Huang, H.; Jafarbeglou, F.; Dunlop, M.

Microbial optogenetic tools can regulate gene expression with high spatial and temporal precision, offering excellent potential for single-cell resolution studies. However, bacterial optogenetic systems have primarily been deployed for population-level experiments. It is not always clear how these tools perform in single cells, where stochastic effects can be substantial. In this study, we focus on optogenetic Cre recombinase and systematically compare the performance of three variants (OptoCre-REDMAP, OptoCre-Vvd, and PA-Cre) for their population-level and single-cell activity. We quantify recombination efficiency, expression variability, and activation dynamics using reporters which produce changes in fluorescence or antibiotic resistance following light-induced Cre activity. Our results indicate that optogenetic recombinase performance can be reporter-dependent, suggesting that this is an important consideration in system design. Further, our single-cell analysis reveals highly heterogeneous activity across cells. Although general trends match expectations for mean levels of light-dependent recombination, we found substantial variation in this behavior across individual cells. In addition, our results show that the timing of recombinase activity is highly variable from cell to cell. These findings suggest critical criteria for selecting appropriate optogenetic recombinase systems and indicate areas for optimization to improve the single-cell capabilities of bacterial optogenetic tools.