| Abstract [eng] |
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. Therefore, the goal of this work was to create a new plasmids vector and to elaborate effective method for its transfer to the cells of geobacilli. First, two new plasmids isolated from wild Geobacillus spp. strains were characterized. One of them, pGTG5, was found to be related mostly to some poorly characterized plasmids and together with these plasmids it could constitute a new family of bacterial plasmids. To date, pGTG5 is the smallest plasmid identified in bacteria belonging to the genus Geobacillus. Another plasmid analyzed in this work, pGTD7, is a member of well known pUB110 plasmid family and is suitable for construction of new shuttle vectors. The replicon of this plasmid ensures high segregational stability and provides high copy number to it bearing vector. Further, the electrotransformation protocol for the transfer of the vector DNA to Geobacillus stearothermophilus NUB3621R cells was elaborated. The influence of various conditions on electrotransformation was tested and the conditions resulting in highest number of the transformants were determined. In summary, results of this work broaden the assortment of geobacilli vectors and facilitate genetic transformation of one of the most relevant Geobacillus spp. strain, G. stearothermophilus NUB3621R. |
