dc.contributorMonguí Cruz, Álvaro
dc.contributorBrandão, Pedro F.B.
dc.contributorRamírez Rojas, Adán Andrés
dc.contributorGrupo de Estudios para la Remediación y Mitigación de Impactos Negativos al Ambiente - GERMINA
dc.creatorRamírez Rojas, Adán Andrés
dc.date.accessioned2020-02-14T20:08:06Z
dc.date.available2020-02-14T20:08:06Z
dc.date.created2020-02-14T20:08:06Z
dc.date.issued2019-10-17
dc.identifierhttps://repositorio.unal.edu.co/handle/unal/75607
dc.description.abstractLos transposones son secuencias de ADN que pueden ser empleadas como herramientas en biología molecular y biotecnología. Por ejemplo, pueden ser empleados como una alternativa que permite mejorar la expresión heteróloga de genes presentes en ADN episomal que no son reconocidos por los hospederos bacterianos generalmente empleados. En el presente trabajo, se describe el desarrollo de “Mu_TnX”, un transposón de tipo Mu que puede ser empleado en estudios de análisis funcional de genes en diferentes hospederos bacterianos. Mediante herramientas computacionales, se realizó el diseño de la secuencia del transposón y se definió la estrategia para su ensamblaje. Utilizando metodologías de síntesis de ADN, PCR y clonación tradicional, se llevó a cabo la construcción de este. Se validó el funcionamiento de esta herramienta mediante la evaluación de su capacidad de transposición in vitro sobre un control de actividad empleando cepas de Escherichia coli y Pseudomonas putida. La capacidad de transposición in vitro fue comprobada al recuperar clones en las cepas evaluadas que exhibieron resistencia a los antibióticos empleados como marcadores de selección y la capacidad de inducir la expresión de genes se evidenció en clones que expresaron la proteína verde fluorescente bajo inducción del compuesto 3-metil benzoato. Aunque no se logró comprobar la misma capacidad en ADN metagenómico, Mu_TnX es la primera herramienta genética de este tipo capaz de funcionar en hospederos de diferentes especies, y se espera que su desarrollo aporte a los estudios de metagenómica funcional para el descubrimiento de genes y compuestos novedosos.
dc.description.abstractTransposons are DNA sequences that can be used as tools in molecular biology and biotechnology. For example, they can be used as an alternative to improve heterologous expression of genes present in DNA that are not recognized by the generally used bacterial hosts. This work describes the development of "Mu_TnX", a Mu type transposon that can be used in functional studies of genes in different bacterial hosts. Using computational tools, the design of the transposon sequence was carried out and the strategy for its assembly was defined. Mu_TnX transposon was assembled using methodologies of DNA synthesis, PCR and traditional cloning. The functioning of this tool was validated by evaluating its in vitro transposition capacity on a performance control plasmid using strains of Escherichia coli and Pseudomonas putida. In vitro transposition capacity was verified by recovering clones in the evaluated strains that exhibit resistance to the antibiotics used as selection markers. The ability to induce the expression of genes was observed in clones expressing the green fluorescent protein under induction of 3-methyl benzoate. Although the same activity could not be proven in metagenomic libraries, Mu_TnX is the first genetic tool of this kind that can be used in different bacterial hosts. It is expected that the further development of this tool will contribute to studies in functional metagenomics and the discovery of novel genes and compounds
dc.languagespa
dc.publisherDepartamento de Química
dc.publisherUniversidad Nacional de Colombia - Sede Bogotá
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dc.rightsAtribución-NoComercial 4.0 Internacional
dc.rightsAcceso cerrado
dc.rightshttp://creativecommons.org/licenses/by-nc/4.0/
dc.rightsinfo:eu-repo/semantics/openAccess
dc.rightsDerechos reservados - Universidad Nacional de Colombia
dc.titleDesarrollo de una herramienta genética para favorecer la expresión de genes provenientes de la diversidad microbiana
dc.typeDocumento de trabajo


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