Rapid Microfluidic Assay for Bacterial Electrotransformation

Electroporation results from exposure of cells to electric fields of sufficient strength to disrupt the plasma membrane. The local trans-­�membrane voltage (TMV) significantly increases during exposure of cells to external electric fields. When the local TMV exceeds a critical threshold, pores are created on the cell membrane, allowing transport of ions and macromolecules (e.g. DNA/RNA) across the membrane. There is vast empirical literature establishing protocols to increase the electrocompetency (capacity for DNA uptake by electroporation) of cells, a process that is currently time consuming and lacks real-­�time feedback. Despite the tremendous need, there are currently no protocols for improving bacterial electrocompetency without relying upon time-­�consuming empirical experimental processes.
Here, we present a rapid microfluidic platform capable of determining electric field thresholds for electroporation. In our microfluidic platform, a converging microchannel generates a linear gradient of electric field. Fluorescence-­�encoded DNA plasmids or nucleic acid stains permeate cell membranes in regions where the electric field is sufficiently high. We correlate the fluorescent region of bacteria with the range of electric fields that results in electroporation. Analysis of the fluorescence images from a single experiment provides electroporation conditions that would otherwise require hundreds of experiments conducted in parallel.
Our microfluidic platform quantitatively characterizes the electric field required for successful electroporation of microorganisms, one of the parameters for determining electrocompetency. The assay is based on a PDMS microfluidic channel with an active electroporation region spanning 2-­�4 mm that enables the generation of high electric fields (5-­�15 kV/cm) at applied voltages of 1000-­�3000 V. This geometry has been optimized to yield a linear variation of electric field strength, facilitating correlation of cell location within the chip and local electric field.
Under typical operation, the inlet to the device is connected via flexible tubing and a hollow stainless steel needle to a syringe pump for controlled delivery of the cell-­â?dye mixture to the microfluidic device. The outlet is connected via a needle and tubing to an external reservoir, enabling the sample to be collected for downstream analysis. The needles serve as the electrical contacts required to deliver the electric field from the MicroPulserTM (Bio-­â?Rad, Hercules, CA) to the sample within the channel. The assay utilizes SYTOX® (Life Technologies, Grand Island, NY), a dye which cannot penetrate the intact cell envelope and only fluoresces once it binds to intracellular DNA; therefore, it is an effective marker for determining electroporation conditions. We have utilized numerical simulations in COMSOL® (Burlington, MA) to develop a â??rulerâ? that allows us to determine the electric field that correlates with the onset of electroporation. The simulations allow us to quantitatively determine the electric field range in regions of dye uptake.

We envision this rapid microfluidic platform as a tool to enable and optimize genetic transformation of
intractable or previously challenging microbial chassis. This platform systematically samples a continuous spectrum of electric fields; differing from the traditional trial and error approach with discrete steps. Results of this study will broaden the scope of bacteria available for applications in synthetic biology and genetic engineering.

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