Optogenetic selection for dynamic phenotypes in bacteria

The goal of this project is to develop methods for screening and selecting individual bacteria based on dynamic, time-varying traits, such as identifying cells that exhibit a rapid response to an environmental change. Screening and selection are powerful tools that enable efficient identification and isolation of cells with specific properties, however most current approaches, such as those based on flow cytometry, only observe cells at a single time point. This "snapshot" view precludes assessment and selection based on dynamic features, such as the speed of a response, the ability of a cell to adapt, or a cell's growth rate. To achieve the goal of developing selection methods suited for isolating cells with dynamic traits, the researchers will use light-inducible constructs and spatially patterned light illumination to activate expression of antibiotic resistance genes in targeted cells. The researchers will demonstrate the potential of screening for dynamic phenotypes by conducting comprehensive experiments to assess temporal responses in promoters that are activated under nutrient stress. These scientific activities are complemented by educational outreach initiatives. These include partnering with the STEM Pathways program to host events for high school students including a coding hackathon on image processing and a mini-jamboree about synthetic biology design. In addition, the project provides training opportunities for undergraduate students. The primary source of technical innovation in this project is the development of tools for optogenetic selection of cells. Specifically, the researchers will develop and optimize genetic constructs where light can be used to trigger expression of an antibiotic resistance gene. Two complementary genetic tools will be developed, based on a blue-light inducible version of Cre recombinase and a red/green light inducible CcaS- CcaR optogenetic system. The researchers will optimize light intensities and exposure times to turn on drug resistance for cells growing within a microfluidic device. Using an assay to select cells based on expression of a fluorophore, the researchers will quantify the sensitivity and specificity of the approach. The ultimate utility of these tools is in their ability to select cells with dynamic phenotypes to generate novel biological insight. To test this potential, the researchers will focus on (p)ppGpp-responsive promoters, efficiently measuring temporal and stochastic properties of promoters with a pooled library approach coupled with dynamic selection. Overall, the project will introduce optogenetic tools for selection using light and will demonstrate the power of these tools to reveal new insights into how individual bacteria cope with environmental stress. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.