Quantitative Measurement of Synthetic Repression Curves Reveals Design Challenges for Genetic Circuit Engineering...

Despite the fact that microbes in natural environments spend most of their time in growth arrest, we understand little about how this physiological state affects the performance of engineered genetic circuits. Here, we measure repression curves from a library of genetic NOT gates at single-cell resolution in Escherichia coli under both active growth and growth arrest to systematically investigate how growth arrest affects circuit behavior. We find that the impact of growth arrest on circuit performance is almost entirely dominated by a >100-fold reduction in unrepressed expression levels. Growth arrest caused gene expression noise to increase only moderately and had minimal impact on the sensitivity and sharpness of the repression curves. Our work shows both that conventional genetic circuit design paradigms are currently insufficient to develop circuits that can function properly under growth arrest, and that addressing the reduction in just a single performance parameter would be sufficient to resolve this problem. This work expands our understanding of bacterial gene regulation under growth arrest and lays the groundwork for new design paradigms that will be essential to ensure the safe and reliable performance of synthetic biology systems in real-world environments.