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and Centrifugation. Appl Environ Microbiol 1995, 61:1805–1809.PubMed mTOR inhibitor Competing interests Financial competing interests: GR and BB are employed at Biótica from which test for Legionella detection was supplied. The author(s) declare that there are no competing interests. Non-financial competing interests: The authors declare that there are no non-financial competing interests. Authors’ contributions GR and RF conceived the study. IS, BB, GR designed the experiments. RF and GR wrote the paper. IS, BB, SM performed experiments and analyzed data. RF and EB helped with research design. IS, SM, RF, GR helped with manuscript discussion. IS provided samples. RF, EB helped to draft the manuscript. All authors have read and approved the final manuscript.”
“Background Disruption of a target gene is essential for revealing the functions of the gene and/or its product exhibiting

an organism’s phenotype, and this process is equally applicable to microbes. The approaches used to disrupt a target gene can be divided into marked and unmarked mutation methods. The marked method requires the integration of a selectable marker, such as an antibiotic resistance gene, into a target gene. Although the marker-inserted gene becomes inactive, the marker L-NAME HCl frequently affects the expression of other genes, the so-called polar effect. In addition, marked mutants usually obtain antibiotic resistance, making it difficult to introduce an additional mutation. In contrast, the unmarked method, which is also called a null or in-frame mutation, requires deletion of the open reading frame of a target gene from the microbial chromosome, raises no concern about the polar effect, and leaves no antibiotic resistance that would prevent the introduction of an additional mutation. Therefore, the unmarked method is preferable for gene disruption. Some bacteria can be mutated by a PCR-based method, in which a PCR product of an allele containing a marker is introduced directly into the cell and exchanged for a target gene by homologous recombination, and the marker is subsequently excised in some way when in need of an unmarked mutant [1–3].