Bactiter glo microbial cell viability assay kit

For cycling assays, cells were grown, induced, and washed as described for the oxygen experiments. All cycles were fully induced (1 mM IPTG). The washed cells were used to inoculate pre-warmed fresh media + amp + IPTG to an OD₆₀₀ of 0.2. For measuring MG1655 and MG ΔptsI ATP levels, 20 h LB (+ 30 μg/mL kan for MG ΔptsI) overnight cultures were washed with MOPS 10 mM glucose medium to remove trace LB components and then used to inoculate 20 mL MOPS 10 mM glucose medium in a 250 mL baffled flask to an OD₆₀₀ of 0.05. A higher cell density was necessary for ΔptsI experiments because the ΔptsI mutant will not grow and the cultures were washed and concentrated to an OD₆₀₀ of 0.2. Cultures were grown for 5 hours at 37°C with 250 rpm shaking (one doubling after leaving lag phase for MG1655), then washed and adjusted to an OD₆₀₀ of 0.2 as described above. ATP was measured using the BacTiter-Glo Microbial Cell Viability Assay Kit (Promega) according to the manufacturer's instructions. Briefly, 100 μL of the OD₆₀₀ 0.2 culture was mixed with 100 μL BacTiter-Glo reagent in an opaque 96-well plate. After five minutes at room temperature, luminescence was read on a Synergy H1 Microplate Reader (BioTek Instruments, Inc.). An ATP standard curve was used to calculate ATP concentrations.