Nucleic acid and protein extraction from electropermeabilized E. coli cells on a microfluidic chip

Abstract

Due to the extensive use of nucleic acid and protein analysis of bacterial samples, there is a need for simple and rapid extraction protocols for both plasmid DNA and RNA molecules as well as reporter proteins like the green fluorescent protein (GFP). In this report, an electropermeability technique has been developed which is based on exposing E. coli cells to low voltages to allow extraction of nucleic acids and proteins.The flow-through electropermeability chip used consists of a microfluidic channel with integrated gold electrodes that promote cell envelope channel formation at low applied voltages. This will allow small biomolecules with diameters less than 30 A to rapidly diffuse from the permeabilized cells to the surrounding solution. By controlling the applied voltage, partial and transient to complete cell opening can be obtained. By using DC voltages below 0.5 V, cell lysis can be avoided and the transiently formed pores can be closed again and the cells survive. This method has been used to extract RNA and GFP molecules under conditions of electropermeability. Plasmid DNA could be recovered when the applied voltage was increased to 2 V, thus causing complete cell lysis.

Evaluation and Optimization of Protein Extraction From E. coli by Electroporation

“…Protein extraction by means of electroporation has been implemented to date in batch and flow modes of operation, as well as on microchips to assist protein research (Shiina et al, 2004;Matos et al, 2013;Flisar et al, 2014;Haberl Meglic et al, 2015). The extraction mechanism is based on induction of changes in cell envelope permeability due to exposure to sufficiently strong electric pulses (Kotnik et al, 2010).…”

Pulsed Electric Fields-Assisted Extraction of Molecules from Bacterial and Yeast Cells

“…During recent years, advances in micro-and nano-scale techniques for various analytical and preparative procedures have been remarkably rapid, thus recent study used microfluidics to control the degree of cell opening to obtain a targeted release of biomolecules from the cells (Matos et al 2013). Nucleic acids extracted from E. coli cells were examined and quantified using agarose gel and nanophotometer.…”