Beta-Lactamase Induction (Crude Preparation)

Wednesday, October 01, 2003

Description
REFERENCE: Gootz & Sanders. A.A.C.

Procedure
1. Inoculate regular growth medium with overnight culture of bacteria. (1/20 - 1/50)
2. Grow to OD600 = 0.3 under usual conditions (37oC, shaking).
3. Add sterile antibiotics so the final concentration is 1/2 the MIC of the antibiotic for the bacterium tested. Include a control of no antibiotic for each strain.
4. Grow for another 3 hours. Check final OD600. (Cells should still grow.)
5. Add 1mM sodium azide (final concentration) to stop cell metabolism.
6. Centrifuge cells in Sorvall 7,000 rpm, 15 min. in G3 or 10,000 rpm, 5 min. in SS34.
7. Wash 2X in 5mM sodium Hepes buffer pH 7.2.
8. Resuspend each pellet in small volume of buffer (approx. 2 mls) (or 1/100 of original growth volume).
9. 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.
10. Spin down in ultracentrifuge 70.1 Ti, 23K, 30', 5oC.
11. 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)
12. 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.

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Beta-Lactamase Induction (Crude Preparation)