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Dec 2017

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Implementation of Blue Light Switchable Bacterial Adhesion for Design of Biofilms
通过蓝光控制的细菌粘附进行生物膜设计   

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Abstract

Control of bacterial adhesions to a substrate with high precision in space and time is important to form a well-defined biofilm. Here, we present a method to engineer bacteria such that they adhere specifically to substrates under blue light through the photoswitchable proteins nMag and pMag. This provides exquisite spatiotemporal remote control over these interactions. The engineered bacteria express pMag protein on the surface so that they can adhere to substrates with nMag protein immobilization under blue light, and reversibly detach in the dark. This process can be repeatedly turned on and off. In addition, the bacterial adhesion property can be adjusted by expressing different pMag proteins on the bacterial surface and altering light intensity. This protocol provides light switchable, reversible and tunable control of bacteria adhesion with high spatial and temporal resolution, which enables us to pattern bacteria on substrates with great flexibility.

Keywords: Bacterial adhesion (细菌粘附), Optogenetics (光遗传学), nMag-pMag (nMag-pMag), Photoswitching (光开关), Biofilm (生物膜)

Background

Controlling the biofilm formation is crucial to understand the social interactions between bacteria in naturally occurring biofilm (Flemming et al., 2016). This is also particularly important for the biotechnological application of biofilms in biocatalysis, biosensing and waste treatment (Zhou et al., 2013; Jensen et al., 2016). The biofilm formation always begins with the bacterial adhesion to a substrate, which determines the spatial organization in biofilms (Liu et al., 2016; Nadell et al., 2016). Many strategies have been proposed to control bacterial adhesion such as modifying bacterial surface with bio-orthogonal reactive groups via liposome fusion (Elahipanah et al., 2016), immobilization of adhesive molecules on the substrates (Sankaran et al., 2015; Zhang et al., 2016; Peschke et al., 2017) and conjugating surface tags on bacteria (Poortinga et al., 2000; Rozhok et al., 2005; Lui et al., 2013). Among these, the light responsive approaches provide the highest spatiotemporal control, which is important to precisely control the fine structure of the biofilms. For instance, azobenzene linkers have been used as a photoswitchable tool to reversibly control the bacterial adhesion to substrates by altering the presentation of mannose, which is recognized by the bacterial surface receptor FimH (Voskuhl et al., 2014; Weber et al., 2014; Sankaran et al., 2015). In addition, azobenzene-based molecules have also been used to control bacteria adhesion to mammalian cells (Mockl et al., 2016), bacterial quorum sensing (Van der Berg et al., 2015) and biofilm formation (Hu et al., 2016) with UV-light. One of the major drawbacks of using UV-light is that it is toxic to bacteria. In this protocol, we present a new approach of how to control bacterial adhesion to substrates with blue light based on photoswitchable proteins. Besides being a non-invasive, reversible and tuneable technique to control bacterial adhesion to substrates, it also provides high spatiotemporal control required to form well-defined biofilms. Photoswitchable proteins are commonly used in the field of optogenetics to regulate gene expression, receptor activation and protein localization in cells with visible light (Müller and Weber, 2013; Tischer and Weiner, 2014). These optogenetic systems are very sensitive to visible light, bioorthogonal and noninvasive. Furthermore, these proteins are genetically encoded so they can be sustainably expressed in the cell. Here, we used the blue light responsive proteins, nMag and pMag, as photoswitches control bacterial adhesion. These proteins heterodimerize under blue light (480 nm) and dissociate from each other in the dark (Kawano et al., 2015). The strength and back conversion kinetics of the nMag and pMag interaction are different for the point mutants. The point mutant pMagHigh (and nMagHigh) has a stronger interaction with its binding partners and slower back conversion, while the opposite is true for the mutant pMagFast1 (and nMagFast1) (Zoltowski et al., 2009).

In our method we display the first interaction partner of the photoswitchable proteins, pMagHigh, pMag or pMagFast1 on the surface of E. coli using the circularly permutated OmpX (outer membrane protein X) protein (Daugherty, 2007). The pMag variants are attached through their C-terminal to the OmpX protein. The second interaction partner the photoswitchable protein, nMagHigh, is immobilized through a His6-tag at its C-terminal on a glass substrate with a PEG (polyethylene glycol) coating, which contains a Ni2+-NTA group (Schenk et al., 2014). This setup allows bacteria expressing pMag proteins on their surfaces to adhere to nMagHigh functionalized substrates under blue light when the two proteins interact but not in the dark. (Figure 1)


Figure 1. The engineered E. coli that express pMag proteins on their surface adhere to nMagHigh modified substrates under blue light. In the dark, the pMag-nMag interaction is reversed, which leads to the detachment of the bacteria from the substrate. Reproduced with permission from Chen and Wegner (2017).

Materials and Reagents

  1. Pipette tips (STARLAB, catalog number: S1111-6700 )
  2. 50 ml Falcon tube (Greiner Bio One International, catalog number: 227261 )
  3. 1.5 ml Eppendorf tube (Eppendorf, catalog number: 0030120086 )
  4. 0.2 ml PCR tubes (Thermo Fisher Scientific, catalog number: AB0620 )
  5. 0.45 µm cellulose filter (Carl Roth, catalog number: KH55.1 )
  6. Ni-NTA column (GE Healthcare, catalog number: 17524801 )
  7. 50 ml syringe (VWR, catalog number: 53548-010 )
    Manufacturer: Air-Tite Products, catalog number: 4850001000 .
  8. 20 x 20 mm glass slides (VWR, catalog number: 631-0122 )
    Notes: The glass slide is used as the glass surface for the protein functionalization and bacterial experiments. 
  9. Parafilm (Sigma-Aldrich, Bemis, catalog number: P7668 )
  10. Aluminum foil (Carl Roth, catalog number: 1770.1 )
  11. 35 mm Petri dish (SARSTEDT, catalog number: 82.1135.500 )
  12. Dialysis tubing (Repligen, Spectrum, catalog number: 132592 )
  13. Plasmid pB33eCPX (Addgene plasmid) (Addgene, catalog number: 23336 )
  14. GFP and mCherry pTrc99A plasmids (Prof. Victor Sourjik lab, Chen and Wegner, 2017)
  15. nMagHigh pET-21b(+) plasmid (Genescript, Chen and Wegner, 2017)
  16. nMagHigh-eCPX plasmid (homemade, Chen and Wegner, 2017)
    Note: The nMagHigh gene is inserted between the KpnI and SacI cutting sites of pB33eCPX. 
  17. pMag-eCPX, pMagHigh-eCPX, pMagFast1-eCPX plasmids (homemade, Chen and Wegner, 2017)
    Note: The different pMag variants are generated by point mutagenesis from the nMagHigh-eCPX plasmid using QuikChange II Site-Directed Mutagenesis Kit.
  18. E. coli K12 MG1655 (DSMZ, catalog number: 18039 )
  19. BL21(DE3) competent E. coli (homemade, Chen and Wegner, 2017)
  20. DH5α competent E. coli (homemade, Chen and Wegner, 2017)
  21. PBS Tablets (Thermo Fisher Scientific, GibcoTM, catalog number: 18912014 )
  22. Mowiol (Sigma-Aldrich, catalog number: 81381 )
  23. LB medium (Carl Roth, catalog number: X968.3 )
  24. Ampicillin (Carl Roth, catalog number: HP62.2 )
  25. IPTG (Sigma-Aldrich, catalog number: I6758 )
  26. PMSF (Sigma-Aldrich, catalog number: P7626 )
  27. DTT (Sigma-Aldrich, catalog number: D0632-10G )
  28. PEG-azide (homemade)
  29. Triethylamine (Sigma-Aldrich, catalog number: T0886 )
  30. Toluene, anhydrous (Alfa Aesar, catalog number: 41464-AK )
  31. EDTA (Sigma-Aldrich, catalog number: 798681 )
  32. NiCl2 (Sigma-Aldrich, catalog number: 339350 )
  33. Chloramphenicol (Sigma-Aldrich, catalog number: C0378 )
  34. L-arabinose (Sigma-Aldrich, catalog number: A3256 )
  35. Paraformaldehyde, reagent grade (Sigma-Aldrich, catalog number: P6148 )
  36. Tris Base (Sigma-Aldrich, catalog number: T1503 )
  37. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  38. Imidazole (Sigma-Aldrich, catalog number: I2399 )
  39. L-Ascorbic acid (Sigma-Aldrich, catalog number: A5960 )
  40. NTA-alkyne (homemade, Schenk et al., 2014)
  41. PEG azide (homemade, Schenk et al., 2014)
  42. Copper sulfate (CuSO4) (Sigma-Aldrich, catalog number: 451657 )
  43. 30% H2O2 (Carl Roth, catalog number: 8070.4 )
  44. H2SO4 (Sigma-Aldrich, catalog number: 30743)
  45. TEM (Transmission electron microscopy) grid (Ted Pella, catalog number: 1GC150 )
  46. Centrifuge tubes 500 ml and 50 ml (Thermo Fisher Scientific, catalog numbers: 3141-0500 , 3119-0050 )
  47. Methanol (Fisher Scientific, catalog number: 10224490 )
  48. N2 gas (Westfalen)
  49. Ethyl acetate (VWR, catalog number: 23882.321 )
  50. Picodent twinsil 22 (Picodent, catalog number: 13001000 )
  51. Buffer A (see Recipes)
  52. Buffer B (see Recipes)
  53. Click reaction solution (see Recipes)
  54. Riranha solution (see Recipes)

Equipment

  1. Dumont #7 Tweezers (Carl Roth, catalog number: K344.1 )
    Note: Dumont #7 Tweezers is used to pick up the glass slides.
  2. 0.1-2.5 μl, 0.5-10 μl, 10-100 μl, 100-1,000 μl Pipettes (Eppendorf, catalog numbers: 3123000012 , 3123000020 , 3123000047 , 3123000063 )
  3. Vortexer (neoLab, catalog number: 7-0092 )
  4. Microcentrifuge (VWR, model: Micro Star 17, catalog number: 521-1646 )
  5. High-speed centrifuge (Beckman Coulter, model: Avanti® J-26S )
  6. Rotors for high-speed centrifuge (Beckman Coulter, models: JA-10 , JA-25.50 )
  7. Incubator (VWR, catalog number: 444-0732 )
  8. Sonicator (OMNI, model: Sonic Ruptor 400 )
  9. Invert fluorescence microscope (Leica Microsystems, model: Leica DMi8 )
  10. Ultrasonic cleaner (BANDELIN electronic, model: Sonorex Super RK 31 )
  11. Blue LED panel (Albrillo, model: LL-GL003 )
  12. OD Meter (Biochrom, BioWave, model: WPA CO8000 )
  13. Nanodrop (Thermo Fisher Scientific, model: NanoDropTM 8000

Software

  1. ImageJ
  2. Originlab
    Note: Oringinlab is used for data analysis.

Procedure

  1. nMagHigh Protein Expression and purification
    1. Transform nMagHigh with a C-terminal His6-tag in a pET-21b(+) expression plasmid into BL21(DE3) E. coli cells using a standard protocol.
    2. Prepare a starter culture from a single colony in 10 ml LB with 50 μg/ml ampicillin. Incubate overnight at 37 °C at 250 rpm.
    3. Transfer 10 ml overnight culture into 1 L LB with 50 μg/ml ampicillin and grown at 37 °C at 200 rpm till OD600 = 0.6-0.8. When the desired OD600 is reached, add 500 µl IPTG (1 M stock, 500 µM final concentration) to the culture and incubate overnight at 16 °C at 250 rpm.
    4. Pellet bacteria at 6,371 x g at 4 °C for 8 min using the high-speed centrifuge with the JA-10 rotor (500 ml centrifuge tube). Discard the supernatant. All the following steps must be done on ice.
    5. Resuspend the pellet in 20 ml Buffer A (Recipe 1) containing supplemented with 1 mM PMSF (100 mM stock in methanol) and 1 mM DTT (1 M stock in water). Transfer the suspension into a 50 ml Falcon tube.
    6. Lyse bacteria by sonication. Settings for tip sonicator: 50% frequency, 40% power, 10 min. Keep the Falcon on ice during sonication. Transfer lysate into plastic high-speed centrifuge tubes, Centrifuge at 17,418 x g, 4 °C for 30 min using the JA-25.50 rotor (50 ml centrifuge tube).
    7. Filter the supernatant through 0.45 µm cellulose filter twice.
    8. Equilibrate Ni-NTA column (column volume = 5 ml) with 20-30 ml Buffer A + 1 mM DTT.
    9. Load clarified cell lysate on to the Ni-NTA column by passing the lysate twice dropwise using a 50 ml syringe.
    10. Wash Ni-NTA column with 10 ml Buffer A + 1 mM DTT and 50 ml (47.5 ml Buffer A + 2.5 ml Buffer B + 1 mM DTT) (Recipe 2).
    11. Elute with 10 ml Buffer B + 1 mM DTT. Discard the first 3 ml and collect the last 7 ml which contain the protein.
    12. Wash the column once more with Buffer B (50 ml, without DTT) and then Buffer A (50 ml, without DTT). Store Ni-NTA column in the fridge.
    13. Put the protein solution in dialysis bags (MW cut-off = 3,500 Da) and dialyze in 2 L Buffer A with stirring at 4 °C for 3 h. Replace the Buffer A with a fresh one and dialyze another 3 h at 4 °C. Collect the protein solution and store in -80 °C. Protein concentration is measured by Nanodrop.

  2. Functionalization of glass surfaces with nMagHigh
    1. Label the upper right corner of glass slides (20 x 20 mm) to distinguish the upper side. Clean glass surfaces in freshly prepared Piranha solution for 1 h, rinse three times with Milli-Q water and dry in an N2 stream.
    2. For the PEGylation reaction, immerse surfaces in a solution of PEG-azide (10 mg PEG-azide, MW = 3,500 g/mol) and a drop of triethylamine in 50 ml dry toluene and kept at 79 °C overnight under an N2 atmosphere in a closed container. Note that surfaces should not be touching each other.
    3. Wash the surfaces first with ethyl acetate for 5 min by sonication (Ultrasonic frequency: 35 kHz), then with methanol for 5 min by sonication and dry in an N2 stream.
    4. Prepare the humid chambers for click reaction by putting wet tissue paper in the lid of a Petri dish and cover it with the clean side of the parafilm.
    5. Prepare the click reaction solution (for six surfaces) (Recipe 3).
    6. Put 100 µl of click reaction solution on the parafilm in the Petri dish.
    7. Place one glass slide upside down on each the click reaction solution droplets (marked surface should be in contact with the click reaction solution). Incubate at room temperature for 2 h.
    8. After click reaction, wash the glass slides with 1) 50 mM EDTA in Buffer A for 5 min to remove Cu2+, and 2) Buffer A twice for 5 min.
    9. Incubate the slides with 100 µl 100 mM of NiCl2 on a parafilm at room temperature for 5 min. Ni2+ complexes to the NTA groups on the surface for His-tag binding. Wash the slides twice with Buffer A to remove excess Ni2+.
    10. Place 100 µl nMagHigh protein solution (10 µM, in Buffer A) droplets on a parafilm and put the glass slides upside down on the droplets. Incubate at room temperature for 30 min.
      Note: The glass surfaces not incubated with nMagHigh protein will be used as negative control in D.
    11. Wash the slides twice with sterile PBS and keep them in the PBS before use.
      Note: The nMagHigh functionalized glass slides should be freshly prepared for the bacterial experiments. 

  3. Bacteria preparation
    1. Co-transform E. coli K12 MG1655 with the pMagHigh-eCPX or pMag-eCPX or pMagFast1-eCPX plasmid (chloramphenicol resistant) and the GFP pTrc99A plasmid (ampicillin resistant) and selected on an LB-agar plate with 35 μg/ml chloramphenicol and 50 μg/ml ampicillin.
    2. Inoculate a single colony into 5 ml LB medium containing 35 μg/ml chloramphenicol and 50 μg/ml ampicillin and incubate overnight at 37 °C at 250 rpm. Wrap all the tubes in aluminum foil.
    3. Add 500 μl of the overnight cultures into fresh 20 ml LB medium containing 35 μg/ml chloramphenicol and 50 μg/ml ampicillin and culture for 2 h at 37 °C, 250 rpm. Wrap all the tubes in aluminum foil.
    4. When the OD600 = 0.5, add 0.04% m/v L-arabinose to induce the expression of the pMag-eCPX proteins and 0.5 mM IPTG to induce the production of the fluorescent protein and incubate the cultures at 25 °C, 250 rpm for 4 h.
    5. Spin down the bacteria at 1,500 x g for 10 min and wash with PBS twice. Finally, re-suspend the bacteria in PBS to OD600 = 1.0.

  4. Bacterial adhesion assays
    1. Use glass surfaces functionalized with nMagHigh protein by the method mentioned above and keep them in PBS in the dark. Use a surface that was not incubated with nMagHigh protein as a negative control to assure that the functionalization with the PEG worked.
    2. Place each glass surface in a 35 mm Petri dish. Add 3 ml of bacterial solution (the bacteria express pMagHigh or pMag or pMagFast1) with OD600 = 1.0 in PBS to each Petri dish.
    3. Place one set of surfaces with bacteria under blue light illumination (blue LED panel, 640 µW/cm2) for 1 h at room temperature. Place the second set of surface in the dark for 1 h.
    4. Wash the surfaces gently three times with PBS. Fix the bacteria with 4% paraformaldehyde for 20 min and mount them with Mowiol. Acquire nine fluorescent images in the GFP channel for each surface on the inverted fluorescence microscope (DMi8, Leica) through a 20x objective. (Figure 2)
    5. Analyze the numbers of bacteria on the surfaces using the particle analyzer tool in ImageJ.


      Figure 2. Fluorescence images of E. coli displaying pMagHigh which adhere on nMagHigh functionalized substrates under blue light but not in the dark. The bacteria are labeled with GFP for detection. Reproduced with permission from Chen and Wegner (2017).

  5. Bacterial attachment kinetics
    1. Functionalize five Glass surfaces with nMagHigh protein by the method mentioned above.
    2. Place each glass surface in a 35 mm Petri dish. Add 3 ml of pMagHigh-eCPX bacteria in PBS (OD600 = 1.0) to each Petri dish.
    3. Incubate the surfaces under blue light illumination (blue LED panel, 640 µW/cm2) for different time (10, 30, 60, 120 to 180 min) at room temperature.
    4. Wash each surface gently three times with PBS. Fix the bacteria with 4% paraformaldehyde and mount them with Mowiol. Acquire nine fluorescent images in the GFP channel (Ex = 488 nm, Em = 510 nm) for each surface on the inverted fluorescence microscope (DMi8, Leica) through a 20x objective.
    5. Analyze the numbers of bacteria on the surfaces using the particle analyzer tool in ImageJ.
    6. Quantify the attachment kinetics of the pMag-eCPX and pMagFast1-eCPX bacteria the same way following steps from E1 to E5 with corresponding bacteria.

  6. Bacterial attachment adjusted by blue light illumination intensity
    1. Use six glass surfaces functionalized with nMagHigh protein prepared as mentioned above.
    2. Place each glass surface in a 35 mm Petri dish. Add 3 ml of pMagHigh-eCPX bacteria in PBS (OD600 = 1.0) to each Petri dish.
    3. Incubate the surfaces under blue light illumination with different intensity (0, 3.2, 32, 320, 640 or 3,200 µW/cm2) for 1 h at room temperature.
    4. Wash the surfaces gently three times with PBS. Fix the bacteria with 4% paraformaldehyde and mount them with Mowiol. Acquire nine fluorescent images in the GFP channel for each surface on an inverted fluorescence microscope (DMi8, Leica) through a 20x objective. Analyze the number of bacteria on the surfaces using the particle analyzer tool in ImageJ.

  7. Bacterial detachment kinetics
    1. Use six glass surfaces functionalized with nMagHigh protein prepared by the method mentioned above.
    2. Place each glass surface in a 35 mm Petri dish. Add 3 ml of pMagHigh-eCPX bacteria in PBS (OD600 = 1.0) to each Petri dish and incubate under blue light illumination (blue LED panel, 640 µW/cm2) for 1 h at room temperature.
    3. Move the Petri dishes to the dark for 0, 10, 30, 60, 120 or 240 min, respectively.
    4. Wash each surface gently three times with PBS. Fix the bacteria with 4% paraformaldehyde and mount them with Mowiol. Acquire nine fluorescent images in the GFP channel for each surface on an inverted fluorescence microscope (DMi8, Leica) through a 20x objective. Analyze the number of bacteria on the surfaces using the particle analyzer tool in ImageJ.

  8. Multiple attachment and detachment cycles
    1. Use five glass surfaces functionalized with nMagHigh protein prepared by the method mentioned above.
    2. Place each glass surface in a 35 mm Petri dish. Add 3 ml of pMagHigh-eCPX bacteria in PBS (OD600 = 1.0) to each Petri dish and incubate under blue light illumination (blue LED panel, 640 µW/cm2) for 1 h attachment at room temperature.
    3. Wash the first surface gently three times with PBS. Fix the bacteria with 4% paraformaldehyde and mount them with Mowiol. Acquire images imaged as described above.
    4. Meanwhile, keep the other four surfaces in the dark for 2 h for bacterial detachment.
    5. Wash the second surface gently three times with PBS and fix the bacteria with 4% paraformaldehyde.
    6. Meanwhile, place the remaining three surfaces under blue light (blue LED panel, 640 µW/cm2) for 1 h for attachment.
    7. Repeat the Steps H3 to H6 for another cycle. For each surface, acquire nine fluorescent images in the GFP channel on an inverted fluorescence microscope (DMi8, Leica) through a 20x objective. Analyze the number of bacteria on the surfaces using the particle analyzer tool in ImageJ.

  9. Bacterial patterning
    1. Co-transform E. coli K12 MG1655 with pMagHigh-eCPX (chloramphenicol resistant) and mCherry pTrc99A plasmid (ampicillin resistant) plasmids. Prepare bacteria as described above.
    2. The glass surface with nMagHigh protein immobilization is stuck to the bottom of a 35 mm Petri dish (with a round hole in 15 mm diameter). 3 ml pMagHigh-eCPX bacteria solution with OD600 = 1.0 is added into the Petri dish and kept in the dark.
      Note: Picodent twinsil 22 is used as a glue to stick the glass surface to the Petri dish bottom.
    3. The glass surface is locally illuminated with blue light through photomask (TEM grid without support film) for 1 h on an inverted fluorescence microscope (DMi8, Leica) through a 10x objective.
    4. The photomask is removed and the glass surface is gently washed by removing 2 ml solution and adding 2 ml PBS into the Petri dish. Repeat the washing steps for three times. Don’t let the surface dry.
    5. The surface is imaged by the inverted fluorescence microscope (DMi8, Leica) through a 10x objective. (Figure 3)


      Figure 3. Photopatterning of pMagHigh displaying bacteria to nMagHigh functionalized glass surfaces. A) Bright field and B) fluorescent images of bacteria patterns. The bacteria are labeled with mCherry for detection. Reproduced with permission from Chen and Wegner (2017).

Data analysis

Data information can be access via https://pubs.acs.org/doi/abs/10.1021/acssynbio.7b00197.

Notes

  1. All the experiments are performed in the dark room. 
  2. Bacteria are cultured in the dark to avoid the activation of the photosensitive protein.

Recipes

  1. Buffer A (1x)
    50 mM Tris-HCl, pH 7.4
    300 mM NaCl (17.5 g for 1 L buffer)
  2. Buffer B (1x)
    Buffer A
    250 mM Imidazole (17 g for 1 L buffer)
  3. Click reaction solution (for six surfaces)
    473 µl MilliQ water
    60 µl 1 M Tris-HCl at pH 8.5
    60 µl 1 M L-Ascorbic acid (prepare fresh: 102 mg L-Ascorbic acid in 741 µl MilliQ water)
    0.9 µl 25 mM NTA-alkyne
    6 µl 100 mM CuSO4 (should be added last; when added, locally a brownish color appears. After mixing, the solution becomes colorless)
    Mix the solution by vortexing
  4. Riranha solution
    3:1 (v/v) conc. H2SO4:H2O2 (30%)
    20 ml H2O2 (30%)
    60 ml H2SO4

Acknowledgments

This work is part of the MaxSynBio consortium, which is jointly funded by the Federal Ministry of Education and Research (BMBF) of Germany and the Max Planck Society (FKZ 031A359L). F.C. would like to thank the Chinese Scholarship Council for a doctoral fellowship. The GFP and mCherry plasmids were a gift from Prof. Victor Sourjik and the CPX plasmid was a gift from Prof. Patrick Daugherty (Addgene plasmid # 23336). Our thanks go to Stefan Schumacher for his help with the figures. This protocol is adapted from previous work (Chen and Wegner, 2017). We would like to thank the editor of ACS Synthetic Biology for the permission to reprint the figures.

Competing interests

There are no conflicts of interest.

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简介

在空间和时间上高精度地控制细菌粘附到基底对于形成明确的生物膜是重要的。 在这里,我们提出了一种方法来设计细菌,使其在蓝光下通过光可切换蛋白质nMag和pMag特异性地粘附在基底上。 这为这些交互提供了精妙的时空遥控。 工程菌在表面上表达pMag蛋白,以便它们可以在蓝光下与nMag蛋白固定化的基质粘附,并在黑暗中可逆地分离。 该过程可以重复开启和关闭。 此外,通过在细菌表面表达不同的pMag蛋白质并改变光强度可以调节细菌粘附性质。 该协议提供了可高度空间和时间分辨率的细菌粘附的光可切换,可逆和可调控制,这使我们能够以极大的灵活性在基底上图案化细菌。

【背景】控制生物膜形成对于了解细菌在自然发生的生物膜中的社会相互作用至关重要(Flemming et。,2016)。这对生物膜在生物催化,生物传感和废物处理中的生物技术应用也特别重要(Zhou等人,2013; Jensen等人,2016)。生物膜的形成始终始于细菌与底物的粘附,这决定了生物膜中的空间组织(Liu等人,2016; Nadell等人,2016)。已经提出了许多策略来控制细菌粘附,例如通过脂质体融合利用生物正交反应基团修饰细菌表面(Elahipanah等,2016),将粘附分子固定在基质上(Sankaran等,等),2015; Zhang等人,2016; Peschke等人,2017)和缀合细菌上的表面标签(Poortinga et al。,2015;等,2000; Rozhok等人,2005; Lui等人,2013)。其中,光响应方法提供了最高的时空控制,这对精确控制生物膜的精细结构非常重要。例如,偶氮苯连接物已被用作可光切换的工具,以通过改变被细菌表面受体FimH识别的甘露糖的呈递来可逆地控制细菌对底物的粘附(Voskuhl等人,2014年; Weber et al。,2014; Sankaran等人,,2015)。另外,基于偶氮苯的分子也被用于控制细菌对哺乳动物细胞的粘附(Mockl等人,2016),细菌群体感应(Van der Berg等人 >,2015)和生物膜形成(Hu等人,2016)。使用紫外线的主要缺点之一是对细菌有毒。在这个协议中,我们提出了一种新的方法,如何控制基于光可切换蛋白质的蓝光对基底的细菌粘附。除了是一种非侵入性,可逆和可调节的技术来控制细菌对底物的粘附,它还提供了形成明确定义的生物膜所需的高时空控制。光可切换蛋白通常用于光遗传学领域,以调节可见光细胞中的基因表达,受体激活和蛋白质定位(Müller和Weber,2013; Tischer和Weiner,2014)。这些光遗传学系统对可见光,生物正交和非侵入性非常敏感。此外,这些蛋白质是遗传编码的,因此它们可以在细胞中持续表达。在这里,我们使用蓝光响应蛋白nMag和pMag作为光控开关来控制细菌粘附。这些蛋白质在蓝光下(480nm)异源二聚化并在黑暗中彼此分离(Kawano等人,2015)。对于突变体,nMag和pMag相互作用的强度和回转动力学是不同的。点突变体pMagHigh(和nMagHigh)与其结合伴侣有更强的相互作用,反向转换更慢,而突变体pMagFast1(和nMagFast1)则相反(Zoltowski等人,2009年)。

在我们的方法中,我们在E表面上显示光可切换蛋白质的第一个相互作用伙伴pMagHigh,pMag或pMagFast1。使用循环置换的OmpX(外膜蛋白X)蛋白(Daugherty,2007)。 pMag变体通过它们的C端连接到OmpX蛋白上。第二个相互作用配偶体光可切换蛋白质nMagHigh通过His6-标签固定在玻璃基底上的C-末端上,其具有PEG(聚乙二醇)涂层,其包含Ni2 + -NTA (Schenk et al。 ,2014)。当两种蛋白质相互作用但不在黑暗中时,该设置允许细菌在其表面上表达pMag蛋白质在蓝光下粘附于nMagHigh官能化底物。 (图1)



图1.设计的 E。大肠杆菌表达pMag蛋白的表面在蓝光下与nMagHigh修饰的底物粘附。在黑暗中,pMag-nMag相互作用被逆转,导致细菌从底物分离。经Chen和Wegner(2017)许可转载。

关键字:细菌粘附, 光遗传学, nMag-pMag, 光开关, 生物膜

材料和试剂

  1. 移液器吸头(STARLAB,目录号:S1111-6700)

  2. 50 ml Falcon管(Greiner Bio One International,目录号:227261)

  3. 1.5 ml Eppendorf管(Eppendorf,产品目录号:0030120086)
  4. 0.2ml PCR管(Thermo Fisher Scientific,目录号:AB0620)
  5. 0.45μm纤维素过滤器(Carl Roth,目录号:KH55.1)
  6. Ni-NTA柱(GE Healthcare,目录号:17524801)
  7. 50毫升注射器(VWR,目录号:53548-010)
    制造商:Air-Tite Products,产品目录号:4850001000。
  8. 20 x 20 mm玻璃载玻片(VWR,目录号:631-0122)
    注意:载玻片用作蛋白质功能化和细菌实验的玻璃表面。  
  9. Parafilm(Sigma-Aldrich,Bemis,目录号:P76 68)
  10. 铝箔(卡尔罗斯,目录号:1770.1)
  11. 35毫米培养皿(SARSTEDT,目录号:82.1135.500)
  12. 透析管(Repligen,Spectrum,目录号:132592)
  13. 质粒pB33eCPX(Addgene质粒)(Addgene,目录号:23336)
  14. GFP和mCherry pTrc99A质粒(Victor Sourjik实验室,Chen和Wegner,2017)
  15. nMag高pET-21b(+)质粒(Genescript,Chen和Wegner,2017)
  16. nMagHigh-eCPX质粒(国产,Chen和Wegner,2017)
    注意:nMagHigh基因插入到pB33eCPX的KpnI和SacI切割位点之间。 
  17. pMag-eCPX,pMagHigh-eCPX,pMagFast1-eCPX质粒(国产,Chen和Wegner,2017)
    注意:使用QuikChange II定点诱变试剂盒通过nMagHigh-eCPX质粒的点突变产生不同的pMag变体。
  18. 电子。大肠杆菌K12 MG1655(DSMZ,目录号:18039)
  19. BL21(DE3)胜任 E。 (自制的,陈和Wegner,2017)
  20. DH5α感受态E. (自制的,陈和Wegner,2017)
  21. PBS片剂(Thermo Fisher Scientific,Gibco TM,目录号:18912014)
  22. Mowiol(Sigma-Aldrich,目录号:81381)
  23. LB培养基(Carl Roth,目录号:X968.3)
  24. 氨苄西林(Carl Roth,目录号:HP62.2)
  25. IPTG(Sigma-Aldrich,目录号:I6758)
  26. PMSF(Sigma-Aldrich,目录号:P7626)
  27. DTT(Sigma-Aldrich,目录号:D0632-10G)
  28. PEG-叠氮化物(自制)
  29. 三乙胺(Sigma-Aldrich,目录号:T0886)
  30. 无水甲苯(Alfa Aesar,目录号:41464-AK)
  31. EDTA(Sigma-Aldrich,目录号:798681)
  32. NiCl 2(Sigma-Aldrich,目录号:339350)
  33. 氯霉素(Sigma-Aldrich,目录号:C0378)
  34. L-阿拉伯糖(Sigma-Aldrich,目录号:A3256)
  35. 多聚甲醛,试剂级(Sigma-Aldrich,目录号:P6148)
  36. Tris碱(Sigma-Aldrich,目录号:T1503)
  37. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  38. 咪唑(Sigma-Aldrich,目录号:I2399)
  39. L-抗坏血酸(Sigma-Aldrich,目录号:A5960)
  40. NTA-炔(国产,Schenk等人,2014)
  41. PEG叠氮化物(自制,Schenk等人,2014)
  42. 硫酸铜(CuSO 4)(Sigma-Aldrich,目录号:451657)
  43. 30%H 2 O 2(Carl Roth,目录号:8070.4)
  44. H 2 SO 4(Sigma-Aldrich,目录号:30743)
  45. TEM(透射电子显微镜)网格(Ted Pella,目录号:1GC150)
  46. 离心管500ml和50ml(Thermo Fisher Scientific,目录号:3141-0500,3119-0050)
  47. 甲醇(Fisher Scientific,目录号:10224490)
  48. N 2气(Westfalen)
  49. 乙酸乙酯(VWR,目录号:23882.321)
  50. Picodent twinsil 22(Picodent,目录号:13001000)
  51. 缓冲区A(见食谱)
  52. 缓冲区B(见食谱)
  53. 点击反应解决方案(请参阅食谱)
  54. Riranha解决方案(请参阅食谱)

设备

  1. 杜蒙#7镊子(卡尔罗斯,目录号:K344.1)
    注:Dumont#7镊子用于拿起载玻片。
  2. 0.1-2.5μl,0.5-10μl,10-100μl,100-1,000μl移液器(Eppendorf,目录号:3123000012,31200000020,3123000047,3123000063)
  3. 涡旋(neoLab,目录号:7-0092)
  4. 微量离心机(VWR,型号:Micro Star 17,目录号:521-1646)
  5. 高速离心机(Beckman Coulter,型号:Avanti J-26S)
  6. 用于高速离心机的转子(Beckman Coulter,型号:JA-10,JA-25.50)
  7. 孵化器(VWR,目录号:444-0732)
  8. Sonicator(OMNI,型号:Sonic Ruptor 400)
  9. 倒置荧光显微镜(Leica Microsystems,型号:Leica DMi8)
  10. 超声波清洗器(BANDELIN electronic,型号:Sonorex Super RK 31)
  11. 蓝色LED面板(Albrillo,型号:LL-GL003)
  12. OD仪(Biochrom,BioWave,型号:WPA CO8000)
  13. Nanodrop(Thermo Fisher Scientific,型号:NanoDrop TM 8000) 

软件

  1. ImageJ
  2. Originlab
    注意:Oringinlab用于数据分析。

程序

  1. nMag高蛋白表达和纯化
    1. 用pET-21b(+)表达质粒中的C端His6标签将nMagHigh转化到BL21(DE3)E中。使用标准方案培养大肠杆菌细胞。
    2. 从含有50μg/ ml氨苄青霉素的10ml LB中的单个菌落中制备发酵剂培养物。在37℃以250转/分孵育过夜。
    3. 将10ml过夜培养物转移至含有50μg/ ml氨苄青霉素的1L LB中并在37℃以200rpm生长至OD 600 = 0.6-0.8。当达到所需的OD 600时,向培养物中加入500μlIPTG(1M储备液,500μM终浓度)并在16℃以250rpm孵育过夜。
    4. 使用具有JA-10转子的高速离心机(500ml离心管)在4℃下6,371×gg的球团细菌8分钟。丢弃上清液。以下所有步骤都必须在冰上完成。
    5. 在含有补充1mM PMSF(100mM储液的甲醇溶液)和1mM DTT(1M水溶液)的20ml缓冲液A(配方1)中重悬沉淀。将悬浮液转移到50毫升Falcon管中。
    6. 通过超声波溶解细菌。 tip sonicator的设置:50%频率,40%功率,10分钟。超声处理过程中保持猎鹰冰。将溶胞产物转移到塑料高速离心管中,使用JA-25.50转子(50ml离心管),在17,418×g,4℃下离心30分钟。
    7. 通过0.45μm纤维素过滤器过滤两次上清液。
    8. 用20-30ml缓冲液A + 1mM DTT平衡Ni-NTA柱(柱体积= 5ml)。
    9. 通过使用50ml注射器使裂解物两次滴加将澄清的细胞裂解物加载到Ni-NTA柱上。
    10. 用10ml缓冲液A + 1mM DTT和50ml(47.5ml缓冲液A + 2.5ml缓冲液B + 1mM DTT)洗涤Ni-NTA柱(配方2)。
    11. 用10ml缓冲液B + 1mM DTT洗脱。丢弃前3毫升,收集最后7毫升含有蛋白质。
    12. 用缓冲液B(50毫升,不含DTT)再用缓冲液A(50毫升,不含DTT)再次清洗柱子。将Ni-NTA色谱柱存放在冰箱中。
    13. 将蛋白质溶液放入透析袋(截留分子量= 3,500Da)中,并在2L缓冲液A中在4℃搅拌3小时进行透析。用新鲜的缓冲液A替换并在4°C透析另外3小时。收集蛋白质溶液并储存在-80°C。通过Nanodrop测量蛋白质浓度。

  2. 用nMagHigh功能化玻璃表面
    1. 标记载玻片的右上角(20 x 20 mm)以区分上侧。在新鲜配制的食人鱼溶液中清洁玻璃表面1小时,用Milli-Q水冲洗三次并在N 2流中干燥。
    2. 对于PEG化反应,将表面浸入PEG-叠氮化物(10mg PEG-叠氮化物,MW = 3,500g / mol)和一滴三乙胺在50ml无水甲苯中的溶液中并在N 下保持在79℃过夜在密闭的容器中> 2个大气。请注意,表面不应该相互接触。
    3. 首先用乙酸乙酯通过声处理(超声波频率:35kHz)清洗表面5分钟,然后通过超声处理用甲醇清洗5分钟并在N 2 / N 2流中干燥。

    4. 准备潮湿的室进行点击反应,将湿纸巾放在培养皿的盖子上,并用封口膜的清洁面覆盖。
    5. 准备点击反应解决方案(六个表面)(配方3)。

    6. 在培养皿中的石蜡膜上放100μl点击反应溶液。
    7. 将一个载玻片颠倒放置在每个点击反应溶液液滴上(标记的表面应与点击反应溶液接触)。在室温下孵育2小时。
    8. 点击反应后,用1)缓冲液A中的50mM EDTA洗涤载玻片5分钟以除去Cu 2 +,2)缓冲液A两次5分钟。
    9. 在室温下在封口膜上用100μl100mM的NiCl 2 2孵育载玻片5分钟。 Ni 2 +与表面上的NTA基团复合以结合His标签。用缓冲液A清洗载玻片两次以除去多余的Ni2 +。
    10. 将100μlnMagHigh蛋白溶液(10μM,在缓冲液A中)滴在封口膜上并将玻璃载玻片倒置在液滴上。在室温下孵育30分钟。
      注:未与nMagHigh蛋白孵育的玻璃表面将在D中用作阴性对照。

    11. 使用无菌PBS冲洗载玻片两次,并将它们保存在PBS中。
      注意:nMagHigh功能化载玻片应该为新的细菌实验做好准备。  

  3. 细菌准备
    1. 共同转换 E。使用pMagHigh-eCPX或pMag-eCPX或pMagFast1-eCPX质粒(氯霉素抗性)和GFP pTrc99A质粒(氨苄青霉素抗性)在大肠杆菌K12 MG1655上进行筛选,并在具有35μg/ ml氯霉素和50μg/ ml氯霉素的LB-琼脂平板上进行选择μg/ ml氨苄青霉素。
    2. 将单菌落接种到含有35μg/ ml氯霉素和50μg/ ml氨苄青霉素的5ml LB培养基中并在37℃以250rpm孵育过夜。
      用铝箔包裹所有的管子
    3. 将500μl过夜培养物添加到含有35μg/ ml氯霉素和50μg/ ml氨苄青霉素的新鲜20ml LB培养基中,并在37℃,250rpm下培养2小时。
      用铝箔包裹所有的管子
    4. 当OD 600 = 0.5时,添加0.04%m / v L-阿拉伯糖以诱导pMag-eCPX蛋白和0.5mM IPTG的表达以诱导荧光蛋白的产生并将培养物在25℃,250rpm,4小时。
    5. 将细菌在1,500×g下旋转10分钟并用PBS洗涤两次。最后,将PBS中的细菌重新悬浮至OD 600 = 1.0。

  4. 细菌粘附分析
    1. 用上述方法使用nMagHigh蛋白质功能化玻璃表面,并将它们保存在黑暗中的PBS中。使用不与nMagHigh蛋白孵育的表面作为阴性对照以确保PEG的功能化起作用。
    2. 将每个玻璃表面放在35毫米培养皿中。向每个培养皿中加入3ml在PBS中OD 600 = 1.0的细菌溶液(细菌表达pMagHigh或pMag或pMagFast1)。
    3. 在室温下,在蓝光照明下(蓝色LED面板,640μW/ cm 2),将一组表面与细菌放置1小时。将第二组表面放置在黑暗中1小时。
    4. 用PBS轻轻洗涤表面三次。用4%多聚甲醛固定细菌20分钟,并将它们装入Mowiol。通过20倍物镜在倒置荧光显微镜(DMi8,Leica)上的每个表面上获取GFP通道中的9个荧光图像。 (图2)

    5. 使用ImageJ中的粒子分析器工具分析表面上的细菌数量。


    图2.荧光图像 E。在蓝光下但不在黑暗中显示粘附在nMagHigh官能化底物上的pMagHigh。细菌用GFP标记以用于检测。经Chen和Wegner(2017)许可转载。

  5. 细菌附着动力学
    1. 用上述方法使nMagHigh蛋白质功能化五个玻璃表面。
    2. 将每个玻璃表面放在35毫米培养皿中。向每个培养皿中加入3ml pMagHigh-eCPX细菌的PBS(OD 600 = 1.0)。
    3. 在室温下在蓝光照明下(蓝色LED面板,640μW/ cm 2 2 )孵育不同时间的表面(10,30,60,120到180分钟)。
    4. 用PBS轻轻洗涤每个表面三次。用4%多聚甲醛固定细菌并将它们装入Mowiol。通过倒置荧光显微镜(DMi8,Leica)上的每个表面获得GFP通道中的9个荧光图像(E <=> = 488nm,Emin = 510nm) 20倍的目标。

    5. 使用ImageJ中的粒子分析器工具分析表面上的细菌数量。
    6. 使用相应的细菌,按照从E1到E5的步骤,以相同的方式量化pMag-eCPX和pMagFast1-eCPX细菌的附着动力学。

  6. 通过蓝光照明强度调节细菌附着
    1. 使用如上所述制备的用nMagHigh蛋白质功能化的六个玻璃表面。
    2. 将每个玻璃表面放在35毫米培养皿中。向每个培养皿中加入3ml pMagHigh-eCPX细菌的PBS(OD 600 = 1.0)。
    3. 在室温下用不同强度(0,3.2,32,320,640或3,200μW/ cm 2 )在蓝光照明下孵育表面1小时。
    4. 用PBS轻轻洗涤表面三次。用4%多聚甲醛固定细菌并将它们装入Mowiol。通过20倍物镜在倒置荧光显微镜(DMi8,Leica)上获取每个表面的GFP通道中的9个荧光图像。使用ImageJ中的粒子分析器工具分析表面上的细菌数量。

  7. 细菌脱离动力学
    1. 使用由上述方法制备的nMagHigh蛋白质功能化的六个玻璃表面。
    2. 将每个玻璃表面放在35毫米培养皿中。向每个培养皿中加入3ml pMagHigh-eCPX细菌的PBS(OD 600 = 1.0),并在蓝色光照下(蓝色LED板,640μW/ cm 2) )在室温下1小时。

    3. 。将培养皿分别移至0,10,30,60,120或240分钟
    4. 用PBS轻轻洗涤每个表面三次。用4%多聚甲醛固定细菌并将它们装入Mowiol。通过20倍物镜在倒置荧光显微镜(DMi8,Leica)上获取每个表面的GFP通道中的9个荧光图像。使用ImageJ中的粒子分析器工具分析表面上的细菌数量。

  8. 多次附着和分离周期
    1. 使用由上述方法制备的nMagHigh蛋白质功能化的五个玻璃表面。
    2. 将每个玻璃表面放在35毫米培养皿中。向每个培养皿中加入3ml pMagHigh-eCPX细菌的PBS(OD 600 = 1.0),并在蓝色光照下(蓝色LED板,640μW/ cm 2) )在室温下连接1小时。
    3. 用PBS轻轻洗涤第一个表面三次。用4%多聚甲醛固定细菌并将它们装入Mowiol。采集如上所述的图像。
    4. 同时,将其他四个表面在黑暗中保持2小时以进行细菌分离。
    5. 用PBS轻轻洗涤第二个表面三次,然后用4%多聚甲醛固定细菌。
    6. 同时,将其余三个表面置于蓝色光下(蓝色LED面板,640μW/ cm 2 )1小时以进行连接。
    7. 重复步骤H3到H6进行另一个循环。对于每个表面,通过20倍物镜在倒置荧光显微镜(DMi8,Leica)上获取GFP通道中的9个荧光图像。使用ImageJ中的粒子分析器工具分析表面上的细菌数量。

  9. 细菌图案
    1. 共同转换 E。使用pMagHigh-eCPX(氯霉素抗性)和mCherry pTrc99A质粒(氨苄青霉素抗性)质粒转化大肠杆菌K12 MG1655。如上所述准备细菌。
    2. 将具有nMagHigh蛋白质固定的玻璃表面粘贴到35mm培养皿的底部(具有15mm直径的圆孔)。将OD 600 = 1.0的3ml pMagHigh-eCPX细菌溶液加入培养皿并保持在黑暗中。
      注意:Picodent twinsil 22可以用来粘贴玻璃表面到培养皿底部。
    3. 通过10倍物镜在倒置荧光显微镜(DMi8,Leica)上通过光掩模(没有支撑膜的TEM网格)在玻璃表面局部照射1小时。
    4. 去除光掩模,并通过去除2ml溶液并将2ml PBS加入培养皿中轻轻洗涤玻璃表面。重复洗涤三次。不要让表面变干。
    5. 通过倒置荧光显微镜(DMi8,Leica)通过10倍物镜对该表面进行成像。 (图3)


      图3. pMagHigh显示细菌到nMag的光图案化高功能化玻璃表面。A)明场和B)细菌图案的荧光图像。用mCherry标记细菌进行检测。经Chen和Wegner(2017)许可转载。

数据分析

数据信息可以通过 https://pubs.acs.org/doi/abs /10.1021/acssynbio.7b00197 。

笔记

  1. 所有的实验都在黑暗的房间里进行。&nbsp;

  2. 在黑暗中培养细菌以避免激活光敏蛋白质。

食谱

  1. 缓冲区A(1x)
    50mM Tris-HCl,pH7.4
    300mM NaCl(1L缓冲液17.5g)
  2. 缓冲区B(1x)
    缓冲区A
    250mM咪唑(1g缓冲液17g)
  3. 点击反应解决方案(六个表面)
    473μlMilliQ水
    pH 8.5的60μl1M Tris-HCl
    60μl1M L-抗坏血酸(新制备:741μlMilliQ水中的102mg L-抗坏血酸)
    0.9μl25mM NTA-炔烃
    6μl100mM CuSO 4(最后加入;加入时,局部出现棕色,混合后溶液变成无色)。
    通过涡流混合解决方案
  4. Riranha解决方案
    3:1(v / v)浓度。 H 2 O 4 SO 4:H 2 O 2(30%)
    20毫升H 2 O 2(30%)
    60毫升H 2 SO 4。

致谢

这项工作是由德国联邦教育和研究部(BMBF)和马克斯普朗克协会(FKZ 031A359L)联合资助的MaxSynBio联盟的一部分。足球俱乐部感谢中国国家留学基金委博士生奖学金。 GFP和mCherry质粒来自Victor Sourjik教授,CPX质粒来自Patrick Daugherty教授(Addgene质粒#23336)。我们要感谢Stefan Schumacher对数字的帮助。该协议是从以前的工作改编(陈和Wegner,2017)。我们要感谢ACS Synthetic Biology的编辑允许重印这些数字。没有利益上的冲突。

参考

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引用:Chen, F. and Wegner, S. V. (2018). Implementation of Blue Light Switchable Bacterial Adhesion for Design of Biofilms. Bio-protocol 8(12): e2893. DOI: 10.21769/BioProtoc.2893.
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Wenjun Sun
Nanjing Tech University
We did not find the specific sequence information of pMagHigh-eCPX and nMagHigh through reading the article. If the author is convenient, I hope the author can provide us with specific sequence information and map. Email: 1121950040@qq.com or 201862118021@njtech.edu.cn. We are expecting a reply.
2020/10/12 13:05:50 回复