Synthetic Biology

Biography

Biography: Gustavo Stadthagen

Abstract

Sonoporation is an ultrasonic cavitation-dependent method for gene delivery, mostly applied to higher eukaryotic cells and tissues. Cavitation in the vicinity of cells induces pore formation in the cell envelope and mechanical internalization of extracellular DNA. Its application to biotechnology useful organisms is less developed. Here we evaluated the CaviBoxR sonoporation device for plasmid delivery into the yeast Kluyveromyces lactis and the bacterium Escherichia coli BL21, commonly employed organisms for bio-production. CaviBoxR tightly controls cavitation through combination of multiple ultrasound beams under strong negative pressures. We evaluated the effect of different cavitation conditions (intensity, duration, duty cycle) and cell suspension media composition on cell viability and functional plasmid uptake. Optimal DNA delivery into K. lactis kept in YPD culture medium and incubated with linearized plasmid before sonoporation was achieved at CI 14 for 60 seconds with minimal effects on cell viability. Transformation efficiency was comparable to conventional chemical transformation of this host. Plasmid uptake by E. coli occurred in LB culture medium supplemented with 100 mM CaCl2 at CI 12-14 for 20 seconds. Under the same cavitation conditions, transformation was 100 times more efficient when bacteria were suspended in a solution of 25 mM MgCl2, 100 mM CaCl2 and 10% Glycerol. These first results demonstrate the feasibility of DNA delivery into bacteria and yeast species of biotechnological interest by sonoporation, directly in culture medium with minimal manipulation. Improvement of this method holds the potential of simple parallel transformation of large strain collections and or recombinant DNA libraries.