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β-lactamase activity assay

QW Qinqin Wang
SW Shaodong Wei
AS Ana Filipa Silva
JM Jonas Stenløkke Madsen
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A colorimetric assay based on the hydrolysis of nitrocefin was used to determine the total intracellular and extracellular β-lactamase activity in LB broth cultures (grown for 24 h) [23]. 500 µl of 24-h-grown LB broth culture was centrifuged at 3000 × g for 15 min, and the obtained supernatant was used to measure the activity of extracellular β-lactamases. To measure intracellular activity, the pellet was resuspended in 500 µl PBS containing 1 mg/ml lysozyme and 2 mM EDTA, and then incubated at 37 °C for 1 h. Thereafter, 2 µl of sample was mixed with 198 µl PBS containing 20 µg nitrocefin in a 96-well plate, and placed in a spectrophotometer to detect and record the OD490 value every 5 min. All measurements were repeated three times.

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