Microfluidic Electroporation Techniques to Accelerate Discovery in Synthetic Biology

One major limitation of genetic engineering and synthetic biology is the inability to incorporate genetic material into many bacteria due to the challenge of permeating the cell envelope while maintaining high cell viability. There are millions of species of bacteria on the planet, yet we can only genetically engineer several dozen. New technologies are needed to unlock the bacterial kingdom’s potential to impact human life and solve many challenges of interest to mankind. In this presentation we will show recent work utilizing novel microfluidic approaches to enable electroporation-based genetic transformation for applications in synthetic biology. 

Electroporation results from exposure of cells to external electric fields of sufficient strength to transiently disrupt the plasma membrane.  It is often used for intracellular delivery of molecules such as drugs, proteins, DNA, or RNA. Recently, flow-through microfluidic electroporation platforms with channels of uniform cross-sectional area have demonstrated genetic transformation with uniform electric fields. However, this process has limited utility since in many cases the optimal conditions (e.g., field strength) for genetic transformation are unknown (as in difficult-to-transfect or intractable organisms). A platform to rapidly and quantitatively determine optimal conditions for electroporation-based transformation will accelerate the development of new chassis organisms for synthetic biology while advancing the fundamental science behind electroporation.

We have developed a flow-through electroporation platform that enables experimental characterization and/or genetic transformation by removing the electric field as a variable in the genetic transformation process. The novel microfluidic device generates a linear electric field gradient that allows for spatial evaluation of the electric field required for electroporation, without using discrete experimental steps. Our rapid microfluidic platform is capable of determining electric field thresholds for electroporation in a single experiment. Fluorescence-encoded DNA plasmids or nucleic acid stains permeate cell membranes in regions where the electric field is sufficiently high to induce membrane permeabilization.  We then correlate the fluorescent region in the channel with the range of electric fields that results in successful electroporation, effectively analyzing a continuous spectrum of experimental conditions in a single experiment. Both gram-positive (Corynebacterium glutamicum and Mycobacterium smegmatis) and gram-negative (Escherichia coli BL21) strains have been tested in our device. The electric field thresholds for electroporation of C. glutamicum (5.20 ± 0.20 kV/cm), M. smegmatis (5.56 ± 0.08 kV/cm), and E. coli BL21 (3.65 ± 0.09 kV/cm) were determined using SYTOX^® as the fluorescent marker.

We envision this microfluidic platform as a tool to rapidly optimize genetic transformation of intractable or previously challenging bacteria. This study focuses on prokaryotes but many of the processes developed could be applied to yeast or mammalian cells as well. This platform systematically samples a continuous spectrum of electric fields in a single experiment, 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.