Beta-Lactamase Induction (Crude Preparation)
Categorized: Antibiotics and Antimicrobial Peptides
Gootz & Sanders. A.A.C.
- Inoculate regular growth medium with overnight culture of bacteria. (1/20 – 1/50)
- Grow to OD600 = 0.3 under usual conditions (37oC, shaking).
- Add sterile antibiotics so the final concentration is 1/2 the MIC of that antibiotic for that bacteria. Include a control of no antibiotic for each strain.
- Grow for another 3 hours. Check final OD600. (Cells should still grow.)
- Add 1mM NaAz (final concentration) to stop cell metabolism.
- Centrifuge cells in Sorvall 7K, 15′ in G3 or 10K, 5′ in SS34.
- Wash 2X in 5mM Hepes pH 7.2.
- Resuspend each pellet in small volume of buffer (approx. 2 mls) (or 1/100 of original growth volume).
- Place each sample in small plastic test tube. Keeping samples on ice, sonicate each one 3X for 1 minute bursts, using small probe at 35%. Alternatively: add polymyxin B to a final concentration of 1mg/ml or French Press in the small cell.
- Spin down in ultracentrifuge 70.1 Ti, 23K, 30′, 5oC.
- Carefully pipet off supernatant. This is the crude B-lactamase extract. Store: short term: 4oC (loses activity with time) long term: -20oC (loses activity with freeze-thawing)
- By doing protein assays and nitrocefin assays on each sample, you can then determine the specific activity. See “permeability Calculations: 10 easy steps” for calculations of specific activity.