Breaking membrane barriers

Microfluidic device could quicken DNA insertion into bacteria

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CAMBRIDGE, Mass.—When attempting to get the cells of an organism to accept foreign DNA in the genetic engineering process, scientists often use electroporation to expose cells to an electric field. Under the right conditions, the process can open up pores within the cell membrane to facilitate the flow of DNA, but the process is time-consuming.
Engineers at the Massachusetts Institute of Technology (MIT) have developed a microfluidic device that could help scientists to speed up the first step in genetic engineering by finding the range of electric potentials that will harmlessly and temporarily open up membrane pores to admit DNA. The device could be used on any microorganism or cell, reducing work that could take months or years to a few days.
Cullen Buie, the Esther and Harold E. Edgerton Associate Professor of mechanical engineering at MIT, and his colleagues, postdoc Paulo Garcia, graduate student Zhifei Ge and lecturer Jeffrey Moran, published their results in the journal Scientific Reports. The research was supported, in part, by the Defense Advanced Research Projects Agency, a U.S. military agency more commonly known as DARPA, and the National Science Foundation.
“The device evolved from the need in industry and academia to better understand electroporation, the process of disrupting the membrane of cells with pulsed electric fields,” explains Garcia. “Scientists and researchers have recognized that many cells are difficult to transfect or intractable, so we set out to develop a useful platform that will help scientists identify the sweet spot for successful transformation. Microfluidic devices are powerful, because one can have tremendous spatial resolution of electric fields and fluid flow. We designed the channel geometry to become an ‘electroporation ruler’ with high temporal resolution when used in combination with fluorescent dyes.”
The microfluidic device consists of a channel—created using soft lithography—that narrows in the center. When an electric field is applied to the device, the channel’s geometry causes the field to exhibit a range of electric potentials, the highest being at the channel’s narrowest region.
The MIT researchers ran several strains of bacterial cells through the device, exposed them to an electric field and added a fluorescent marker that lights up in the presence of DNA. Cells that were successfully permeated by the electric field would let in the fluorescent marker, which would light up in response to the cell’s own genetic material. To identify the magnitude of the electric potential that was able to open a cell membrane, the researchers marked the location of each fluorescent cell along the channel.
The researchers successfully permeated strains of E. coli and Mycobacterium smegmatis, a bacterium in the same family as the organism that causes tuberculosis. The membranes of the latter are reported to be extremely difficult to penetrate.
According to Garcia, the device can accomplish identification of the electric field required for the onset of electroporation when used in combination with fluorescent dyes. Additionally, it can be used to transform cells with DNA for genetic engineering and synthetic biology applications. The implication is the ability to determine the parameters for successful transformation in a rapid manner that will potentially accelerate discovery of new medicines, production of biomaterials and generation of biofuels, among other biotech applications.
Garcia says that the microfluidic device is unique in three different ways. “The channel geometry was designed to generate a variable electric field distribution with a linear correlation to each location along the channel geometry,” he explains. “This translates into the ability to evaluate hundreds of electric fields simultaneously in one experiment as opposed to the traditional cuvette experiments in which only one electric field can be evaluated at a time.”
Secondly, he says, the device was “designed to achieve successful electroporation of bacterial cells, which, to the best of our knowledge, has not been achieved before in a microfluidic device.” Because these cells are roughly 10 times smaller than mammalian cells and require about 10 times stronger electric fields, there are important design and technical considerations.
“The device can also operate in a flow-through manner that allows for continuous transformation of bacterial cells and collection for downstream analysis,” Garcia adds. “This is a platform that could be scaled and automated for allowing compatibility with established industrial processes in genetic engineering, synthetic biology and DNA synthesis.”
Garcia concludes: “We are currently focusing on the scientific potential as opposed to the commercial potential, since we are still optimizing channel designs and performing robust studies across additional bacterial strains. We do know that the commercial potential could be significant because of the ability to rapidly and significantly improve several different biotech industries, but we are currently focusing on the science.”

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