How Bacterial Growth-Phase Reshapes Synthetic Gene Circuit Function

Abstract

The development of biological engineering tools have improved the ability to embed novel functions into living organisms. Concurrently, our understanding of the interface between synthetic genetic circuits and their host has accelerated in the past decade, enabling the development of context-aware predictive models. However, long-term circuit function remains difficult to predict when designs operate in field-relevant conditions. Specifically, while synthetic genetic circuits are typically characterized during exponential growth, these must operate for extended periods of time in nutrient-limited conditions and where bacteria enter stationary growth phase. In this work, we construct a mathematical model that captures the dynamics of a genetic circuit as cells transition into stationary growth phase. To achieve this, we construct synthetic genetic circuits with fluorescent reporter output and measure plasmid DNA copy number, mRNA level, protein level, and live cell count over the course of one week in stationary growth phase. We find that transcription rates plummet shortly after entering stationary phase but settle at a basal value that depends on both the promoter and ribosome binding site. Translation rates also decline in stationary phase, but protein accumulation due to lack of cell division can increase protein levels well beyond exponential phase. Our model recapitulates these findings and is capable of predicting the output of new genetic circuits. This model can serve as a tool to determine how a genetic circuit design may change on longer time scales where cells enter stationary growth phase.