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 inducible 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. In experiments, we quantified the recombination efficiency of Bxb1 by monitoring expression of green fluorescent protein (GFP) as a reporter. Interestingly, the experiments conducted show that the growth phase of the culture plays an important role in recombination efficiency. Forcing recombination events mediated by Bxb1 to occur during the stationary phase, instead of the exponential phase as usual, results in significant increases in recombination efficiency. Thus, lower concentrations of Bxb1 achieve higher recombination efficiencies when these events occur during the stationary phase, compared to higher concentrations of Bxb1 when recombination occurs during the exponential phase.