(387g) A High-Throughput Platform for Electrotransformation of E. coli

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 C_DNA = 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
(2.97x10⁸ CFU/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.