Materials and Reagents

1. Glass test tubes (approximately 10 x 90 mm)
   
2. E. coli MWC215 (Sm^R ndh::Cm^R) or GR70N (wild type or mutated) cells grown under aeration
   
3. Luria Broth or other medium suitable for aerobic growth of E. coli culture
   
4. Antibiotics, as required for particular E. coli strain
   
5. Lysozyme
   
6. Ethylenediaminetetraacetic acid (EDTA)
   
7. Magnesium sulfate (MgSO₄)
   
8. Dithiothreitol (DTT)
   
9. Phenylmethylsulfonyl fluoride (PMSF)
   
10. Ethanol
    
11. Liquid N₂
    
12. Electric potential-sensitive probe Oxonol VI (Sigma-Aldrich, catalog number: 75926 )
    
13. Ammonium sulfate, (NH₄)₂SO₄
    
14. Decylubiquinone (Sigma-Aldrich, catalog number: D7911 )
    
15. Potassium cyanide (KCN)
    
16. Nicotinamide adenine dinucleotide reduced (NADH)
    
17. Rolliniastatin
    
18. Ubiquinone 1 (Sigma-Aldrich, catalog number: C7956 )
    
19. Adenosine triphosphate (ATP)
20. Aurovertin B (Sigma-Aldrich, catalog number: A5297 )
    Note: This product has been discontinued.
    
21. Tris-HCl
    
22. Sucrose
    
23. HEPES
    
24. Potassium hydroxide (KOH)
    
25. Potassium chloride (KCl)
    
26. Magnesium chloride (MgCl₂)
    
27. 3-(N-morpholino)propanesulfonic acid (MOPS)
    
28. 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP)
    
29. The medium (see Recipes)
    
30. Buffer A (see Recipes)
    
31. Buffer B (see Recipes)
    
32. Buffer C (see Recipes)
    
33. Buffer D (see Recipes)
    

Equipment

1. 200 ml Erlenmeyer flasks
   
2. 2 L Erlenmeyer flasks
   
3. Shaker, such as Sertomat R/Sertomat HR or other suitable
   
4. Centrifuges, such as (Thermo Fisher Scientific, model: Sorvall RC 5C Plus )
   
5. Rotors (Thermo Fisher Scientific, Thermo Scientific^TM, models: SS-34 , SLA 3000 ; Beckman Coulter, model: Ti70 )
   
6. Sonicator (Emerson, model: Branson Sonifier Cell Disruptor B15 ) or other with the tip diameter 10-12 mm
   
7. UV/VIS spectrophotometer (i.e., Ocean Optics, model: HR2000 )
   

Procedure