Engineered temperature compensation in a synthetic genetic clock

Synthetic biology promises to revolutionize biotechnology by providing the means to reengineer and reprogram cellular regulatory mechanisms. However, synthetic gene circuits are often unreliable, as changes to environmental conditions can fundamentally alter a circuit's behavior. One way to improve robustness is to use intrinsic properties of transcription factors within the circuit to buffer against intra- and extracellular variability. Here, we describe the design and construction of a synthetic gene oscillator in Escherichia coli that maintains a constant period over a range of temperatures. We started with a previously described synthetic dual-feedback oscillator with a temperature-dependent period. Computational modeling predicted and subsequent experiments confirmed that a single amino acid mutation to the core transcriptional repressor of the circuit results in temperature compensation. Specifically, we used a temperature-sensitive lactose repressor mutant that loses the ability to repress its target promoter at high temperatures. In the oscillator, this thermoinduction of the repressor leads to an increase in period at high temperatures that compensates for the decrease in period due to Arrhenius scaling of the reaction rates. The result is a transcriptional oscillator with a nearly constant period of 48 min for temperatures ranging from 30 degrees C to 41 degrees C. In contrast, in the absence of the mutation the period of the oscillator drops from 60 to 30 min over the same temperature range. This work demonstrates that synthetic gene circuits can be engineered to be robust to extracellular conditions through protein-level modifications.