Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

1. Preparation of PEG-DMA Hydrogels

1. Select an appropriate measuring/mixing container (flask, beaker, etc.) for the application. Clean it thoroughly using detergent and water, and then wipe it with lint-free tissue wipers.
2. Wear gloves to avoid contaminating the glassware with oils from fingertips. Rinse the container with enough deionized water to remove detergent residue.
3. Wipe the container with lint-free tissue to get rid of the water, then spray with isopropyl alcohol (IPA, 99.5%) and wipe until clean. Place it in a vacuum chamber to allow all IPA to completely evaporate. Clean the rest of the lab equipment used in the hydrogel forming process with distilled water.
4. To prepare a 40% (w/v) PEG-DMA hydrogel solution, weigh 4 g of the poly(ethylene-glycol) dimethacrylate (PEG-DMA; MW = 1,000 g/mol) polymer using a laboratory scale.
5. Place the weighed PEG-DMA into flask and heat using a sonicator bath at 45-55 °C until the solid PEG-DMA has liquefied. During the process, cover the opening of the flask with Parafilm/wax paper to keep water out.
6. Once the PEG-DMA has liquefied, add buffer solution (500 mM KCl, 10 mM MOPS, pH 7.0) until total volume reaches ~10 ml (enough for several experiments over a six months period).
7. Add the curing agent at 0.5% (w/v). In this case, add 0.05 g of the curing agent to the 10 ml mixture. Place the flask back into the sonicator bath and allow the components to dissolve in solution (around 10 min, 250 watts).

NOTE: Once the curing agent has been added to the solution, the hydrogels will cure (solidify) if exposed to any light source for a sufficient amount of time. To help combat this, wrap the vial/container with black tape and store it in a dark place. This solution can be stored for several weeks at room temperature (22 °C).

2. Preparation of Liposomes

1. Prepare 10 ml of a 2 mg/ml lipid solution by adding 10 ml of buffer (500 mM KCl, 10 mM MOPS, pH 7.0) to 20 mg of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) synthetic lipids purchased as lyophilized powder. Make sure both lipid vesicles and buffer solution are thoroughly mixed (the mixture should look homogenous and hazy when everything is dissolved).
2. Freeze (-20 °C) and completely thaw the new lipid mixture for a total of six times. Let the mixture thaw at room temperature, never in a heated environment.
3. Using a commercially available extruder, extrude the lipids by forcing the whole lipid suspension first through a 0.4 μm polycarbonate membrane filter and then six times through a 0.1 μm membrane filter. This process yields particles with diameters near 100 nm (equal to the pore size of the filter).

NOTE: Other lipids and lipid ratios can be prepared using this method. Liposomes should be stored at 4 °C for several weeks.

3. MscL Isolation and Reconstitution

1. Streak out a temperature agarose plate, containing 100 μg/ml ampicillin, E. coli MJF465 cells with a pB10b plasmid carrying the V23T MscL gene extended with a 6-His tag on the 3' end (C-terminus). Allow the plate to culture overnight (12-16 hr) at 37 °C in a stationary incubator. The plasmid is selected for and retained in cells with 100 μg/ml ampicillin in standard LB medium. The next day place 20 ml of LB media with 100 μg/ml ampicillin in a culturing vesicle (a 50 ml flask or what is available to hold the culture). Take the plate that was cultured overnight and select a colony from the plate to transfer (inoculate) to the prepared 20 ml LB media with a sterile inoculation stick. Allow the 20 ml culture to grow overnight (12-16 hr) at 37 °C at 250 rpm in a shaking incubator.
2. Decant the 20 ml overnight culture into 2-4 L of LB medium. Ampicillin is no longer required. Shake the flasks in a shaking incubator at 250 rpm at 37 °C until OD₆₀₀ reaches 0.5. Add Isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.6 mM and let the culture go for another hour (to OD₆₀₀ = 0.8-1.0).
3. Put the flasks on ice to chill the cultures and then collect the bacteria by centrifugation. Use six 400 ml conical tubes (or as many as allowed by the rotor used) and centrifuge for 5-8 min at 7,438 x g which is enough to pellet the bacteria. Decant the supernatant, and repeat the procedure until all the cells from the media are harvested. The number of spins required varies based on the amount of culture that has been grown and the rotor used. For a 2 L culture it is only needed to spin down the cells once. Transfer all harvested cells into a single centrifuge tube.
4. Resuspend the cell pellets in ~20 ml of French press buffer (100 mM KPi and 5 mM MgCl₂, pH 7.4). The suspension should be dense (like cream or milk). Immediately before French-pressing the suspension add the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 2 mM and mix vigorously.
5. French-press the culture, in a 35 ml French-pressure cell, at 10,000 to 16,000 psi. Spin down the suspension to separate out the unbroken cells at 7,438 x g, 10 min at 4 °C.
6. Put the supernatant in a separate tube, and add to it lysozyme and DNase (0.2 mg/ml each). Let supernatant tumble for 10 min at room temperature.

NOTE: DNase is optional; it reduces the viscosity for the high-speed centrifugation. Lysozyme is critical; it digests the remnants of cell wall and helps increase the yield of the membrane extraction done with a mild non-denaturing detergent.

7. Distribute the supernatant mix into two ultracentrifuge tubes and spin them at 106,883-153,911 x g (depending on the rotor) at 4 °C for 40 min. After centrifugation the supernatant is decanted and the brownish pellet at the bottom (the total membrane fraction) can be frozen in the tube for long-term storage (- 80 °C) or used for immediate protein purification.
8. Prepare 0.5-1 L of High Imidazole buffer: 100 mM NaCl + 500 mM imidazole, titrate to pH 7.2-7.4 with concentrated HCl. Note that imidazole is a good buffering substance by itself.
9. Prepare 0.5-1 L of Low Imidazole buffer: 100 mM NaCl, + 15 mM imidazole, by appropriately diluting the buffer above with 100 mM NaCl. No pH adjustment is required.
10. Take 100-150 ml of each buffer in separate bottles, and add 1% (w/v) b-octyl glucopyranoside (OG). Stir the solution well and filter it through a 0.22 μm filter. These solutions are the low- and high-imidazole chromatography solutions.
11. Prepare Extraction buffer, take 50 ml of Low-imidazole buffer, and add 3% (w/v) OG and filter the buffer.
12. Use membrane pellets of 0.5-2 g wet weight for protein isolation. Add 5-7 ml of extraction buffer, resuspend the pellet, and homogenize it in a 30 ml hand-driven glass-piston homogenizer. With 5-10 gentle strokes make a homogeneous suspension without lumps. Use caution, shear stress is known to cause protein denaturation.
13. Spin down the insoluble particles (mid-range centrifuge, fixed-angle rotor, 38,478-68,405 x g, at 4 °C for 15 min). Meanwhile, take 3 ml of Ni NTA beads (6 ml of suspension) and wash them once with the low-imidazole buffer (w/o OG) by shaking them in a 15 ml screw-cap tube. Let the beads settle on the bottom (~ 5-7 min) or spin them down at 129-201 x g for one minute at 4 °C. Once the pellet is formed, carefully decant the supernatant by hand and repeat the procedure. Equilibrate the beads with 2-3 ml of 3% OG extraction buffer.
14. Mix the homogenized mixture (membrane pellet and extraction buffer) from 3.12 with 3-3.5 ml Ni NTA beads. Let the mix tumble in a screw-cap tube for 60 min (batch-loading). Spin the beads down at 201 x g (30 sec), decant the supernatant by hand, and wash the beads with 1% OG low-imidazole buffer once. Pellet the beads as in 3.13 again and resuspend them in 20-30 ml of fresh low-imidazole buffer.
15. Pack a small column (equipped with an upper flow adapter) with the Ni NTA beads, and let the beads settle by opening the stopcock to let the extract flow through (do not allow the beads to dry). Wash the beads with one aliquot of 10 ml low-imidazole buffer (1% OG) with the stop cock open.
16. Load the chromatography machine with pure low- and high-imidazole buffers about 25 ml of each at the machine defined flow rate (varies per machine); this is done by passing the low-imidazole buffer through the system first. Zero the optical recorder at OD₂₆₀ (baseline). Note that the buffer is quite different from water because low-grade imidazole has impurities which absorb UV. Insert the flow adapter to the column and attach it to the machine.
17. Wash the column again with low-imidazole buffer (at 1 ml/min) until the OD of the flow through comes reaches the baseline (it may take 10-20 ml). This removes the unbound proteins from the column.
18. Apply a linear gradient of imidazole of 20 to 500 mM, for 30 min, at 1 ml/min. Start collecting 4 ml fractions when OD₆₀₀ shows an increase. The first two fractions are full of loosely bound proteins, whereas MscL-6His starts elution at ~40% of the linear gradient. The majority of the protein appears in fractions 3 to 8.
    NOTE: a linear rise of OD will be observed due to the increasing % of imidazole.
19. Pool the fractions 3-4, 5-6, and 7-8 together. Optionally, concentrate the fractions individually. Concentrate the fractions 6-10 fold using centrifugal filters. After a 20 min spin at 804 x g and at 4 °C, carefully resuspend the concentrated protein, the protein tends to stick to the filter.
20. Withdraw 50 μl aliquots, mix them with SDS sample buffer, and check for protein purity using PAGE gel electrophoresis.
    NOTE: MscL will migrate as a fuzzy band of about 17 kDa to the bottom of the gel.
21. Use the concentrated fractions to quantify the protein using a protein assay kit, following the manufacturer's instruction. A typical yield from a 0.8 g membrane pellet is up to 0.2 mg of pure protein in the combined fractions.
22. Reconstitute V23T MscL into DPhPC liposomes through dialysis. Take a 10 mg/ml chloroform solution of DPhPC and aliquot 0.5 ml (i.e. 5 mg of lipid) of it into three disposable round-bottom (12 x 130 mm) glass tubes. Dry the lipid under the stream of nitrogen and remove the remnants of chloroform under vacuum (4-6 hr).
23. Add 15 20 mg of powdered OG to the dry lipid in each tube, dissolve it in 2 ml of dialysis buffer (100 mM KCl, 5 mM KPi, pH 7.2), vortex, and mildly sonicate. The OG-solubilized lipids should form a clear solution.
24. Add concentrated V23T MscL solutions to each tube to achieve 1:100, 1:300 and 1:1,000 protein-to-lipid ratios and vortex well. Cut and wash three pieces (~12 cm long) of dialysis tubing (MWCO 8000, 7.5 mm diameter, have three pairs of numbered clips ready.)
25. Place the solubilized lipid-protein mixtures inside the tubing, carefully close the ends with clips, and dialyze against 2 L of buffer (100 mM KCl, 5 mM KPi, pH7.2) for 48 hr at 4 °C with four changes of the buffer every 12 hr. After dialysis, the proteo-liposomes are ready.

NOTE: The liposome solution can be supplemented with 2 mM of NaN3 (sodium azide) and stored at 4 °C. Avoid freezing.

4. Manufacture of the Oil Reservoir

1. Drill two opposing holes (1.0 mm in diameter) all the way through the wall of a 2 inch diameter, 1.5 cm long acrylic cylinder at 1 cm from the bottom (Figure 1A).
2. Drill two 4 mm holes concentric to the previously drilled 1 mm holes. The depths of the holes should be 1 mm each (make sure not to drill all the way). These holes are made to fit the rubber gaskets.
3. Place and glue two rubber gaskets having 1 mm inner diameters in the bigger holes in order to prevent oil from leaking.
4. Glue the machined cylinder to a 10 cm x 10 cm thin acrylic sheet using any multipurpose epoxy (Figure 1).

On the day of the experiment:

5. Preparation of Electrodes

1. Cut a 7 cm length of two 250 μm-diameter silver wires, and then immerse their tips in bleach for two hours to form a silver-chloride (AgCl) coating. A gray color indicates that an AgCl coating has been formed (Figure 2E).
2. Using a glass cutter, split a 10 cm long, 1/0.58 OD/ID mm borosilicate class capillary into two 5 cm capillaries.
3. Using a 34 gauge microfil needle fill the capillaries with the PEG-DMA hydrogel. To prevent the hydrated hydrogel from swelling out of the capillary, keep a 3 mm clearance at the tips and make sure there are no air bubbles in the capillaries.
4. Insert the Ag/AgCl electrodes into the hydrogel filled capillaries (Figure 2E).
5. Cure the PEG-DMA hydrogel through free-radical photopolymerization upon exposure to UV light for 2 min at 1 W using a UV spot gun.

6. Setting Up the Experiment

NOTE: The experiment is setup under a faraday cage grounded to a ground connection on the patch amplifier.

1. Attach one of the micropipettes to a straight microelectrode holder that has a male connector (Figure 2E).
2. Connect the microelectrode holder to the headstage of the patch amplifier (Figure 2A). To connect the headstage to ground, solder an 18 gauge insulated copper wire to an appropriate connector for the headstage.
3. Mount the headstage on a 3-axis manual micromanipulator. Attach the headstage mounting plate (it should be bought along with the micromanipulator or custom made in a machine shop) to the micromanipulator and then connect the headstage to the mounting plate using appropriate screws.
4. Attach the second micropipette to the linear actuator through a lab-made connector and then mount both on a second micromanipulator (Figure 2B). The micropipettes should be opposing each other, aligned, and horizontally leveled. Note: the brand and style of the manipulators do not matter.
5. In a glass vial, mix 0.1 ml of the DPhPC liposomes with 0.01 ml of the V23T MscL proteoliposome solution.

NOTE: This step is needed to reduce the protein-to-lipid ratio (~0.0002), which is critical to the formation of a stable lipid bilayer.

6. Using a 34 gauge microfil needle, fill the tips of both micropipettes with proteoliposome solution (Figure 3A).
7. Place reservoir on top of an upright microscope, and feed the micropipettes through the opposing 1 mm holes (Figure 2B). NOTE: any microscope could be used as long at the droplets could be clearly seen.
8. Fill reservoir to the surface with Hexadecane (99%). The Hexadecane does not need further purification.
9. To form the spherical droplets at the tip of the micropipettes, use a third 10 μm-diameter borosilicate glass micropipette, mounted on a third micromanipulator, to dispense diluted V23T MscL proteoliposome solution (~0.00052 ml) at the tips of the micropipettes and form the droplets (Figure 3).
10. Control the size of the droplets (by decreasing or increasing the volume) as desired and let them rest for 10 min for the monolayers to form completely (Figure 3).
11. Bring the droplets into contact, bilayer formation will occur within 1 to 2 min.

7. Setting Up the Software and Equipment

1. Prepare the software by turning on the computers, microscope, piezoelectric oscillator controller, function generators, patch amplifier, and the low-noise data acquisition system.
   NOTE: any patch amplifier could be used and the following instructions are specifically for the one we used and which is listed in the materials and equipment list.
2. On the front panel of the patch amplifier, set the "Mode" knobs to VHOLD/IHOLD and V-CLAMP.
3. On the front panel set the "Lowpass" Bessel Filter to 1 kHz and Output Gain to 2.
4. Set the "Configuration" to WHOLE CELL β = 1.
5. Make sure the rest of the knobs are set to zero or in neutral position.
6. Sample all DIB current measurements at 5 kHz with a 1 kHz Bessel anti-aliasing filter.
7. Run software by double-clicking on the icon on the desktop.
8. Click "Configure > Digitizer" to open the "Digitizer" dialog, and then click the "Change" button.
9. In the "Change Digitizer" dialog select "Digidata 1440 Series" from the "Digitizer Type" list.
10. Click the Scan button to detect the digitizer.
11. Click "OK" to exit the "Change Digitizer" dialog, and then click "OK" to exit the "Digitizer" dialog.
12. Click "Configure > Lab Bench".
13. In the Input Signals tab of the lab Bench, select Analog In Å under Digitizer Channels. Set the scale factor to 0.002.

8. Formation of the Lipid Bilayer

1. Using a BNC cable, connect the output of a waveform generator to the external command input front switched (on the rear panel of the data acquisition system). Send a 10 Hz, 500 mV pk-to-pk triangular waveform to the headstage.
2. Using the micromanipulator, move the glass micropipettes horizontally to bring droplets into contact until they slightly touch and wait for bilayer thinning to occur (usually around 1 to 2 min) (Figures 3C and 3D).

NOTE: Progression of the bilayer formation process can be seen visually through the microscope and may be monitored by current measurement (Figure 4).

3. Adjust the bilayer size (~250 μm in diameter) by controlling the position of the droplet mounted on the actuator, using the micromanipulator. Note: the bilayer size could be estimated visually through the microscope. This method makes it easier for the researcher to control the size of the bilayer by easily moving the droplets using the micromanipulators.

9. Dynamic Excitation and MscL Gating

1. Once the bilayer has formed and is stable (i.e. the bilayer is not breaking or conductive), stimulate the droplets by sending a sinusoidal signal using a function generator.
2. To stimulate the MscL protein incorporated in the bilayer, send a sinusoidal waveform with a 175 μm peak-to-peak amplitude, 0.2 Hz frequency, and 50% duty cycle to the piezoelectric servo-controller. (Various types of waveforms could be sent with different amplitudes, frequencies, and duty cycle)

10. Processing and Interpreting Results

1. Save the current measurements, recorded using the data acquisition system, in .ABF format. Import data (in .ABF format) to Matlab using a function file "abfload", then analyze and process the data. The "abfload" file is available for free online.
2. Estimate the tension in the bilayer and areal expansion of the droplets, using videos of the droplet during full actuation cycles that are recorded using an appropriate camera.
3. Process videos in Matlab, by processing individual frames using image processing techniques to estimate the area of the water/oil interface, as well as the angle between the droplets. NOTE: using a 2D frame taken from the video, detect the water-oil interface (i.e. the edge of the droplet) and then estimate the surface area by revolution.