Unveiling Heterogeneity in Optogenetic Circuit Dynamics via Single-Cell Time-Lapse Imaging*

Advancements in gene modification technology have enabled complex tuning of protein expression through gene regulatory circuits. Traditional methods such as flow cytometry or RTqPCR provide single time point data, overlooking temporal heterogeneity in expression kinetics. Efficiently characterizing these kinetics is essential to describing how these systems can be tuned to gain precise control over expression. Here, we perform time-lapse imaging of single mammalian cells engineered with chemical and light-inducible negative feedback gene circuits, allowing us to quantify individual cell expression kinetics. This facilitates the characterization of synthetic gene circuits' response to both induction methods and describes the variability in resulting protein expression across isogenic populations. This technique evaluates how modifications to gene expression circuits contribute to the temporal heterogeneity in expression kinetics. To achieve this, we modify cell culture substrates such that thousands of single cells are aligned onto adhesion patches. Time-lapse imaging is then performed to observe the circuits' expression via a fluorescent reporter. Through automated image analysis, individual cell fluorescent intensity time traces are quantified, and computational methods are used to infer the kinetic rate parameters of the expression dynamics. This method provides the means of quantifying interpopulation heterogeneity in expression kinetic rate parameters.