Ethidium bromide uptake was monitored as described earlier (43 (link)). MabWt, MabΔembC, and MabΔCMab were grown to an OD600 of 0.6–1.0. Bacteria were collected by centrifugation and resuspended in uptake buffer (PBS, 0.05% Tween 80, pH 7.0) to an OD600 of 0.5, followed by the addition of 25 mM glucose for energization. Ethidium bromide (1 μg/mL) was added, followed by real-time measurement in a fluorescence spectrometer (PerkinElmer) with excitation and emission wavelengths set at 520 and 595 nm, respectively. Results were normalized against fluorescence of ethidium bromide and are depicted relative to the highest fluorescence measured. The experiment was performed in triplicate and repeated three times.
Other organizations :
University of Chinese Academy of Sciences, Guangzhou Institutes of Biomedicine and Health, Shenzhen Third People’s Hospital, State Key Laboratory of Respiratory Disease, Chinese Academy of Sciences, University of Science and Technology of China, Hefei Institutes of Physical Science, Anhui University, Vavilov Institute of General Genetics, University of Otago, Maurice Wilkins Centre, University of Auckland, Guangzhou Medical University, First Affiliated Hospital of Guangzhou Medical University, Guangzhou Regenerative Medicine and Health Guangdong Laboratory
Resuspension in uptake buffer (PBS, 0.05% Tween 80, pH 7.0) to an OD_600 of 0.5
Addition of 25 mM glucose for energization
Ethidium bromide concentration (1 μg/mL)
Excitation and emission wavelengths (520 and 595 nm, respectively)
controls
Fluorescence of ethidium bromide (used for normalization)
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