Coupled Perturbations of Gene Circuit Dynamics by Resource Competition and Growth Dilution

Abstract

Resource competition and growth-mediated dilution are major sources of context dependence in synthetic gene circuits. However, their coupled effects on circuit behavior remain insufficiently dissected. This chapter presents a step-by-step experimental workflow for constructing and characterizing inhibitory genetic cascades in E. coli to examine whether and how circuit dynamics depend on these two contextual factors. The protocol details the design and assembly of inhibitory circuits with tunable promoter strength, ribosome binding site strength, and plasmid copy number. Methods are provided for quantifying circuit dynamics using plate-reader fluorescence assays under varying inducer doses, with and without preinduction, and across different growth conditions. Finally, data processing and analysis procedures are outlined to characterize module interdependences and expression dynamics. Collectively, this protocol provides a practical framework for investigating how shared cellular resources and growth processes shape gene circuit behavior and serves as a guide for studying multiple context-dependent effects in synthetic biology.

Keywords: Gene expression dynamics; Growth-mediated dilution; Host-circuit interactions; Inhibitory genetic cascades; Resource competition.