Thermostable polymerases play a significant role in molecular biology and diagnostic practice. The most famous and demanded is Polymerase I from the thermophilic bacterium Thermus aquaticus (Taq-pol). This polymerase at one time made a kind of revolution in the polymerase chain reaction. In this work, we attempted to modify this polymerase by attaching an additional Sso7d protein from Sulfolobus solfataricus to Taq-pol, which provides additional binding to the double-stranded DNA of the template. Sso7d-Taq fusion gene was expressed in BL21(DE3) cells. Optimal conditions were selected for maximum production of modified Sso7d-Taq polymerase. The optimal conditions for the intracellular accumulation of Sso7d-Taq polymerase: activation of the T7 promoter when the optical density of the culture reaches OD600 = 0.8-1.0 by adding IPTG at a concentration of 0.2 mM, followed by incubation of the culture at 37°C for 20-24 hours. Recombinant Sso7d-Taq polymerase has been purified and tested by PCR for thermal stability and elongation time. It was found that the Sso7d-Taq enzyme withstands 5 hour incubation at 95°C and 75 minute incubation at 98°C. Comparative analysis with unmodified Taq DNA polymerase showed that the Sso7d-Taq enzyme reduces the elongation rate by several times - up to 15-13 seconds per 1 kbp. The results obtained indicate the prospects of using Sso7d-Taq DNA polymerase in scientific research and diagnostic practice.
For survival in cold conditions, many organisms have developed unique adaptive mechanisms based on the synthesis of antifreeze proteins, peptides and glycoproteins that prevent ice formation at negative temperatures. These molecules tend to bind ice crystals and lower the freezing point of the solution without the formation of large crystals. Antifreeze proteins (AFP) were found in almost all types of living organisms, including insects, fungus, yeasts, bacteria and plants. The gene of antifreeze protein - glucan endo-1,3-beta-D-glucosidase (ScGlu-3) from Secale cereale was cloned into shuttle vector pPICZαA. The competent cells of yeast Pichia pastoris GS115 were transformed and the producer strain was obtained, which secreted of ScGlu-3 into the culture medium using 3% methanol as the only carbon source. It was found by western blotting that the maximum accumulation of ScGlu-3 in the culture occurs after 48 hours of fermentation on a medium with methanol. Established that rScGlu-3 precipitates at 50-65% of ammonium sulfate.
Antimicrobial agents are used in animal husbandry for the prevention and treatment of diseases in farm animals and have become an indispensable aspect of commercial livestock production. Antimicrobial therapy is well-established as a component of comprehensive preventive measures aimed at minimizing diseases in farm animals and has been incorporated into procedures directed at promoting livestock growth and productivity. Bacillus licheniformis strain T7 is susceptible to the antibiotics clidamycin, rifampicin, erythromycin, ciprofloxacin, tobramycin, tetracycline, penicillin, streptomycin, and chloramphenicol, has good sporogenic properties, grows rapidly on nutrient agar at 30-55 °C, pH 5.5–8.0, and can serve as a test culture in a microbial inhibitor test. Bacillus licheniformis T7 spores were obtained by growing the culture in Difco sporulation medium and then inactivating the vegetative cells at 90 °C for 20 minutes. Endospores of Bacillus licheniformis T7 co-polymerized with nutrient agar and germinated at 55 °C for 3–3.5 h, resulting in a pH shift from 5.5 to >6.5, which can be measured with an acid-base indicator. Bromocresol purple was suggested as a pH indicator for use in a microbiological inhibition test, wherein the presence of antibiotics or other compounds impeding the development of a microbiological culture could be determined by a change in the agar's color. A microtube- and plate-based microbiological inhibitor test prototype has been developed.
For the development of science-intensive technologies in biology, biotechnology and medicine, it is necessary to store valuable DNA samples: genes of enzymes, antigens, proteins for various purposes, and DNA loci in a depository. Storing DNA is inexpensive, and having such a depository will reduce the time spent on research in basic and applied science. The genetically engineered materials bank has been formed at the National center for biotechnology. Now, the bank has 88 samples of cloned genes of enzymes, antigens, proteins and diagnostically significant loci. Plasmid vectors with cloned genes are stored at a temperature -20°С. Besides competent cells of Escherichia coli strain DH5α were transformed with vectors and recombinant strains were isolated. Recombinant strains carrying the required plasmid vectors are stored for cryopreservation at a temperature -80°С.
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