Flexible Programming of Cell-Free Protein Synthesis Using Magnetic Bead-Immobilized Plasmids

The use of magnetic bead-immobilized DNA as movable template for cell-free protein synthesis has been investigated. Magnetic microbeads containing chemically conjugated plasmids were used to direct cell-free protein synthesis, so that protein generation could be readily programmed, reset and reprogrammed. Protein synthesis by using this approach could be ON/OFF-controlled through repeated addition and removal of the microbead-conjugated DNA and employed in sequential expression of different genes in a same reaction mixture. Since the incubation periods of individual template plasmids are freely controllable, relative expression levels of multiple proteins can be tuned to desired levels. We expect that the presented results will find wide application to the flexible design and execution of synthetic pathways in cell-free chassis

Estrategias para la detección directa de ADN genómico en soportes polimericos

[ES] El objetivo del trabajo es la detección directa de secuencias específicas de ADN genómico sin pasar por la etapa de amplificación. Para ello, se plantean dos hipótesis de trabajo. La primera consiste en inmovilizar sondas específicas de diferente tamaño de modo covalente en la superficie polimérica sin alterar las propiedades ópticas de esta, alcanzando densidades de inmovilización elevadas con el fin de aumentar la sensibilidad del ensayo. Como segunda hipótesis se plantea la racionalización y mejora de la fragmentación y el marcaje (biotina, digoxigenina, nanopartículas de oro, etc.) del ADN genómico. La interacción de las dianas marcadas con los receptores anclados en el disco compacto, dará lugar a una señal analítica, dependiente de la concentración de ADN, que será cuantificada por un grabador de DVDs. Los resultados del ensayo propuesto se compararán con los obtenidos mediante amplificación por PCR en términos de sensibilidad, selectividad y versatilidad.[EN] The aim of the work was the direct detection of specific sequences of genomic DNA in hybridization assay without the polymerase chain reaction amplification step. The assays were performed on polymeric surfaces derived from the audio-video industry. In order to achieve this challenge, the work was based on the development of a method for the covalent immobilization of oligonucleotides by means of the chemical surface activation of a compact disc, keeping at the same time its optical and mechanical properties. In addition, a tyramide based signal amplification system was implemented in the hybridization assay for the genomic DNA detection. The principle of the hybridization assay is based on genomic DNA fragmentation and massive biotinylation. These biotinylated fragments interact with the specific immobilized probes on the disk, generating an analytical signal which depends on the biotinylated fragments concentration. This signal is quantified with a DVD player, used as a detector. The results of the proposed assay are compared with those achieved including the PCR amplification step in terms of sensitivity, selectivity and versatility.Lucas Garrote, B. (2015). Estrategias para la detección directa de ADN genómico en soportes polimericos http://hdl.handle.net/10251/56379.TFG

Evaluation of discriminating capability of planar rRNA-based oligonucleotide microchips

Master'sMASTER OF ENGINEERIN