Fig. 4.

Longevity of the cellular machinery within SimCells (protein synthesis, regulation, and degradation) was elucidated by indirectly tracking the cellular availability of ATP/NADH. (A) SimCells could produce luminescence for about 10 d, indicating they had sufficient energy currency to sustain functionality for a significant amount of time. The strain with the glycolysis pathway had a significant energy advantage over the strain without glycolysis in the early days. However, this difference in luminescence production leveled off after day 5. Unpaired (independent) t tests were performed: *P < 0.005, **P < 0.001. (B) Fluorescence detected from unstable mCherry stopped increasing after 10 d, which is likely the point where transcription and translation did not have enough energy to be operational. This corresponds to the availability of ATP as suggested by luminescence production (A). SimCells had detectable levels of unstable mCherry for a long time (28 d) because the activity of proteases is ATP-dependent. Unpaired (independent) t tests were performed: *P < 0.001. Data show means ± SE, n = 4.