Geobacillus stearothermophilus NUB3621R genetic transformation by electroporation

Anotacija

Background. Thermophilic bacteria of genus Geobacillus are valuable microorganisms that can be used in various biotechnological processes such as bioconversion and bioremediation. However, effective employment of geobacilli is impossible without a reliable and convenient methodology for their genetic engineering. In this study, we sought to elaborate an electrotransformation protocol for one of the  most studied Geobacillus strain, G. stearothermophilus NUB3621R. Materials and methods. To determine factors influencing transformation efficiency of the  strain we have changed different conditions of electro-competent cells preparation (cell growth conditions, composition of eletrotransformation media) and parameters of electroporation itself (electric field strength). Results. The best transformation efficiencies reaching 10³ of transformants per μg of DNA were achieved when the cells were grown to OD₅₉₀ of 1.1–1.3 and suspended in hyperosmotic electrotransformation media composed of sorbitol, mannitol and glycerol. Moreover, the  positive effect of MgCl₂ has been demonstrated. The electric field strength of 20–22 kV/cm was most suitable for the transformation of NUB3621R strain. Conclusions. The developed methodology allows transferring plasmid DNA to the cells of the G. stearothermophilus NUB3621R strain by electroporation. Although it is not sufficiently effective for some purposes (such like construction of DNA libraries and direct functional screening for desired enzymes), however, in this study determined conditions can serve as a starting point for further improvement of the electrotransformation protocol.