| Abstract [eng] |
Thermophilic bacteria are extremophilic microorganisms which are capable of growing at high temperatures (>45 ℃). These bacteria are extremely important in biotechnology due to their thermostable enzymes (amylase, xylanase, esterase, etc.) and their use in various processes (biofuel production, bioremediation and biosynthesis of metal nanoparticles). However, the wider application of thermophilic bacteria is prevented by their complicated genetic transformation and few strains are known that can be transformed. Electroporation is one of the most applicable methods of genetic material transfer, which can be applied to thermophilic bacteria. However, in order to apply this method to a specific transformation of a bacterial strain, first it must be optimized. The aim of this work was to optimize the genetic transformation of Parageobacillus thermoglucosidasius DSM 2542T using the electroporation method. A total of 7 parameters were optimized: growth medium (2SPYNG), growth phase (beginning of exponential phase, OT600  2.0), electric field strength (23 kV/cm), plasmid amount (2 L, 100 ng/L), regeneration temperature (52 ℃), regeneration (TGP) and selective media (TGP). The maximum transformation efficiency obtained was 4,8 ( 0,9)  103 CFU/µg DNA. The efficiency of genetic transformation of P. thermoglucosidasius DSM 2542T using a square electric field electroporator was also evaluated. The optimal electroporation conditions for maximum transformation efficiency were electric field strength (20 kV/cm), duration of electric field pulses (1 ms) and number of pulses (3). The obtained maximum transformation efficiency was 5,3 ( 0,7)  103 CFU/µg DNA. The selection of transformed thermophilic bacteria was also carried out using the optimized electroporation protocol of P. thermoglucosidasius DSM 2542T. It was found that out of 7 strains of thermophilic bacteria belonging to the genera Geobacillus, Parageobacillus and Anoxybacillus, only Geobacillus thermoleovorans DSM 5366T strain was able to be transformed. |
