| Abstract [eng] |
The availability of total genome sequence information from a variety of bacterial genera has facilitated a dramatic increase in loss of function analysis technologies for pathogenic species of clinical relevance. Elucidation of essential metabolic and biosynthetic pathways in pathogenic organisms is critical for identification of new antimicrobial targets. From a pharmaceutical standpoint, it is the ability to rapidly identify essential genes where loss of function is coincident with loss of viability that is driving force behind genomics-based targets validation. The aim of this work was to construct the mutant‘s pool of Helicobacter pylori J99 for identification of nonessential genes and to prepare large amount of DNA tags representing sites for nonessential genes. To achieve this goal we constructed representable H.pylori J99 DNA library which was composed from 240,000 independent clones and overlap H.pylori J99 genome many times. In parallel, special DNA cassette containing chloramphenicol acetyl transferase gene (cat) which confers the chloramphenicol resistance to the H.pylori was constructed and inserted into cloned H.pylori J99 DNA fragments. At least 12,000 different clones containing correct insertions of DNA cassette were selected. Then total plasmid preparation was used for genetic transformation of H.pylori J99 strain. Cells that survived the allelic exchange were selected on agar media containing chloramphenicol. As allelic exchange results DNA cassette insertions were transferred from recombinant plasmids into genome of H.pylori J99 resulting at least 100,000 mutants. The DNA tags representing cassette insertion sites were produced using new technology developed in our laboratory. |
