Resource Competition and Growth Dilution Modulate Synthetic Gene Cascade Dynamics

In synthetic biology, a key goal is to design robust and stable genetic circuits with accurate and predictable behavior. Modularity is a central principle in circuit design, enabling the construction of complex systems from smaller, well-characterized parts. However, resource competition presents a major obstacle to modularity by disrupting gene expression dynamics. Here, we constructed and characterized a library of inhibitory genetic cascades with varied promoter strengths, RBS strengths, and plasmid backbones. We found that increasing the expression of the downstream module could unexpectedly lead to a reduction in the expression of the upstream regulatory module by competing for shared cellular resources. These results indicate that resource limitations can transform a unidirectional inhibitory cascade into an unintended feedback loop. In addition, we found that growth-mediated dilution can reshape gene expression patterns, further influencing circuit dynamics. Together, these findings underscore the critical roles of both resource competition and growth dilution in shaping the behavior of synthetic gene circuits.