml)
            400毫升ddH 2 O O
            1.43g H 3 3 3
            900mg MnCl 2·4H 2 O
            110mg ZnSO 4·7H 2 O
            195mg Na 2 MoO 4·2H 2 O 39.5mg CuSO 4·5H 2 O
            24.7mg Co(NO 3 3)2·6H 2 O
            将ddH 2 O加入500 ml
            使用注射器和过滤器过滤灭菌
            储存于4°C
          5. 0.6%w / v柠檬酸铁铵溶液(50ml)
            300毫克柠檬酸铵柠檬酸铵 将ddH 2 O添加到50 ml
            使用注射器和过滤器过滤灭菌
            储存于4°C
          6. 1 M TES缓冲液pH 8.0(50ml)
            30毫升ddH 2 O - / - 11.45g 2 - [(2-羟基-1,1-双(羟甲基)乙基)氨基]乙磺酸(TES)
            用NaOH调节至pH 8.0 将ddH 2 O添加到50 ml
            使用注射器和过滤器过滤灭菌
            储存于4°C
          7. 30%w / v Na 2 S 2 O 3 O 3溶液(50ml)
            15g Na 2 S 2 O 3&lt; 3&gt;
            将ddH 2 O添加到50 ml
            使用注射器和过滤器过滤灭菌
            储存于4°C
          8. 40%w / v D(+) - 葡萄糖溶液(50ml)
            20克D(+) - 葡萄糖
            将ddH 2 O添加到50 ml
            使用注射器和过滤器过滤灭菌
            在1.5ml Eppendorf管中分装,并在4°C下储存
          9. 100x BG11培养基(500 ml)
            74.79g NaNO 3
            3.75g MgSO 4·7H 2 O·
            1.80g CaCl 2·2H 2 O O
            0.30克柠檬酸
            0.28ml 0.5M Na 2 EDTA pH8.0
            将ddH 2 O加入500 ml
            高压灭菌并储存在4°C
          10. 2x BG11培养基(1,000ml)
            900毫升ddH 2 O - / - 20 ml 100x BG11培养基 2ml 3%w / v K 2 HPO 4·3H 2 O溶液
            2ml 2%w / v Na 2 CO 3溶液
            2ml痕量金属混合液
            将ddH 2 O添加到998ml
            高压灭菌器
            2ml 0.6%w / v柠檬酸铁铵溶液
            在室温下存放
          11. 1x BG11培养基(1,000ml)
            900毫升ddH 2 O - / - 10 ml 100x BG11培养基 10ml 1 M TES缓冲液pH 8.0
            1ml 3%w / v K 2 HPO 4·3H 2 O溶液
            1ml 2%w / v Na 2 CO 3 溶液
            1毫升痕量金属混合溶液
            将ddH 2 O添加到999 ml
            高压灭菌器
            1ml 0.6%w / v柠檬酸铁铵溶液
            在室温下存放
          12. 1%w / v琼脂溶液(200 ml) 2克琼脂,神户我
            将ddH 2 O加入200 ml
            高压灭菌和新鲜使用
          13. 0.6%w / v琼脂糖溶液(100ml)
            0.6g UltraPure TM 琼脂糖
            将ddH 2 O添加到100 ml
            高压灭菌和新鲜使用
          14. 宏观光滑板培养基(80 ml)
            0.8 ml 1 M TES缓冲液pH 8.0
            0.8ml 30%w / v Na 2 S 2 O 3溶液
            0.4ml 40%w / v D(+) - 葡萄糖溶液
            39 ml 2x BG11培养基
            39 ml熔融1%w / v琼脂溶液
            混合和倾倒非常轻轻地避免任何气泡和表面的不均匀性
            避免干燥板
          15. 显微光滑片培养基(50ml)
            0.5 ml 1 M TES缓冲液pH 8.0
            0.5ml 30%w / v Na 2 S 2 O 3 O 3溶液
            0.25 ml 40%w / v D(+) - 葡萄糖溶液
            24.375毫升2x BG11培养基
            24.375ml熔融的0.6%w / v琼脂糖溶液
            使用移液辅助剂轻轻混合和倾倒,以避免任何气泡和表面不均匀 避免干燥板

          致谢

          该协议基于Schuergers等人(2016)和Schuergers等人(2015)中描述的方法。用于细胞追踪的BacteriaMobilityQuant软件由Tiago Esteves和Maja Temerinac-Ott(Schuergers等人,2016年)开发。 BG11培养基根据Rippka等人制备(1979)。这项工作由德国研究基金会(GSC-4,Spemann研究生院)的卓越计划资助。

          参考

          1. Agostinelli,C.and Lund,U.(2013)。&nbsp; R包”圆形“:循环统计(版本0.4-7)。
          2. Rippka,R.,Deruelles,J.,Waterbury,JB,Herdman,M.and Stanier,RY(1979)。&lt; a class =“ke-insertfile”href =“http://mic.microbiologyresearch.org/内容/ journal / micro / 10.1099 / 00221287-111-1-1“target =”_ blank“>蓝细菌的纯培养物的通用分配,应变历史和性质。 Gen Microbiol 111):1-61。
          3. Schuergers,N.,Lenn,T.,Kampmann,R.,Meissner,MV,Esteves,T.,Temerinac-Ott,M.,Korvink,JG,Lowe,AR,Mullineaux,CW和Wilde,A.(2016) 。蓝藻使用微光学感测光线方向。 < em> eLIFE (5):e12620。
          4. Schuergers,N.,Nurnberg,DJ,Wallner,T.,Mullineaux,CW and Wilde,A.(2015)。&nbsp; PilB定位与蓝藻中的抽搐运动方向相关。 PCC 6803。微生物学161(Pt 5):960-966。
          5. Thiel,T.,Bramble,J.and Rogers,S。(1989)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/2513249” target =“_ blank”>固体培养基上蓝细菌生长的最佳条件 FEMS Microbiol Lett 52(1-2):27-31。
          6. Trautmann,D.,Voss,B.,Wilde,A.,Al-Babili,S.和Hess,WR(2012)。&lt; a class =“ke-insertfile”href =“http://www.ncbi .nlm.nih.gov / pubmed / 23069868“target =”_ blank“>蓝细菌中的微量分解:重新测序集胞藻的运动子序列。 PCC 6803。 DNA Res 19(6):435-448。
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引用:Jakob, A., Schuergers, N. and Wilde, A. (2017). Phototaxis Assays of Synechocystis sp. PCC 6803 at Macroscopic and Microscopic Scales. Bio-protocol 7(11): e2328. DOI: 10.21769/BioProtoc.2328.
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