Characterization of recombinase activity across cellular growth phases.

Recombinases are essential enzymes in synthetic biology and biomedical research, enabling site-specific DNA modifications for applications such as gene therapy, the generation of transgenic models, and the construction of genetic circuits. Recombination efficiency depends on several factors, including intracellular recombinase concentration and the growth phase of the host cells. Although recombination is typically studied during exponential growth, the effects of stationary-phase dynamics on recombinase activity remain poorly understood. In this study, we examine how bacterial growth phase influences recombinase-mediated DNA modifications, using the serine recombinase Bxb1 as a model. We engineered a genetic system in Escherichia coli to quantify intracellular Bxb1 levels and to measure recombination efficiency across different growth phases. Our results reveal a quasi-linear relationship between recombinase concentration and recombination efficiency during exponential growth, up to a saturation point. Notably, recombination continues in the stationary phase following recombinase induction in exponential phase, despite the decline in plasmid gene expression. Cells that undergo recombination during the stationary phase show significantly higher recombination efficiencies upon re-entering exponential growth than those maintained in exponential phase throughout. These findings highlight the importance of induction timing in optimizing recombinase-based genetic modifications. Specifically, inducing recombinase expression just before the onset of stationary phase can enhance recombination efficiency while minimizing the need for high expression levels. Moreover, the observed quasi-linear relationship during exponential growth provides a framework for tuning gene expression with precision. Overall, this work offers new insights into leveraging bacterial growth dynamics to improve the design and control of synthetic genetic systems.