Design and optimization of genetic transformation method for thermophilic gram-positive bacteria

The genus Geobacillus is a thermophilic, gram-positive, spore-forming bacterium. The ability of this bacteria genus to grow within the temperature range between 45°C to 70 °C shows their great advantage over mesophilic bacteria cultures. These valuable microorganisms can be used in many different biotechnological and industrial processes such as bioconversion or bioremediation. However, the development of many bacteria strains that could be used in industry has been hampered by the lack or low efficiency genetic transformation systems. The aim of this work was to design and optimize genetic transformation method for Geobacillus lituanicus DSM 15325T, G. stearothermophilus 15 and Aeribacillus pallidus 8 strains. To optimize the transformation conditions, was attempted to select suitable composition growing medium for the preparation of electrocompetent cells, regeneration and transformation. Moreover, using different parameters of the electric field, we tried to select the most suitable electric field strength for efficient transformation of the strains mentioned above. It is also important to determine the effect of transforming plasmid concentration and its methylation type on the efficiency of transformation. The studies showed that for preparation of electrocompetent cells and transformants regeneration of G. stearothermophilus 15 is the most suitable mTGP medium and for a growth of transformants is acceptable NB medium. The most advisable electric field strength for this strain electrotransformation is 17 kV/cm, 500 μs or 14 kV/cm, 1 ms. For efficient electrotransformation of the G. sternothermophilus 15, is preferably to use pUCG18 plasmid, excreted from the same strain, and its appropriate concentration is 6 μg/mL. Thus, the transformation of G. stearothermophilus 15 might be very useful in further studies of the genes, encoding the bacteriocin synthesis in this strain. It could be also used for recombinant protein expression derived from different thermophilic strains. The efficient transformation system for G. lituanicus and A. pallidus were not achieved