| The study of bacteria essentially that of E. coli for gram negative bacteria and that of Bacillus subtilis for gram-positive bacteria, allowed to have a basic data on the physiology, biochemistry and pathogenicity of bacteria.This study of microorganisms finds its importance in the search of knowledge that can help in the fight that the man leads against pathogens agents that destabilize his health or simply his well-being but also knowledge which enables a clear understanding of the functioning of the microorganism as an integrated system.Currently this study goes beyond the phenotypic or genotypic basic characteristics, and stands at the genome level to understand the individual involvement of each gene to a specific position and the study of organisms as systems.Thus, with the genomic data for various microorganisms which are currently available in the gene banks and for which the number is increasing at an exponential rate; the great challenge for present and future will no longer be based to know the genomes of different organisms, but to know the modes of operation, the various interactions that lead to physiological and pathological function which belongs to an organism.The study of the gene function has also known its development since the time of Mendel up to the present. Thus, these days, the study of gene function by the modification or disruption of its sequence allows detecting with great accuracy the function of the lacked or modified gene; the reverse genetics became an important and a straightforward approach to study gene function, individually or collectively.Different techniques have been developed, that include the site-directed mutagenesis (which is localized and takes into account a few nucleotides of a sequence of gene); the gene knockout, partially or completely, by making a replacement using a free DNA fragment or by using plasmids carrying the inactivation sequence.Recentlty, the Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E.coli K-12 through homologous recombination using linear PCR products. In the thesis, E.coli BW 25113 mutants were created by changing the localization of genes on the genome using an improved method which contains both inactivation and reinsertion steps. Genes were inactivated by having the ORF and their regulator region replaced with a kanamycin cassette flanked by FLP recognition target sites. The site for insertion was selected based on the intergenic distance and non-coding regions, thus the new neighboring genes sequences were not affected. The Insertion was carried out by designing primers armed with homologous sequences to the insertion site; the primers contained the enzyme restriction sites in order to ligate the gene PCR product with the FRT-kan-FRT marker prior to the electroporation. The PCR reactions, cultures in the media supplemented with appropriate antibiotics, comparative gene expression study were carried out to confirm the knockout, the insertion and the expression of the genes in their new location. This procedure and the created E.coli mutants can be efficiently used for the study of bacterial genomics, especially in systems biology to understand the relation between the genes loci on the genome and their expression alongside new neighboring genes and others with the same functional group. The data thus generated can increase our knowledge on the organization of the bacteria. |
