Defining protein-protein interactions between red? and red? from ? bacteriophage = Definiendo...
Contenido de la obra
Contenido de la obra
- Autor: Bohórquez Suárez, Laura Catalina
- Título: Defining protein-protein interactions between red? and red? from ? bacteriophage = Definiendo interacciones proteina-proteina entre red? y red? del bacteriofago ?
- Publicación original: 2013
- Descripción física: PDF
The central system for generating genetic diversity and protecting the genomic integrity of replicating cells, from bacteria to humans, involves the homologous recombination (HR) machinery. It involves the process of exchange of molecules between DNA strands using the homology sequences. Because these machineries are highly efficient and precise it has become a useful analytical tool for the bacterial geneticists, thus, the engineering recombinant DNA molecules by homologous recombination, method called recombineering, is functional for sequence deletions, substitutions, or insertions.
Under some conditions, the phage-mediated recombination system can be provided in E. coli by the ? phage Red enzymes, Red? and Red?. It involves a processive 5’ to 3’ exonuclease or a member of helicase family, namely Red?, which generates single stranded DNA (ssDNA) regions that are substrates for the single strand annealing protein (SSAP), i. e. Red?, which promotes joining of the DNA molecules by joining the recently replicated complementary sequences. The motivation of this study is to contribute to the understanding of the physical and functional coupling of the two enzymes Red? and Red?, as well as understanding the influence of including the DNA into the reaction, which acts as the substrate.
Consequently, the experiments were designed and performed incorporating in vitro techniques not only for finding protein-protein interactions, such as co-immunoprecipitations and size exclusion chromatography, but also for analyzing the exonuclease activity of Red?. Also, the interaction of DNA with Red enzymes by means of the mobility shift assays was evaluated. In parallel, in vivo tests were made with the objective of defining the stoichiometry of the intracellular concentrations of both proteins during the co-expression in E. coli. In order to reveal more about the physical coupling of Red system’s components the preliminary data of a fluorescence correlation spectroscopy assay is described with the aim of determining whether the presence of Red? has an effect on Red?’s dwell-time on its substrate.
- Notas de reproducción original: Digitalización realizada por la Biblioteca Virtual del Banco de la República (Colombia)Notas:
- Resumen: Bacteriofago ?; Protein-protein interacccion; Homologous Recombination; Ingenieria de recombinacion; Interaccion proteina-proteina; Recombinacion homóloga; Recombineering; ? Bacteriophage
- © Derechos reservados del autor
- Forma/género: tesis
- Idioma: inglés
- Institución origen: Biblioteca Virtual del Banco de la RepúblicaMaterias: