Materials and Reagents

1. 1.5 ml microcentrifuge tubes (Fisher, catalog number: 05-408-129)

2. Pipettes and tips (Eppendorf, catalog number: 2231300002)

3. Drummond calibrated micropipettes (100 µl) (Drummond Scientific Company, catalog number: 2-000-100)

4. PCR tubes (Fisher, catalog number: 14-230-225)

5. 50 ml centrifuge tubes (Corning, catalog number: 430829)

6. Cell culture tubes (Fisher Scientific, catalog number: 14-959-1B)

7. Serological pipettes (Midsci, catalog numbers: TP94010, TP94005)

8. Various sizes of Baffled cell culture flasks (Thermo Scientific, catalog numbers: 41100250, 41100250)

9. 10 ml syringes (Becton Dickinson (BD), catalog number: 302995)

10. E. coli OverExpress C43 (DE3) competent cells (Lucigen, catalog number: 60442-1)

11. pQE82L expression vector

12. Liquid nitrogen

13. Luria Broth Media (RPI Research Products, catalog number: L24400)

14. Ampicillin Sodium Salt (Sigma Aldrich, catalog number: A0166)

15. Cephalexin hydrate (Alfa Aesar, catalog number: J63672-06)

16. Isopropyl β-D-1-thiogalactopyranoside (Gold Bio, catalog number: I2481C)

17. Sucrose (Sigma-Aldrich, catalog number: S0389)

18. Tris base (Sigma-Aldrich, catalog number: 1070897600)

19. Ready-Lyse Lysozyme solution (Lucigen, catalog number: R1804M)

20. OmniCleave Endonuclease (Lucigen, catalog number: OC7850K)

21. Ethylenediaminetetraaceticacid (Sigma- Aldrich, catalog number: E9884)

22. Magnesium Chloride (Sigma-Aldrich, catalog number: M8266)

23. Potassium Chloride (Sigma-Aldrich, catalog number: 529552)

24. Calcium Chloride (Sigma-Aldrich, catalog number: C8106)

25. Potassium Hydroxide (Sigma-Aldrich, catalog number: 221473)

26. Hydrochloric acid (Acros organics, catalog number: AC450550025)

27. LB medium (see Recipes)

28. Stop buffer (see Recipes)

29. Pipette solution (Intracellular solution) (see Recipes)

30. Bath solution (see Recipes)

Equipment

1. -80°C freezer

2. Water bath

3. Incubating shaker with temperature control (New Bruinswick Scientific, Model C25KC classic series incubator shaker)

4. Spectrophotometer/Nanodrop

5. Autoclave

6. Centrifuge (Eppendorf, model: 5810R)

7. Deionized water (18.2 µΩ resistance)

8. pH meter (Thermo Scientific Orion Star A214)

9. Electrophysiology setup:

   Air table (TMC)

   Fluorescence microscope (Olympus, model: IX70)

   Micromanipulator (Scientifica, model: ACCi UI)

   Digidata 1440A (Axon Instruments)

   Multiclamp (Axon Instruments)

   Perfusion system (home-made gravity perfusion system)

   Chamber (Warner Instruments, catalog number: RC26)

10. Incubator for bacterial growth

11. Pipette puller (Sutter Instruments, model: P-97)

12. Phase contrast Microscope (Invitrogen, model: EVOS M5000)

Software

1. pCLAMP and Clampfit (Molecular devices)

Procedure

1. Making the spheroplasts

   1. Bacterial transformation

      1. Take an aliquot (40 µl) of E. coli OverExpress C43 (DE3) competent cells from -80°C freezer and place on ice for approximately 5 min to let it thaw.

      2. Then, add 1 ng of plasmid containing gene of interest. We used pQE82L expression vector containing MthK-IR (Inactivation removed calcium activated potassium channel) gene to the bacteria and gently flick the tube to mix them well. Let it incubate on ice for about 30 min.

      3. After incubation, perform heat-shock on the DNA-competent cell mixture by placing in a 42°C water bath for 45 s and then put the tube back on ice for another 5 min before spreading it on LB Agar plates containing appropriate antibiotic (ampicillin in this case).

      4. Incubate the plate at 37°C overnight.

   2. Bacterial growth

      1. The next day (16 h after transformation), pick a single colony from the plate and inoculate it into 20 ml LB medium with antibiotic held in a 60 ml cell culture flask.

      2. Incubate the culture in a shaker at 37°C and 220 rpm. After 3 h, check the optical density (OD₆₀₀) of the culture periodically (every 20-30 min) with a spectrophotometer or nanodrop until OD₆₀₀ reaches ~0.7.

   3. Cephalexin treatment

      1. Pre-heat 55 ml of autoclaved LB medium at 42°C. Once the culture reaches OD₆₀₀ = 0.7, place the culture on ice, quickly take 5 ml of this liquid culture and add to 55 ml of pre-heated LB medium containing antibiotic. Add 50 μg/ml cephalexin.

      2. Put the culture in an incubator at 42°C and reduce the speed of the shaker to 120 rpm (‘snake-like’ E. coli filaments are really fragile).

      3. Remove 100 μl of the culture every 20 min and observe under a microscope to check the efficiency of cephalexin treatment (Figure 1A).

      4. Based on our experience, this process usually takes about 2 h. Once the snake-like filaments are longer than 100 µm, leave the culture at room temperature. Drop the temperature of the shaker to 37°C (typically takes about 15 min).

      5. Add 0.8 mM IPTG to the culture and place it back in the incubator at 37°C and 120 rpm for another hour.

   4. Spheroplast preparation

      1. Following induction, divide the 60 ml cell culture in equal volumes into three 50 ml centrifuge tubes. Centrifuge the culture at low speed 453 × g at 4°C for 15 min. Discard the supernatant carefully, as the bacteria does not attach to the bottom of the tube firmly. Place the three tubes with bacterial pellets on ice and resuspend each of the pellets gently in 3 ml of 1 M sucrose.

      2. Add 240 μl 1 M Tris, pH 8.0, 67,500 units of Ready-Lyse Lysozyme solutions (Lucigen), 1,600 units OmniCleave Endonuclease (Lucigen), and 20 μl of 500 mM EDTA to the resuspended cells and incubate at room temperature. After 10 min of incubation, add 1 ml of stop buffer (see Recipes) to one of the three tubes and mix by gently swirling the tube. Place the tube on ice. Similarly, add the stop solution to the other two tubes after 20 min and 40 min of incubation, respectively, and mix by gently swirling the tubes. Place all tubes on ice immediately after adding the stop buffer. Take 20 μl of the solution from each spheroplast preparation and examine under the microscope to identify the optimal spheroplasts preparation out of the three, as shown in Figure 1B.

      3. Under a phase contrast microscope, the well prepared spheroplasts appear like dark gray circles (Figure 1C). Choose the preparation with the highest number of dark gray spheroplasts. Aliquot the spheroplasts into PCR tubes (50 μl per tube), flash freeze in liquid nitrogen, and store at -80°C until further use. If stored at -80°C, the spheroplasts can be used for recording for 6 months.

         
         

      
      Figure 1. Illustration of spheroplasts preparation. A. An image of E. coli cell culture before cephalexin treatment. The scale bar equals 50 µm. B. Illustration of ‘snake-like’ bacterial filaments after 2 h of 50 μg/ml cephalexin treatment under phase contrast microscope. Scale bar is 100 µm. C. Phase contrast image showing spheroplasts after the digestion of E. coli outer membrane with lysozyme and EDTA. The dark gray circle (red arrow) is a good spheroplast and can form giga-ohm seals while the transparent one (blue arrow) is not suitable for patching. The scale bar indicates 50 µm.

      
      

2. Patch clamping the spheroplasts

   1. Solutions: Bacteria have a higher cytosolic osmolarity compared to cell lines such as HEK293 or X. Laevis oocytes. In our experiments, we recorded currents from Mthk, which is a calcium-activated potassium channel, and therefore the pipette solution (Intracellular solution) (see Recipes) and the bath solution (see Recipes) have differernt calcium concentrations. The osmolarity for different concentrations of calcium should be adjusted with sucrose accordingly.

   2. Glass electrodes: We chose 100 µl Drummond calibrated micropipettes. The micropipettes are made of N51A borosilicate glass and have an internal diameter of 1.3 mm and outer diameter of 1.68 mm. Pull the pipettes with an electrode puller (Sutter Instruments Model P97), so that the bubble number (described below) is between 5.0-5.5.

   3. As the E. coli spheroplasts are fairly small in size (5-10 µm), the tip size of the glass electrodes needs to be approximately 1 μm, which is too small to be accurately evaluated under a light microscope. Thus, to calibrate the tip size of the glass electrodes, which is extremely important for the success rate of patching spheroplasts, ‘bubble number’ should be used as a standard (Mittman et al., 1987). Pull a glass electrode and connect it to a 10 ml syringe with an aspirator for forming an airtight seal as shown in Figure 2A. Submerge the micropipettes into methanol to about 1 cm deep and slowly press the plunger from 10 ml graduation until a fine stream of bubbles appears in the methanol (Figure 2B, 2C). The volume read out at this moment is the ‘bubble number’ (or the volume of air remaining in the syringe is the bubble number) of the micropipette and this number should ideally be between 5.0 to 5.5. In our experience, a ‘bubble number’ between 5.5 to 6 is also usable (see Video 1). If the bubble number is outside this range, the electrode pulling program should be modified accordingly until the tip size falls in this range.

      
      

      
      Figure 2. Bubble number measurement. A. Illustration of a 10 ml syringe connected with an aspirator. B. Submerge the tip of the micropipette into a vial containing methanol with the plunger set at 10 ml. C. Apply positive pressure on the plunger. Watch for the first appearance of bubbles in the methanol. The corresponding reading on the syringe is the bubble number. Pipettes with a bubble number between 5.0 and 5.5 are ideal for patching spheroplasts.

      
      
      
      Video 1. Bubble number measurement.

      
      

   4. Spheroplast patching

      Note: It is very important to keep the glass tip clean to form a giga-ohm seal, so a positive air pressure should be applied before the electrode is immersed into the bath solution.

      Spheroplasts do not stick to the bottom of the glass surface of the chamber. If the positive pressure applied is too large, then the spheroplasts are pushed away. To overcome this problem, use a very low positive pressure (5 to 10 millibars of pressure is sufficient). Alternatively, release the pressure right before you approach the spheroplast. Another related issue is that it is difficult to see the dent on the spheroplast caused by the positive pressure from the glass electrode tip; while we do not have a solution for this, with some practice researchers can get a feeling of when to apply a negative pressure to form a giga-ohm seal with the electrode.

      
      

3. Finding a good spheroplast

   When imaged with a phase contrast microscope, a small percentage of spheroplasts will appear to have lost their interior phase contrast. Based on our experience, these types of spheroplasts could not form a giga-ohm seal with the glass electrode.

   Forming an inside-out patch: Once the giga-ohm seal is formed, move the glass microelectrode away from the bottom of the chamber (Caution: do not move it out of the bath solution). Apply a sharp but gentle movement on the head-stage by flicking it. This would apply a dramatic shear force between the tip of the microelectrode and the spheroplast. Sometimes this step will cause a loss of the giga-ohm seal.

Recipes

1. LB medium

   25 g granulated LB

   1 L of ddH₂O

2. Stop buffer

   0.8 M sucrose

   80 mM Tris, pH 8.0

   12 mM MgCl₂

3. Pipette solution (Intracellular solution)

   150 mM KCl

   20 mM MgCl₂

   15 mM Tris, pH 8.0

   0.1 mM CaCl₂

   450 mM sucrose

4. Bath solution

   150 mM KCl

   20 mM MgCl₂

   15 mM Tris, pH 8.0

Acknowledgments

We would like to thank Dr. V.A. Klenchin for his help on various aspects of this protocol. We would like to thank Dr. Sergei Sukharev at the University of Maryland for his help with patch clamping bacterial spheroplasts. We thank National Institutes of Health (NS-116850, NS-101723, NS-081293, GM131662), McDonnell Center for Neuroscience, CIMED and Department of Anesthesiology for research support. This protocol was derived from the original research paper “Activation of the archaeal ion channel MthK is exquisitely regulated by temperature” (Jiang et al., 2020).

Competing interests

There are no conflicts of interest or competing interests.

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