Millisecond duration pulses for flow-through electro-induced protein extraction from E. coli and associated eradication.

Pulsed electric fields are used to induce membrane permeabilization on cells. In the case of species with cell wall (yeasts, microalgae), it was previously shown that when the pulse duration was several ms long, this resulted in a cytoplasmic soluble protein slow leakage. In this work, we show that a similar consequence can be obtained with different strains of E. coli. Experimental evidences of a resulting wall alteration are described. Pre-industrial flow process pilots are used. As the membrane electropermeabilization can be irreversible by applying a proper choice of the pulse parameters, this approach is used for bacterial inactivation in flow process. It is observed that sub-millisecond pulse trains are more cost effective than longer ones.

Flow process for electroextraction of total proteins from microalgae.

Classical methods for protein extraction from microorganisms, used for large-scale treatments such as mechanical or chemical processes, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow-process yeast electroextraction proved that pulsed electric field technology allows us to preserve the integrity of released cytosolic proteins by keeping intact vacuole membranes. Furthermore, large volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electroextract total cytoplasmic proteins from microalgae (Nannochloropsis salina and Chlorella vulgaris). Given that induction of electropermeabilization is under the control of the target cell size, as the mean diameter for N. salina is only 2.5 µm, we used repetitive 2-ms-long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24-h incubation period in a salty buffer. The amount of total protein released was evaluated by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris under milder electrical conditions, as expected from their larger size. This innovative technology designed in our group should become familiar in the field of microalgae biotechnology.

Electric field orientation for gene delivery using high-voltage and low-voltage pulses.

Electropermeabilization is a biological physical process in response to the presence of an applied electric field that is used for the transfer of hydrophilic molecules such as anticancer drugs or DNA across the plasma membranes of living cells. The molecular processes that support the transfer are poorly known. The aim of our study was to investigate the effect of high-voltage and low-voltage (HVLV) pulses in vitro with different orientations on cell permeabilization, viability and gene transfection. We monitored the permeabilization with unipolar and bipolar HVLV pulses with different train repetition pulses, showing that HVLV pulses increase cell permeabilization and cell viability. Gene transfer was also observed by measuring green fluorescent protein (GFP) expression. The expression was the same for HVLV pulses and electrogenotherapy pulses for in vitro experimentation. As the viability was better preserved for HVLV-pulsed cells, we managed to increase the number of GFP-expressing cells by up to 65% under this condition. The use of bipolar HVLV train pulses increased gene expression to a higher extent, probably by affecting a larger part of the cell surface.