282874 Investigating the Effects of Dynamic External Stimuli On Single Cell Fitness and Gene Expression in Escherichia Coli
In this work, we study the bacterial cellular response to time-dependent external stimuli in single living cells. We developed a microfluidic platform for single cell analysis that allows for dynamic control of well-defined environmental growth conditions and culture media. Using this platform, we studied the effect of small molecule inducers on gene expression in the lacoperon using fluorescent reporter proteins and cell growth rates as a proxy of cellular fitness. We applied temporally varying inducer concentrations by translating single cells between two adjacent fluid streams containing either growth medium or growth medium and inducer. We observed that single cell gene expression depends on growth rate and frequency of exposure to inducer concentrations. Single cell induction experiments are compared to control experiments with and without continuous fluid flow in microfluidic channels.
A microfluidic-based hydrodynamic trap recently developed in our lab facilitates single cell analysis for these experiments. The hydrodynamic trap enables confinement and manipulation of single cells in free solution using the sole action of fluid flow. Automated feedback control is integrated into the device using an “on-chip” valve, which allows for precise confinement of cells in free solution. Using this device, cells are confined at a fluid stagnation point generated in a cross-slot microfluidic geometry, thereby enabling non-perturbative trapping of cells for long time scales.
Using optical microscopy, we observe single Escherichia coli cells growing and dividing both at room temperature and at 370C, and cell division rates in the microfluidic trap compare favorably to the growth rates of E. coli measured in bulk studies. We demonstrated proof-of-principle induction of T5 promoter system with IPTG using the hydrodynamic trap. We are currently using this system to research the effect of alternating inducer concentrations at different frequencies on gene expression in order to determine a cutoff frequency for transcription of the lac operon. We anticipate that the microfluidic-based trap is an ideal platform to study cellular regulation and gene network dynamics of single living cells in free solution.
- Tanyeri M., Johnson-Chavarria E.M. and Schroeder C.M., "Hydrodynamic Trap for Single Particles and Cells", Applied Physics Letters, 96, 224101 (2010)
- Johnson-Chavarria E.M., Tanyeri M. and Schroeder C.M., "A Microfluidic-based Hydrodynamic Trap for Single Particles", Journal of Visualized Experiments, 47, (2011).
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