Precise, Single-Cell Optogenetic Induction of Antibiotic Resistance for Selection of Escherichia coli

Optogenetic control, or the use of light to control gene expression, is a valuable tool for both elucidating and influencing cell behavior. Advantages of optogenetic tools over chemical induction include high temporal and spatial precision, quickly reversible kinetics, and ease of integration with computational control. Here, we use the CcaSR two-component system, which activates transcription in the presence of green light and represses transcription in the presence of red light, to control expression of a tetracycline resistance gene both in bulk culture and at the single-cell level. We show that cells expressing this system are reliably killed when exposed to red light in the presence of inhibitory levels of tetracycline, while cells exposed to green light continue to grow at a high rate. Using this system in combination with real-time cell segmentation and feature extraction, it is possible to target cells for selection based on desired phenotypic traits, such as size, expression of a fluorophore, or a certain growth rate. We present results demonstrating successful selection based on expression of a fluorophore and progress towards recovering cells from the device, which can then be used for additional rounds of selection or for other purposes. The ability to dynamically target a large number of individual cells based on arbitrary traits will make this system useful for selecting cells based on features that are not easily identified by conventional means, such as dynamic behavior over the course of many hours. Additionally, the high precision and reliability of the described antibiotic resistance induction setup make it well-suited for a number of other potential applications, including exploring the dynamics of antibiotic resistance mechanisms or controlling the growth rate of cultures.