Single Cell Response to Periodic Environmental Stimuli using a Microfluidic Bioreactor

Abstract

Recent studies have reported the ability of biological systems to implement low-pass filters to distinguish high frequency noise in environmental stimuli from lower frequency input signals. This cellular adaptation is critical for survival in fluctuating environmental conditions, yet we still lack a complete understanding of this phenomenon. Over the last several years, microfluidic-based techniques have been developed to study inducible gene expression at the single cell level, albeit without the ability to control external stimuli with precise methods. Most are limited by long diffusive timescales to alternate environmental concentrations. In this work, we report a microfluidic-based platform for single cell analysis that provides dynamic control over periodic, time-dependent culture media. Single cells are confined in free solution by the sole action of gentle fluid flow, thereby enabling non-perturbative trapping of cells for long time scales. Using fluorescent reporter proteins and cell growth rates as a proxy of cellular fitness, we investigated the effect of small molecule inducers on gene expression of the lac operon in Escherichia coli. Single cell division rates in the microfluidic trap compare favorably to growth rates at room temperature and 37oC measured in batch culture. We observed that single cell gene expression depends on the correlation between growth rate and frequency of exposure to inducer concentrations. In addition, we performed diffusion experiments of TetR:EYFP on a TetO binding array by rapidly switching to concentrations of aTc. Overall, this microfluidic bioreactor provides a direct method for sustaining periodic environmental conditions, measuring growth rates, and detecting gene expression of single cells suspended in free solution.