Nov 2017



Microfluidics-Based Analysis of Contact-dependent Bacterial Interactions

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Bacteria in nature live in complex communities with multiple cell types and spatially-dependent interactions. Studying cells in well-mixed environments such as shaking culture tubes or flasks cannot capture these spatial dynamics, but cells growing in full-fledged biofilms are difficult to observe in real time. We present here a protocol for observing time-resolved, multi-species interactions at single-cell resolution. The protocol involves growing bacterial cells in a near monolayer in a microfluidic device. As a demonstration, we describe in particular observing the dynamic interactions between E. coli and Acinetobacter baylyi. In this case, the protocol is capable of observing both contact-dependent lysis of E. coli by A. baylyi via the Type VI Secretion System (T6SS) and subsequent functional horizontal gene transfer (HGT) of genes from E. coli to A. baylyi.

Keywords: Microfluidics (微射流技术), Horizontal gene transfer (HGT) (水平基因转移), Type VI secretion system (T6SS) (Ⅵ型分泌系统), Natural competence (自然感受态), Antibiotic resistance (抗生素耐药性), Acinetobacter (不动杆菌属), Biofilm (生物膜), Microbial ecology (微生物生态学)


Spatially-dependent interactions between different species of bacteria likely occur ubiquitously in nature, but they can be difficult to observe. One example is enhancement of horizontal gene transfer (HGT) by contact-dependent, in situ lysis of a prey cell, which serves as DNA donor, by a naturally competent, predatory, DNA recipient cell. This was only recently observed in Gram-negative bacteria, but it has already been seen in multiple species, and it is thought to be a relatively widespread phenomenon (Borgeaud et al., 2015; Cooper et al., 2017; Veening and Blokesch, 2017; Ringel et al., 2017). Killing-enhanced HGT cannot easily be observed at single-cell resolution in shaking culture tubes, both because single cells cannot be observed over time, and the well-mixed environment prevents spatial structure. These interactions occur in biofilms, but it is difficult to observe and track cells in their interior. Cells pressed between a glass slide and an agar pad are constrained to a two-dimensional spatial structure and can be observed during contact-dependent lysis (LeRoux et al., 2012; Basler et al., 2013). However, this method allows only a limited duration of observation before either nutrients are depleted, stopping cell growth, or growing colonies push up the agar and develop three-dimensional structure. Microfluidics is an ideal solution to these problems, as it continually provides fresh media while washing away excess, growing cells. The commonly used polydimethylsiloxane (PDMS) and glass substrates are rigid enough to maintain cells in an easily visualized monolayer, while still allowing complex dynamics that can approximate biofilm growth (Liu et al., 2015; Humphries et al., 2017). While we specifically describe contact-dependent killing and HGT between A. baylyli and E. coli, this method should also be generalizable to other species and spatially-structured interactions.

Materials and Reagents

  1. Glass coverslips, #1.5 (Fisher Scientific, catalog number: 12-530F )
  2. Plastic weighing boat, hexagonal (Fisher Scientific, catalog number: 02-202B )
  3. Razor blade (Fisher Scientific, catalog number: 12-640 )
  4. 0.5 mm biopsy punch (World Precision Instruments, catalog number: 504528 )
  5. Cutting mat (Harris, GE Healthcare, Whatman, catalog number: WB100020 )
  6. Clear removable tape (e.g., Scotch Magic Tape, catalog number: B0000DH8HQ )
  7. Aluminum foil (e.g., Reynolds Recycled, catalog number: B0028LZ86A )
  8. Squirt bottles containing 70% ethanol, water, methanol, and heptane, respectively (Fisher Scientific, catalog number: 03-409-34 )
  9. Parafilm (optional) (Bemis, catalog number: PM996 )
  10. 15 ml conical tubes (Genesee Scientific, catalog number: 21-103 )
  11. 1.5 ml microcentrifuge tubes (Fisher Scientific, catalog number: 05-408-129 )
  12. Microfluidic mold or chip
    Our layout is shown in Figure 1, and a mask design for photolithography is provided as supplementary information (see Co-culture chip.zip. Design files for a microfluidic chip for co-culturing different species of bacteria. The chip includes several different trap designs and geometries to facilitate experiment optimization). The specific design of a microfluidic chip is not critical, but some key considerations are as follows:
    1. Trapping regions should be large enough to contain enough cells to make interesting dynamics likely, but they should also be small enough to allow sufficient diffusion of nutrients throughout the trap. We used several trap geometries with areas on the order of 104 μm2.
    2. Trap heights should maintain a cell monolayer. We used approximately 0.8-1 μm.
    3. The fluid channels should avoid right angles and dead spaces, which contribute to clogging.
    Note: Our chip was based on the design developed in Danino et al. (2010), with parallel main media channels that have trapping chambers on their sides. We included multiple trap geometries, including some with one edge open to media channels, some with two edges open, some with additional, low-flow wings, and some with media feeder channels at the back, so we could test them all in parallel (Figure 2). The chip also includes a Dial-A-Wave design that allows precise control of the ratios of two different media sources. For single-media experiments as described here, it is sufficient to only use one inlet port, leaving the others un-punched. For further design considerations and details on media switching, see Ferry et al. (2011). If you have a pre-fabricated chip, you do not need items 1-16, and items 18-21 may need to be adjusted as appropriate for use with your chip.

    Figure 1. A microfluidic chip with four rows of traps and 3 media ports to allow dynamic media switching. Channels shown in blue are 20 μm tall, traps shown in green are 0.8-1 μm tall, and an optional layer of feeder channels shown in red are 0.2 μm tall. The three inlets allow precise concentrations of a dynamically varying chemical of choice, via the Dial-A-Wave mechanism. Note that the feeding channel layer and use of more than one inlet are optional.

    Figure 2. A closer view of the four rows of trapping regions. A. The top row contains 100 μm wide traps that alternate between 100 and 200 μm long, open to channels on two sides. B. The second row contains the same trap design with additional wings on the side that experience lower flow. C. The third row of traps is the same as the first, but open on only one side. D. The fourth row of traps is the same as the third, but the back is connected to another media channel by an optional third 0.2 μm tall layer to allow media exchange without allowing cells through. Note that this very low-height layer is technically difficult both to fabricate on a wafer (mold) and to prevent from collapsing on an assembled PDMS chip.

  13. BD 60 ml Luer-Lok syringes (BD, catalog number: 309053 )
  14. Tubing, 0.02" ID, 0.06" OD (Tygon, Murdock Industrial, catalog number: AAD04103 )
  15. Straight luer stubs, 23 ga x 0.50 in (Instech Laboratories, catalog number: LS23S )
  16. Bent luer stubs, 23 ga x 0.50 in, bent 90° (OK International, Metcal, catalog number: 923050-90BTE )
  17. E. coli carrying a plasmid that can transfer from E. coli to A. baylyi
    Note: We used pBAV1k-GFP (Addgene, catalog number: 26702 ), a plasmid with kanamycin resistance and a broad host range origin of replication that can propagate in both species (Bryksin and Matsumura, 2010). Another option would be to use a plasmid with the ColE1 origin of replication, which cannot propagate in A. baylyi, but to insert a GFP gene (and optionally the antibiotic resistance marker) between two flanking regions of A. baylyi genomic homology. This would encourage homologous recombination into the A. baylyi genome, which occurs at higher frequency than transfer of a self-replicating plasmid (Palmen et al., 1993). A good strain of E. coli for use in microfluidics is MG1655 (Coli Genetic Stock Center, catalog number: 6300 ), although other strains will work as well. Common cloning strains with mutations in recA, including dh5-alpha, are not ideal because they grow more slowly.
  18. A. baylyi strain ADP1 (ATCC, catalog number: 33305 ), see also Notes
    Note: Either A. baylyi or E. coli or both should contain a fluorescent marker such as mCherry to visually distinguish between the two in movies. We inserted mCherry into a neutral region of the A. baylyi genome using a modified version of pp2.1-PT5-lacI-gusA-specR-pp2.2-IMBB (Murin et al., 2011; Addgene, catalog number: 30505 ) with the lacI-gusA insert replaced by mCherry.
  19. Tween 20, aka, polysorbate 20 (Sigma-Aldrich, catalog number: P9416-50ML )
  20. Dehydrated LB (Luria-Bertani) broth, Miller (BD, catalog number: 244610 )
  21. 70% ethanol (Fisher Scientific, catalog number: BP8201500 ).
  22. Distilled water
  23. Methanol (Fisher Scientific, catalog number: A412-500 )
  24. Heptane (Fisher Scientific, catalog number: H350-1 )
  25. Antibiotics as appropriate to maintain your cell strains 
    1. For E. coli carrying pBAV1k, use kanamycin at 50 μg/ml. 
    2. For A. baylyi with a marker inserted using a variant of pp2.1-PT5-lacI-gusA-specR-pp2.2-IMBB, use spectinomycin at 17 μg/ml.
  26. Sylgard 184 silicone elastomer kit (i.e., polydimethylsiloxane or PDMS) including both base and curing agent (Ellsworth Adhesives, catalog number: 184 SIL ELAST KIT 0.5KG) (For Recipe 1)
    Manufacturer: Dow Corning, catalog number: 4019862 .
  27. PDMS (see Recipe 1)
  28. LB + Tween 20 media (see Recipe 2)


  1. Tweezers (e.g., Sports Medica, catalog number: B075VSFWSV )
  2. Glass stirring rod for PDMS (e.g., United Scientific Supplies, catalog number: GSR008 )
  3. Vacuum desiccator for degassing PDMS (e.g., Thermo Fisher Scientific, Nalgene polypropylene desiccator with stopcock, catalog number: 5310-0250 )
    Note: We attach this to an in-house vacuum line, but a vacuum pump would also work.
  4. Oven set to 80 °C (e.g., Isotemp 500, Fisher Scientific, catalog number: 13246516GAQ )
  5. UVO cleaner attached to an oxygen tank, used to bond chips to the cover glass (e.g., Jelight, model: 42 )
  6. Dissecting microscope for punching holes in the PDMS (e.g., Amscope, model: SM-4B )
  7. Goose-neck illuminator for use with the microscope (e.g., Amscope, model: LED-6W )
  8. Fume hood
  9. Appropriate personal protective equipment
    Note: Appropriate personal protective equipment should be worn throughout, including a lab coat, nitrile gloves, and safety glasses.
  10. Inverted, fluorescent microscope (We used Nikon TE and TI models)
    Note: An inverted, fluorescent microscope capable of automated imaging, including a CCD camera, a fluorescent light source, and appropriate filters. 
  11. Computer
    Note: A computer connected to the microscope and camera that is running software capable of controlling and receiving data from them, such as Micro-Manager (free and open source) or NIS-Elements, and ImageJ (free) to process image files.


  1. ImageJ (https://imagej.nih.gov/ij/) or its packaged version FIJI (https://fiji.sc/)


  1. Fabricate a microfluidic chip
    Note: This section can be skipped if you have a pre-fabricated microfluidic chip.
    1. Prepare PDMS
      1. Mix PDMS base and curing agent in a weigh boat at a ratio of 10:1; i.e., 36.4 g PDMS base and 3.6 g curing agent. Mix thoroughly with a glass stirring rod. 
      2. Place the boat in the vacuum desiccator to degas the PDMS. When air bubbles are nearly overflowing the weigh boat, break the vacuum momentarily to pop and deflate the bubbles. Continue until there are no more bubbles.
    2. Prepare wafer
      Tear off a piece of aluminum foil and wrap it around the wafer containing your trap molds. Carefully press down, avoiding the mold features, to remove air bubbles (see Figure 3).

      Figure 3. A wafer with molds of microfluidic chips ready for PDMS to be poured on

    3. Pour and cure PDMS
      Pour the degassed PDMS onto the wafer, and place it back into the vacuum desiccator to degas the PDMS as before. When the PDMS is thoroughly degassed, cure it in an 80 °C oven for an hour or overnight. Allow the cured PDMS to cool before proceeding, to avoid damaging your wafer.
    4. Cut and punch chips
      1. Carefully peel the cured and cooled PDMS away from the wafer. Cut out individual chips with a razor blade on a cutting mat. 
      2. Turn the chips feature site up and place under the dissecting microscope. Carefully use the biopsy punch to remove a core all the way through the chip at each media port. Note that an experiment with no media switching only requires punching one inlet and one outlet.
    5. Clean and bond chips
      1. Clean the chips sequentially with 70% ethanol and water, and dry them with a stream of nitrogen gas. Be sure to clean out the ports either with high-velocity gas or by injecting 70% ethanol or water through them using a syringe and luer stub adapter. Immediately place tape over the features of each chip after drying to prevent dust from settling on it.
      2. In a fume hood, clean the coverslips sequentially with heptane, methanol, and distilled water before drying under a stream of nitrogen gas. Immediately place tape over each coverslip after drying to prevent dust from settling on them. 
      3. Open the oxygen line to the UVO cleaner. Place the chips (feature side up) and coverslips inside the UVO cleaner, peel off the tape, and activate the surface for 3 min. Re-close the valve of the oxygen tank.
      4. Quickly and carefully place each chip feature side down onto a coverslip, and then bake at 80 °C for an hour or overnight to bond them.
        Note: If your chips have low height features (like the 1 μm traps on our chips), they can collapse if you press down on the chip. Placing the coverslip on top of the chip instead of the other way around can help protect low features.

  2. Prepare chip and media
    1. Note that our design includes an optional media switcher requiring 3 inlet ports. For an experiment with no media switches, it is sufficient to use only one inlet and one outlet, leaving the remaining inlets un-punched. For details on media switching, see Ferry et al. (2011). For each port, remove the metal part of a bent luer stub adapter. This can be done by soaking the adapter in acetone to dissolve the adhesive, and then pulling out the metal piece with tweezers. Discard the plastic and keep the metal piece.
    2. For each port, attach a straight luer stub adapter to a 50 ml syringe. Attach a length of tubing to the adapter, and insert the metal piece from a bent luer stub adapter into the other end of the tubing.
    3. Prime the tubing of all but one syringe by injecting 1 ml of LB + Tween 20 (Recipe 2) straight down into the bottom of the syringe (see Figure 4). Press the pipet tip against the very bottom of the syringe, and then maintain steady pressure on the plunger until you see LB begin to come out the other end of the tubing. Carefully add another 10 ml of LB into the syringe. Allow fluid to flow through the tubing until you are sure there are no more bubbles in the line, and then tape the end of the tubing to the syringe just above the liquid level to stop the flow. Cover the top of the syringe with tape or parafilm to prevent contamination (see also Figure 5). Note that chilled media will release dissolved gases as it warms, so to avoid bubbles, allow any chilled media to warm to at least room temperature before loading your syringes. See also Video 1, in which the syringe is loaded with food coloring to aid visualization.

      Figure 4. Prime the tubing by injecting media directly into the luer stub, through the bottom of the syringe

      Figure 5. An example of a primed syringe

      Video 1. Loading a syringe. To aid visualization, this syringe was loaded with water dyed green with food coloring.

  3. Prepare cells
    1. Meanwhile, grow 10-15 ml of each strain separately in LB with appropriate antibiotics to mid-exponential phase. For E. coli carrying pBAV1k, use kanamycin at 50 μg/ml.
    2. Harvest each strain by transferring to 15 ml conical tubes and centrifuging for 5 min at 2,000 x g. Room temperature is fine for centrifugation steps.
    3. Resuspend each strain in 1 ml fresh LB, transfer to a 1.5 ml microcentrifuge tube, and harvest again for 3 min at 10,000 x g. This washing step is to remove any residual antibiotics.
    4. Resuspend the two strains at high density – use about 1-3 volumes of LB with Tween 20 for each volume of cell pellet.
    5. Mix the two strains at 3 volumes of E. coli for each volume of A. baylyi. The ratio can be adjusted in subsequent attempts, but E. coli MG1655 are less adhesive than A. baylyi and thus are more likely to be washed out of the traps during and immediately after loading. If using another E. coli strain that is more adhesive, a more equal ratio of the two species may work better. Note also that species ratio can affect the frequency of interactions such as horizontal gene transfer (Cooper et al., 2017).
    6. Load the cell mixture into a prepared syringe and tubing in the same way as for the media syringes above.

  4. Load the chip
    1. For each media port, lower the luer stub below the fluid level in the syringe, hold it until fluid begins flowing out from the end, and insert it into the appropriate port in the chip, as in Figure 6. Do the same for the cell mixture in the waste port. Note that the loading speed can be increased by placing the chip in the vacuum desiccator for at least 20 min before loading.

      Figure 6. A loaded PDMS chip with 2 ports

    2. Place the chip onto the microscope stage, fix it in place, and raise the syringes a few feet above the chip. They can be taped to the wall, or for a more advanced setup see Ferry et al. (2011). Watch under the microscope as the channels fill with media. You want the fresh media and the cells to meet about midway into the trapping region. The extra buffer space is to prevent contaminating the media channel with cells in the next step. Adjust the syringe heights to ensure this. When the media does meet the cells, adjust the syringe heights so there is a slow forward flow from media to waste.
    3. If cells have not loaded well into the traps, flick the waste media line (loaded with cells) with your fingers. Try gently at first, and then harder as needed (see also Video 2). The pressure waves should force cells into the traps, but be careful not to force cells into the upstream media channels.

      Video 2. Flicking the microfluidics lines. A demonstration of how to flick the media lines, which helps initial loading of cells and can temporarily disrupt clogs.

    4. Once sufficient cells of each species are loaded into the traps (see Figure 7 for a representative example of a loaded trap), immediately adjust the syringe heights to obtain a forward flow from media to waste. The flow velocity must be fast enough to avoid clogging, but not so fast as to wash the less sticky E. coli out of the traps. The flow rate can be adjusted up as the cells grow and fill the traps. Both syringes should be above the chip.

      Figure 7. A microfluidic trap loaded with cells. The channel runs along the bottom of the image, A. baylyi cells are shown in red, and E. coli cells are shown in green.

  5. Record data and babysit the experiment
    1. Set your microscope to record one set of images every 3-5 min. You will need to determine the appropriate exposure power and duration to observe cellular fluorescence on your equipment. In general, use the lowest possible exposure that will give reliable data, to avoid damaging the cells. A longer exposure at lower intensity is preferable to a short exposure at high intensity. Imaging multiple stage positions using a motorized stage is ideal, because it increases the likelihood you will capture usable data.
    2. Keep an eye on the experiment to be sure that cells are growing and the channels do not clog. If cells are washing out of the traps, reduce the flow rate by raising the waste syringe or lowering the fresh media syringe. If channels are beginning to clog, or if media is not flowing forward, increase the flow rate by adjusting the syringe heights in the opposite direction.
    3. If channels begin clogging, this generally portends the beginning of the end. Sometimes, the experiment can be given a temporary reprieve by flicking the tubing connecting the syringes to the chip, as in Video 2. However, this can also rearrange the cells within the traps, which can compromise time course data. See also Notes.

Data analysis

Captured still images can be converted into movies using the free program ImageJ, which also comes in a package recursively called FIJI.

  1. Import a folder of images with the command File > Import > Image Sequence. Be sure to select ‘Sort names numerically’. Using a virtual stack (opening with the computer’s virtual memory rather than loading into RAM) will load faster and can be useful for large folders on computers with limited RAM, but annotations often do not work on virtual stacks.
  2. Convert to a hyperstack using the Images > Stacks > Stack to Hyperstack command. Specify the appropriate number of positions, channels, time points, and Z-slices. Select display as composite.
  3. Adjust the color and contrast of each channel using the tools available at Image > Color > Channels Tool and Image > Adjust > Brightness And Contrast.
  4. Optional: If you want to add any annotations, such as time stamps or scale bars, you must convert the multichannel hyperstack to an RGB stack using Image > Type > RGB Color. Be sure to adjust the contrast first, because it will be fixed once the stack has been converted to RGB.
  5. Optional: Add any desired annotations. To add time stamps, use Image > Stacks > Label. To add a scale bar, use Analyze > Set Scale followed by Analyze > Tools > Scale Bar.
  6. Optional: Crop all frames in the movie by selecting the rectangle tool and then Image > Crop. Select only a portion of the time series using Image > Stacks > Tools > Make Substack.
  7. Save your processed movie as a TIFF stack at full quality and/or as a compressed movie using the options in File > Save As. You may need to download a plugin to save as a .avi or .mov file from https://imagej.nih.gov/ij/plugins/. Saving as an animated GIF is another option that is easily shared on social media.
  8. Representative results can be seen in the attached movies for Cooper et al. (2017).


In our hands, A. baylyi was difficult to work with in microfluidics, because it adhered to both the PDMS and glass coverslips of our chips. We attempted several strategies to reduce cellular adhesion, including adding high levels of Tween 20, adding DNase (Das et al., 2010), adding PEG to the PDMS before curing, and deleting the thin pilus gene acuA (Gohl et al., 2006), but none helped significantly.
Interestingly, some of our experiments began well, but then there appeared to be a change in A. baylyi that caused them to become more adhesive to both the surfaces and each other. In a microfluidic device where non-adhesive cells are washed away, the adhesive phenotype is constantly selected for and rapidly dominates once it emerges. The adhesive phenotype may be related in part to recently described genomic instability caused by mobile genetic elements that cause genomic insertions and deletions at relatively high frequencies (Renda et al., 2015). When this instability disrupts production of bioemulsifier, the cells begin to aggregate. A strain of A. baylyi that lacks all insertion elements (Suárez et al., 2017) may work better in microfluidics, but we have not tested it. An alternative explanation may be a developmental switch between bacillar and coccoid phenotypes, with the coccoid phenotype being related to nutrient stress and adhesion (James et al., 1995).
Regardless, while cellular adhesion limited the duration of our experiments, we were able to run them for long enough to reproducibly observe T6SS-dependent killing and subsequent HGT before the channels fully clogged (Cooper et al., 2017).


  1. PDMS
    36.4 g silicone elastomer
    3.6 g curing agent
    Weigh together in a plastic weigh boat and stir thoroughly with a glass stir bar immediately before proceeding to degas and pour onto the wafer
  2. LB + Tween 20
    25 g LB powder
    Distilled water to 1 L
    Tween 20
    Dissolve the LB powder and Tween 20 into distilled water, then filter sterilize. Alternatively, add Tween 20 into pre-made LB liquid and filter sterilize


RMC was supported by a fellowship from The Hartwell Foundation. This work was supported in part by the National Institute of General Medical Sciences (NIGMS): San Diego Center For Systems Biology – P50 GM085764. This protocol was adapted from Cooper et al. (2017).

Competing interests

The authors declare no conflicts of interest.


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  18. Veening, J. W. and Blokesch, M. (2017). Interbacterial predation as a strategy for DNA acquisition in naturally competent bacteria. Nat Rev Microbiol 15(10): 621-629.


自然界中的细菌生活在具有多种细胞类型和空间依赖性相互作用的复杂社区中。 在充分混合的环境中研究细胞,例如摇动培养管或烧瓶,不能捕获这些空间动力学,但是在完全成熟的生物膜中生长的细胞难以实时观察。 我们在这里提出了一种用于观察单细胞分辨率下的时间分辨,多物种相互作用的方案。 该方案涉及在微流体装置中以近单层生长细菌细胞。 作为演示,我们特别描述了观察 E之间的动态相互作用。 大肠杆菌和 Acinetobacter baylyi 。 在这种情况下,该方案能够观察 E的接触依赖性裂解。 大肠杆菌由 A。 baylyi 通过VI型分泌系统(T6SS)和随后的来自 E的基因的功能性水平基因转移(HGT)。 大肠杆菌到 A.baylyi。

【背景】 不同种类细菌之间的空间依赖性相互作用可能在自然界普遍存在,但它们很难观察到。一个例子是通过天然感受的捕食性DNA受体细胞的接触依赖性,原位裂解作为DNA供体的捕食细胞来增强水平基因转移(HGT)。这种情况最近才出现在革兰氏阴性菌中,但已经在多个物种中观察到,并且它被认为是一种相对普遍的现象(Borgeaud et al。,2015; Cooper 等人,,2017; Veening和Blokesch,2017; Ringel et al。,2017)。在振荡培养管中不能以单细胞分辨率容易地观察到杀伤增强的HGT,这是因为随着时间的推移不能观察到单细胞,并且良好混合的环境阻止了空间结构。这些相互作用发生在生物膜中,但很难观察和跟踪其内部的细胞。在载玻片和琼脂垫之间按压的细胞被限制为二维空间结构,并且可以在接触依赖性裂解期间观察到(LeRoux 等人,,2012; Basler 等。 ,2013)。然而,该方法仅允许在营养物耗尽,停止细胞生长或生长菌落推高琼脂并形成三维结构之前的有限持续观察。微流体是解决这些问题的理想解决方案,因为它可以持续提供新鲜培养基,同时洗去多余的细胞。常用的聚二甲基硅氧烷(PDMS)和玻璃基板足够刚性,可以将细胞保持在易于观察的单层中,同时仍然可以使复杂的动力学接近生物膜的生长(Liu et al。,2015; Humphries 等人,2017)。虽然我们具体描述了 A之间的接触依赖性杀戮和HGT。 baylyli 和 E.大肠杆菌,这种方法也应该推广到其他物种和空间结构的相互作用。

关键字:微射流技术, 水平基因转移, Ⅵ型分泌系统, 自然感受态, 抗生素耐药性, 不动杆菌属, 生物膜, 微生物生态学


  1. 玻璃盖玻片,#1.5(Fisher Scientific,目录号:12-530F)
  2. 塑料称重船,六角形(Fisher Scientific,目录号:02-202B)
  3. 剃刀刀片(Fisher Scientific,目录号:12-640)
  4. 0.5 mm活检穿孔器(World Precision Instruments,目录号:504528)
  5. 切割垫(Harris,GE Healthcare,Whatman,目录号:WB100020)
  6. 清除可移动磁带(例如,Scotch Magic Tape,目录号:B0000DH8HQ)
  7. 铝箔(例如,Reynolds Recycled,目录号:B0028LZ86A)
  8. 分别喷洒含70%乙醇,水,甲醇和庚烷的瓶子(Fisher Scientific,目录号:03-409-34)
  9. Parafilm(可选)(Bemis,目录号:PM996)
  10. 15毫升锥形管(Genesee Scientific,目录号:21-103)
  11. 1.5 ml微量离心管(Fisher Scientific,目录号:05-408-129)
  12. 微流体模具或芯片
    1. 诱捕区域应该足够大以容纳足够的细胞以产生有趣的动态,但它们也应该足够小以允许营养物在整个诱捕器中充分扩散。我们使用了几个陷阱几何结构,其面积大约为10 4 μm 2 。
    2. 陷阱高度应保持细胞单层。我们使用大约0.8-1μm。
    3. 流体通道应避免直角和死角,这会导致堵塞。
    注意:我们的芯片基于Danino等人开发的设计。 (2010年),平行的主要媒体渠道,侧面有诱捕室。我们包括多个陷阱几何形状,包括一些边缘向媒体通道打开,一些边缘打开,一些带有额外的低流动翼,有些带有背面的介质馈送通道,因此我们可以并行测试它们(图2)。该芯片还包括Dial-A-Wave设计,可以精确控制两种不同媒体源的比例。对于此处所述的单媒体实验,仅使用一个入口端口就足够了,而其他入口端口则不会打孔。有关介质切换的进一步设计考虑和详细信息,请参阅Ferry等人。 (2011年)。如果您有预制芯片,则不需要第1-16项,并且可能需要根据芯片的需要调整第18-21项。


    图2.四排捕获区域的近视图。 A.顶行包含100μm宽的陷阱,在100到200μm长之间交替,对两侧的通道开放。 B.第二排包含相同的陷阱设计,侧面有额外的机翼,可以降低流量。 C.第三排陷阱与第一排相同,但仅在一侧打开。 D.第四排陷阱与第三排相同,但背面通过可选的第三层0.2μm高的层连接到另一个媒体通道,以允许介质交换而不允许细胞通过。请注意,这种非常低高度的层在技术上难以在晶圆(模具)上制造并且防止在组装的PDMS芯片上坍塌。

  13. BD 60 ml Luer-Lok注射器(BD,目录号:309053)
  14. 管道,0.02“内径,0.06”外径(Tygon,Murdock Industrial,目录号:AAD04103)
  15. 直路鲁尔短截线,23 ga x 0.50 in(Instech Laboratories,目录号:LS23S)
  16. 弯曲鲁尔接头,23 ga x 0.50 in,弯曲90°(OK International,Metcal,目录号:923050-90BTE)
  17. 电子。携带可从 E转移的质粒的大肠杆菌。大肠杆菌到 A. baylyi
    注意:我们使用了pBAV1k-GFP(Addgene,目录号:26702),这是一种具有卡那霉素抗性和广泛宿主范围复制起点的质粒,可在两个物种中繁殖(Bryksin和Matsumura,2010)。另一种选择是使用具有ColE1复制起点的质粒,其不能在A. baylyi中繁殖,而是在A. baylyi基因组同源性的两个侧翼区域之间插入GFP基因(和任选的抗生素抗性标记)。这将促进同源重组进入A. baylyi基因组,其发生频率高于自我复制质粒的转移(Palmen等,1993)。用于微流体的良好的大肠杆菌菌株是MG1655(Coli Genetic Stock Center,目录号:6300),尽管其他菌株也可以起作用。 recA突变的常见克隆菌株,包括dh5-alpha,并不理想,因为它们生长得更慢。
  18. 甲。 baylyi 菌株ADP1(ATCC,目录号:33305),另见注释
    注意:A. baylyi或大肠杆菌或两者都应含有荧光标记,如mCherry,以在视觉上区分电影中的两者。我们使用pp2.1-PT5-lacI-gusA-specR-pp2.2-IMBB的修饰版本将mCherry插入A. baylyi基因组的中性区域(Murin等,2011; Addgene,目录号:30505)将lacI-gusA插入物替换为mCherry。
  19. Tween 20,又名聚山梨醇酯20(Sigma-Aldrich,目录号:P9416-50ML)
  20. 脱水LB(Luria-Bertani)肉汤,Miller(BD,目录号:244610)
  21. 70%乙醇(Fisher Scientific,目录号:BP8201500)。
  22. 蒸馏水
  23. 甲醇(Fisher Scientific,目录号:A412-500)
  24. 庚烷(Fisher Scientific,目录号:H350-1)
  25. 适合维持细胞应变的抗生素 
    1. 对于 E。携带pBAV1k的大肠杆菌,使用50μg/ ml的卡那霉素。 
    2. 对于 A。使用pp2.1-PT5-lacI-gusA-specR-pp2.2-IMBB的变体插入标记的baylyi ,使用17μg/ ml的壮观霉素。
  26. Sylgard 184有机硅弹性体试剂盒(即,聚二甲基硅氧烷或PDMS),包括碱和固化剂(Ellsworth Adhesives,目录号:184 SIL ELAST KIT 0.5KG)(配方1)
  27. PDMS(见食谱1)
  28. LB + Tween 20媒体(见食谱2)


  1. 镊子(例如,运动医学,目录号:B075VSFWSV)
  2. 用于PDMS的玻璃搅拌棒(例如,United Scientific Supplies,目录号:GSR008)
  3. 用于脱气PDMS的真空干燥器(例如,Thermo Fisher Scientific,带有旋塞阀的Nalgene聚丙烯干燥器,目录号:5310-0250)
  4. 烤箱设定为80°C(例如,Isotemp 500,Fisher Scientific,目录号:13246516GAQ)
  5. UVO清洁剂连接到氧气罐,用于将芯片粘合到盖玻片上(例如,Jelight,型号:42)
  6. 解剖显微镜在PDMS中打孔(例如,Amscope,型号:SM-4B)
  7. 用于显微镜的鹅颈照明器(例如,Amscope,型号:LED-6W)
  8. 通风柜
  9. 适当的个人防护装备
  10. 倒置荧光显微镜(我们使用的是Nikon TE和TI型号)
  11. 电脑


  1. ImageJ( https://imagej.nih.gov/ij/ )或其打包版本FIJI(< a href =“https://fiji.sc/”target =“_ blank”> https://fiji.sc/ )


  1. 制造微流体芯片
    1. 准备PDMS
      1. 将PDMS基料和固化剂在称重船中以10:1的比例混合; 即,36.4g PDMS碱和3.6g固化剂。用玻璃搅拌棒彻底搅拌。&nbsp;
      2. 将船放入真空干燥器中以使PDMS脱气。当气泡几乎溢出称重船时,立即打破真空以弹出并使气泡放气。继续,直到没有气泡。
    2. 准备晶圆


    3. 倒入并治愈PDMS
    4. 切割和打孔芯片
      1. 小心地将固化和冷却的PDMS从晶片上剥离。在切割垫上用剃刀刀片切出单个芯片。&nbsp;
      2. 将芯片特征部位向上翻转并放置在解剖显微镜下。小心地使用活检冲头在每个介质端口处通过芯片一直移除芯。请注意,没有介质切换的实验仅需要冲压一个入口和一个出口。
    5. 清洁和粘合芯片
      1. 依次用70%乙醇和水清洗芯片,并用氮气流干燥。务必使用高速气体清洁端口,或使用注射器和鲁尔接头适配器注入70%的乙醇或水。干燥后立即将胶带放在每个芯片的特征上,以防止灰尘沉积在其上。
      2. 在通风橱中,依次用庚烷,甲醇和蒸馏水清洗盖玻片,然后在氮气流下干燥。干燥后立即将胶带放在每个盖玻片上,以防止灰尘沉积在它们上面。&nbsp;
      3. 打开UVO清洁剂的氧气管线。将芯片(特征面朝上)和盖玻片放入UVO清洁剂内,剥离胶带,并将表面激活3分钟。重新关闭氧气罐的阀门。
      4. 快速小心地将每个芯片特征面朝下放在盖玻片上,然后在80°C下烘烤一小时或过夜以粘合它们。

  2. 准备芯片和媒体
    1. 请注意,我们的设计包括一个可选的媒体切换器,需要3个入口。对于没有介质开关的实验,仅使用一个入口和一个出口就足够了,剩下的入口不打孔。有关媒体切换的详细信息,请参阅Ferry 等。 (2011)。对于每个端口,卸下弯曲的鲁尔接头适配器的金属部分。这可以通过将适配器浸泡在丙酮中以溶解粘合剂,然后用镊子拉出金属片来完成。丢弃塑料并保留金属片。
    2. 对于每个端口,将直的鲁尔接头适配器连接到50 ml注射器。将一段管道连接到适配器,然后将金属片从弯曲的鲁尔接头适配器插入管道的另一端。
    3. 将1 ml LB + Tween 20(配方2)直接注入注射器底部,灌注除一个注射器以外的所有注射器(见图4)。将移液管尖端压在注射器的最底部,然后在柱塞上保持稳定的压力,直到看到LB开始从管道的另一端出来。小心地在注射器中加入另外10毫升的LB.让流体流过管道,直到您确定管线中没有气泡为止,然后将管道末端粘在液位上方的注射器上以停止流动。用胶带或封口膜覆盖注射器顶部以防止污染(另见图5)。请注意,冷却介质会在温度升高时释放溶解的气体,因此为了避免产生气泡,在装入注射器之前,请让任何冷却介质加热到至少室温。另请参见视频1,其中注射器装有食用色素以帮助可视化。



      视频1.装入注射器。 为了帮助实现可视性,该注射器装有染成绿色的水和食用色素。

  3. 准备细胞
    1. 同时,在LB中用适当的抗生素分别培养10-15ml每种菌株至指数中期。对于 E。携带pBAV1k的大肠杆菌,使用50μg/ ml的卡那霉素。
    2. 通过转移至15ml锥形管并在2,000 x g 下离心5分钟来收获每个菌株。离心步骤的室温很好。
    3. 将每个菌株重悬于1ml新鲜LB中,转移至1.5ml微量离心管中,并以10,000 x g 再次收获3分钟。该洗涤步骤是除去任何残留的抗生素。
    4. 以高密度重悬两种菌株 - 对于每个体积的细胞沉淀,使用约1-3体积的LB与吐温20。
    5. 将两种菌株在3倍体积的 E下混合。 A的每个体积的大肠杆菌。 baylyi 。可以在后续尝试中调整该比率,但 E.大肠杆菌 MG1655的粘附性低于 A. baylyi 因此更有可能在装载期间和装载后立即从陷阱中冲洗掉。如果使用另一个 E。大肠杆菌菌株更具粘性,两种物种的比例更均匀可能效果更好。还要注意物种比例可以影响水平基因转移等相互作用的频率(Cooper et al。,2017)。
    6. 以与上述培养基注射器相同的方式将细胞混合物装入准备好的注射器和管中。

  4. 加载芯片
    1. 对于每个介质端口,将luer短管降低到注射器中液位以下,保持它直到流体从末端流出,然后将其插入芯片中的相应端口,如图6所示。对电池执行相同操作混合物在废物港口。请注意,在装载前将芯片放入真空干燥器至少20分钟,可以提高装载速度。


    2. 将芯片放在显微镜载物台上,将其固定到位,然后将注射器抬高到芯片上方几英尺处。它们可以贴在墙上,或者用于更先进的设置,请参阅Ferry 等。 (2011)。在显微镜下观察,因为通道充满了媒体。您希望新鲜培养基和细胞在诱捕区域的中途相遇。额外的缓冲空间是为了防止在下一步中使用单元污染介质通道。调整注射器高度以确保这一点。当介质确实与细胞相遇时,调整注射器高度,以便从介质到废物的缓慢向前流动。
    3. 如果细胞没有很好地装入陷阱,请用手指轻弹废液介质管线(装有细胞)。首先轻轻尝试,然后根据需要更加努力(另见视频2)。压力波应该迫使细胞进入陷阱,但要小心不要让细胞进入上游媒体通道。


    4. 一旦将每个物种的足够细胞加载到捕集器中(参见图7以获得负载捕集器的代表性示例),立即调整注射器高度以获得从介质到废物的向前流动。流速必须足够快以避免堵塞,但不能快速洗涤粘性较小的 E。大肠杆菌离开陷阱。当细胞生长并填充陷阱时,可以调节流速。两个注射器都应该在芯片上方。

      图7.装有细胞的微流体陷阱。通道沿着图像的底部运行, A. baylyi 单元格显示为红色, E。大肠杆菌细胞以绿色显示。

  5. 记录数据并照看实验
    1. 将显微镜设置为每3-5分钟记录一组图像。您需要确定适当的曝光功率和持续时间,以观察设备上的细胞荧光。通常,使用尽可能低的曝光量来提供可靠的数据,以避免损坏细胞。较低强度下的较长曝光优于高强度下的短曝光。使用电动载物台对多级位置进行成像是理想的选择,因为它增加了捕获可用数据的可能性。
    2. 密切关注实验,确保细胞生长,通道不会堵塞。如果细胞从捕集器中冲洗掉,则通过升高废弃注射器或降低新鲜介质注射器来降低流速。如果通道开始堵塞,或者介质没有向前流动,则通过调整相反方向的注射器高度来增加流速。
    3. 如果通道开始堵塞,这通常预示着结束的开始。有时,可以通过轻弹将注射器连接到芯片的管道来暂时缓解实验,如视频2中所示。但是,这也可以重新排列陷阱内的细胞,这可能损害时间进程数据。另见注释。



  1. 使用命令 File&gt;导入图像文件夹导入&gt;图像序列。 请务必选择“以数字方式排序名称”。使用虚拟堆栈(使用计算机的虚拟内存打开而不是加载到RAM中)将加载更快,对于RAM有限的计算机上的大文件夹非常有用,但注释通常不适用于虚拟堆栈。
  2. 使用 Images&gt;转换为hyperstack。堆栈&gt; Stack to Hyperstack 命令。指定适当数量的位置,通道,时间点和Z切片。选择显示为复合。
  3. 使用 Image&gt;中提供的工具调整每个通道的颜色和对比度。颜色&gt;渠道工具和图片&gt;调整&gt;亮度和对比度。
  4. 可选:如果要添加任何注释,例如时间戳或比例尺,则必须使用 Image&gt;将多通道超级堆栈转换为RGB堆栈。输入&gt; RGB颜色。请务必先调整对比度,因为一旦堆栈转换为RGB,它就会被修复。
  5. 可选:添加任何所需的注释。要添加时间戳,请使用 Image&gt;堆栈&gt;标签。要添加比例尺,请使用 Analyze&gt;设置比例,然后是分析&gt;工具&gt;比例尺。
  6. 可选:通过选择矩形工具然后图像&gt;裁剪影片中的所有帧。作物。使用 Image&gt;选择部分时间序列。堆栈&gt;工具&gt;制作Substack 。
  7. 使用文件&gt;中的选项将处理后的影片保存为完整质量的TIFF堆栈和/或压缩影片。保存为。您可能需要从 https:// imagej下载插件以保存为.avi或.mov文件。 nih.gov/ij/plugins/ 。保存为动画GIF是另一种可在社交媒体上轻松共享的选项。
  8. 代表性的结果可以在Cooper 等人的附带电影中看到。 (2017)。


在我们手中, A. baylyi 很难在微流体技术中使用,因为它粘附在我们芯片的PDMS和玻璃盖玻片上。我们尝试了几种降低细胞粘附的策略,包括添加高水平的吐温20,添加DNase(Das 等,2010),在固化前向PDMS添加PEG,并删除薄的菌毛基因 acuA (Gohl et al。,2006),但没有一个显着帮助。
有趣的是,我们的一些实验开始很好,但是 A似乎发生了变化。 baylyi 使它们对表面和彼此变得更加粘合。在微流体装置中,非粘性细胞被冲走,粘合剂表型被不断选择并且一旦出现就迅速占主导地位。粘附表型可能部分地与最近描述的由移动遗传元件引起的基因组不稳定性相关,所述移动遗传元件在相对高的频率下引起基因组插入和缺失(Renda 等人,2015)。当这种不稳定性破坏生物乳化剂的产生时,细胞开始聚集。 A的菌株。没有所有插入元素的baylyi (Suárez et al。,2017)在微流体学方面可能效果更好,但我们还没有测试过。另一种解释可能是细菌和球菌表型之间的发育转换,球菌表型与营养应激和粘附有关(James et al。,1995)。
无论如何,虽然细胞粘附限制了我们实验的持续时间,但我们能够运行它们足够长的时间,以便在通道完全堵塞之前重复观察T6SS依赖性杀伤和随后的HGT(Cooper et al。,2017 )。


  1. PDMS
  2. LB + Tween 20


RMC得到了哈特韦尔基金会的奖学金支持。这项工作部分得到了圣地亚哥系统生物学中心的支持。该协议改编自Cooper 等。 (2017)。作者宣称没有利益冲突。


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Copyright Cooper et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Cooper, R., Tsimring, L. and Hasty, J. (2018). Microfluidics-Based Analysis of Contact-dependent Bacterial Interactions. Bio-protocol 8(16): e2970. DOI: 10.21769/BioProtoc.2970.
  2. Cooper, R., Tsimring, L. and Hasty, J. (2017). Inter-species population dynamics enhance microbial horizontal gene transfer and spread of antibiotic resistance. Elife 6: e25950.

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