Real-Time Optogenetic Control of Intracellular Protein Concentration in Microbial Cell Cultures

Real-time light-driven temporal control of gene expression and protein concentration in S. cerevisiae
Justin Melendez, Benjamin Oakes, Michael Patel, Marcus Noyes and Megan McClean
Department of Biomedical Engineering, University of Wisconsin, Madison
Studies of signaling and transcriptional networks are limited by the tools currently available to systematically perturb and interrogate such networks. Standard genetic tools such as deletion, overexpression, and mutation are effective for identifying network components but less suitable for understanding kinetics and identifying feedback interactions. Here we describe the development of a system for real-time light-inducible control of gene expression in S. cerevisiae. Controlling transcription enables fine-tuned temporal modulation of protein levels useful for interrogating genetic networks.
Light-induction is achieved through fusion of the Arabidopsis thaliana proteins cryptochrome 2 (CRY2) and its interaction partner (CIB1) to appropriate DNA-binding and activation domains (1). CRY2 and CIB1 naturally dimerize on blue-light exposure, bringing the DNA-binding domain and the activation domain together to drive expression of the desired gene. We characterize the kinetics, off-target effects, and dose-response of this induction system and develop improved fusion proteins to allow for specific and controllable induction.
To implement real-time control we developed an integrated robotics system. Cells from a steady-state chemostat culture are continuously imaged for fluorescence. Information from this imaging is used to adjust the inducing light levels to vary the concentration of the fluorescently-tagged protein. By controlling the light-intensity, pulse frequency, and pulse duration we are able to maintain a steady- state protein concentration of our choosing. By modulating the inducing light we can produce dynamic protein level perturbations such as pulses and oscillations. This tool allows us to flexibly perturb protein levels to interrogate the kinetics and structure of genetic networks.
(1) Kennedy, MJ et al 2010 Nature Methods 7: 973-975