472497 A High-Throughput Platform for Electrotransformation of E. coli

Tuesday, November 15, 2016: 2:30 PM
Embarcadero (Parc 55 San Francisco)
Paulo A. Garcia and Cullen R. Buie, Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA

Synthetic biology holds the potential for solving many pressing challenges for mankind and our planet. However, one technical challenge in any area that relies upon genetic manipulation of cells is the low throughput encountered in many forms of genetic transformation. We developed a new continuous flow microfluidic system for bacterial genetic electrotransformation using pulsed electric fields. The proposed microfluidic platform has the potential to outperform the state-of-the-art cuvette electroporation by several orders of magnitude, based on its increased throughput.

Mammalian cell electroporation in microfluidic devices has demonstrated significantly improved transfection efficiency and higher cell viability in comparison to bulk, cuvette electroporation. The increase in transfection efficiency in flow-through microfluidic devices uses a fraction of the experimental sample and lower voltages, maintaining high transfection efficiency and high cell viability. Despite substantial advances in transfection of mammalian cells in microfluidic devices, an electrotransformation platform for high-throughput processing of bacteria without requiring physical microbe modifications (e.g. magnetic beads and/or oil droplets) has not been developed. A microfluidic platform for continuous electrotransformation of bacteria (nominal size ~1 μm) that achieves maximum pulsed electric field ~15 kV/cm is presented here. The microfluidic channel employs a non-uniform constriction to generate high electric fields to induce bacterial electrotransformation without generating lethal Joule heating. In this device, cells experience a time-dependent electric field that is hydrodynamically controlled, allowing cells to experience conditions that would be challenging to achieve with standard electronics.

Escherichia coli DH5a in exponential phase (MIT, Boyer Lab) was used to demonstrate the high-throughput electrotransformation platform. Pulsed electric fields (2.5 kV and 5-ms square pulses with a 20 % duty cycle) were delivered in the presence of DNA-coding (Parts Registry K176011) for ampicillin resistance and green fluorescent protein (GFP) at a final DNA concentration of CDNA = 1 ng/μL. The electroporation buffer consisted of 10 % (v/v) glycerol supplemented with 0.05 % (v/v) Tween 20 in order to mitigate cell-to-cell agglomeration. Each experimental sample (100 μL) was driven at 0 μL/min (1.97x108 CFU/μgDNA), 250 μL/min (2.26x108 CFU/μgDNA), or 500 μL/min (2.97x10CFU/μgDNA) and resulted in a residence time (pulse duration) within the constriction < 5 ms. We achieved high transformation efficiencies with a throughput increase of up to three orders of magnitude compared to the state-of-the-art cuvette electroporation. This work facilitates high throughput electrotransformation of microorganisms, accelerating development of genetically engineered microbes for important industrial applications. Electrotransformation of microbes is an essential part of many scientific fields including the study of pathogenic microbes, metabolic engineering, synthetic biology, and human microbiome for therapeutic applications. With further development, a flow through microfluidic electrotransformation platform will be realized for discovery of electroporation conditions for difficult-to-transfect or intractable microbes. We envision that the proposed microfluidic technology will reduce electrotransformation techniques that can take months down to a couple hours.

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