CHARACTERIZATION OF RECOMBINASE ACTIVITY ACROSS CELLULAR GROWTH PHASES

Recombinases, which are enzymes that catalyze targeted DNA modifications, hold significant potential in synthetic biology. Their capacity to precisely manipulate genetic material enables the construction of complex genetic circuits that can be dynamically reconfigured in response to environmental stimuli. Such capabilities are essential for developing synthetic organisms tailored for specific functions, including biosensing, bioremediation, and pharmaceutical production. Therefore, characterizing the dynamics of recombinases is crucial for the innovative design of cellular devices. A deeper understanding of how recombinases interact with DNA in various conditions can improve the efficiency and control of genetic modifications, thereby enhancing both the functionality and reliability of synthetic biological systems. This study presents a detailed examination of the dynamics and efficiency of the serine recombinase Bxb1, focusing on its behavior under controlled expression in Escherichia coli. It highlights the significant influence of cellular growth phases (exponential and stationary) on the efficiency of recombinase-mediated gene expression. Our findings show that recombinase activity is maintained during stationary phase, which is critical to ensure ongoing recombination without the need for the continuous presence of an inducer. In experiments, we quantified the recombination efficiency of Bxb1 by monitoring expression of green fluorescent protein (GFP) as a reporter. Optimal expression of Bxb1, which maximized the recombination efficiency, occurred during exponential phase. However, once the culture reached stationary phase, accumulated Bxb1 continued to facilitate recombination, although GFP expression levels plateaued due to reduced cellular activity. These insights are relevant for synthetic biology applications where precise control of genetic functions is necessary.