The study of biology has undergone a fundamental change due to advancements in genetic engineering, DNA synthesis and DNA sequencing technologies. As opposed to the traditional dissective mentality of discovering genes via genetics, describing genetic behaviors through biochemistry, and then drawing diagrams of functional networks, researchers now have the potential (albeit limited) to construct novel biological molecules, networks, and even whole organisms with user-defined specifications. We have engineered novel catalytic DNAs (deoxyribozymes) with the ability to 'read' an input DNA sequence and then 'write' (by ligation) a separate DNA sequence which can in turn be detected sensitively. In addition, the deoxyribozymes can read unnatural (synthetic) nucleotides and write natural sequence information. Such simple nanomachines could find use in a variety of applications, including the detection of single nucleotide polymorphisms in genomic DNA or the identification of difficult to detect (short) nucleic acids such as microRNAs. As an extension of in vitro biological engineering efforts, we aimed to construct novel signal transduction systems in vivo. To this end, we used directed evolution to generate a catalytic RNA (ribozyme) capable of creating genetic memory in E. coli. In the end we evolved an RNA which satisfied the conditions of our genetic screen. Rather than maintaining genetic memory, however, the RNA increased relative cellular gene expression by minimizing the translational burden it imposed on the host cell. Interestingly, detailed mutational analysis of the evolved RNA led us to new studies on the relationship between ribosome availability and stochasticity in cellular gene expression, an effect that had frequently been alluded to in the literature, yet never examined. We have also taken a more canonical approach to the forward engineering of biological systems with unnatural behaviors. To this end, we designed a protein-based synthetic genetic circuit that allows a community of E. coli to function as biological film, capable of capturing and recapitulating a projected light pattern at high resolution (theoretically 100 mexapixels). The ability to control bacterial gene expression at high resolution could be used to ‘print’ complex bio-materials or deconvolute signaling pathways through precise spatial and temporal control of regulatory states. / text

El aumento de la actividad minera en el Perú hace necesario el desarrollo de tecnologías rápidas y económicas de detección de contaminantes para su monitoreo y control. Implementando conocimientos de biología molecular y de la regulación génica podemos construir un circuito genético sintético que posibilite el monitoreo de sustancia toxicas que generen estrés oxidativo como son los compuestos cianurados. El objetivo de esta investigación es desarrollar un circuito genético sintético conformado por el promotor de la proteína del shock por fagos (psp) de Escherichia coli y las secuencia codificante del gen de la proteína verde fluorescente (GFP). La construcción de dicho circuito se logró usando estrategias de clonamiento por topoisomerasas y clonamiento clásico con enzimas de restricción, se usó la reacción en cadena de la polimerasa (PCR) para confirmar que todos los segmentos del circuito estén presentes en el vector. Los estudios preliminares de la actividad del nuevo circuito se realizaron transformando genéticamente células competentes de E. coli. La observación de dichas bacterias muestra una expresión de GFP continua, lo que indica que el circuito sintético está siendo activado sin estar en presencia de agentes de estrés oxidativo, lo que suponía una posible interacción con otros sistemas de regulación de estrés en la célula. Due to the increase of mining activity in Peru new technologies that can detect and monitor hazardous pollutants in a faster and cheaper way must be developed. Implementing molecular biology knowledge about genetic regulation we are able to construct a synthetic genetic circuit that can allow the monitoring of toxic substances that generate oxidative stress such us cyanide compounds. The objective of this research is to develop a synthetic genetic circuit from the promoter of the phage shock protein operon from E. coli and the complementary DNA of the green fluorescent gene (GFP). The construction of the circuit was achieved using classic cloning strategies with restriction enzymes and also more advanced strategies such us topoisomerase cloning, the polymerase chain reaction (PCR) was used to confirm the presence of all the desire segments in the vector. Preliminary studies of the circuit activity were carried out by genetically transforming competent E. coli cells. The observation of the bacteria shows a continuous expression of GFP without any inducer, this indicates that the synthetic circuit is being activated through a possible interaction with other stress response pathway.