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

Probiotics are live microorganisms that can confer health benefits to the host. They have long been consumed through fermented foods. While the specific mechanisms of probiotics are largely unclear, there is evidence that their beneficial effects may be attributed to the microbes’ ability to modify the gastrointestinal (GI) environment, to modulate host immune response, or to produce natural products that directly inhibit pathogens in the gut. With the increasing awareness of the important functions that the gut microbiota plays in affecting host heath, probiotics may no longer just stay as simple dietary supplements, but become a promising approach to disease management. With recent advances in synthetic biology, novel functions can be introduced into these “good” microbes to provide additional benefits. Genetically engineered bacteria have been developed to specifically target pathogens or effectively deliver therapeutics to the GI tract. However, there are significant limitations to the existing systems developed. For example, the engineered pathogen sensors largely rely on the similarity between the host and the pathogens, the therapeutics delivery systems are usually constrained by the molecular structures, and the majority of the works have been limited to laboratory settings. In this dissertation, I present a system we have developed based on a food-grade probiotic, Lactococcus lactis, and demonstrate a synthetic biology methodology that could be applied to build biosensors of other pathogens or environmental signals, as well as a generalizable peptide delivery vehicle to the GI tract. I will present my work in three parts. (1) The discovery of an effective antagonistic effect of L. lactis against the infectious diarrheal disease cholera, and elucidation of the mechanism with an infant mouse model. (2) The development of a diagnostic circuit in L. lacits that enables in situ detection of the pathogen and easy readout through fecal sample analysis. (3) The design of a generalizable therapeutic peptide delivery system utilizing the endogenous secretion pathway of L. lacits. Overall, my work exploits the natural and engineered benefits of the probiotic L. lactis and demonstrates its use in the intestinal disease diagnosis and therapy. / 2019-02-20T00:00:00Z

With effective engineering using synthetic biology approaches, plant-based platforms could conceivably be designed to minimize the production costs and wastes of high-value products such as medicines, biofuels, and chemical feedstocks that would otherwise be uneconomical. Additionally, modern agricultural crops could be engineered to be more productive, resilient, or restorative in different or rapidly changing environments and climates. To achieve these complex goals, information-processing genetic devices and circuits containing multiple interacting parts that behave predictably must be developed. A genetic Boolean AND logic gate is a device that computes the presence or absence of two inputs (signals, stimuli) and produces an output (response) only if both inputs are present. Here, we optimized individual genetic components and used synthetic protein heterodimerizing domains to rationally assemble genetic AND logic gates that integrate two hormonal inputs in whole plants. These AND gates produce an output only in the presence of both abscisic acid and auxin, but not when either or neither hormone is present. Furthermore, we demonstrate the AND gate can also integrate two plant stresses, cold temperature and bacterial infection, to produce a specific response. The design principles used here are generalizable, and therefore multiple orthogonal AND gates could be assembled and rationally layered to process complex genetic information in plants. In addition to bioproduction, these layered logic gates may also be used in circuits to probe fundamental questions in plant biology such as hormonal crosstalk.

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.

<p dir="ltr">More than 60% of all antifungal, antimicrobial, and anti-cancer drugs come from natural sources, such as plants, fungi, and bacteria; we call these nature-derived drugs Natural Products (NPs). One ‘super producer’ of NPs is the bacterial genus <i>Streptomyces</i>, with over 70% of therapeutic NP sourced from <i>Streptomyces</i>. These chemical weapons are regulated by sensitive signaling systems that respond to environmental conditions by activating ‘NP gene cluster’ expression, enzymes that biosynthesize NPs. Essential components of these signaling systems are Cluster Situated Regulators (CSRs); specific regulatory proteins of this class are transcription factors which can repress NP gene cluster expression until they sense a chemical signal. Previous research identified gamma-butyrolactones (GBLs), butenolides (BNs), and methylenomycin furans (MMFs) -small, low-concentration signaling molecules that affect morphological development- as ligands to a multitude of CSRs. These hormones are capable of inducing NP expression, thereby triggering production of therapeutically relevant NPs such as avermectin or streptomycin. This regulatory strategy is bioinformatically predicted to regulate potentially hundreds of novel NPs. My research objective is to elucidate signaling systems of <i>Streptomyces</i> which induce or enhance NP expression. To execute this, I first collaborated with organic chemists to derive a hybrid synthetic/biocatalytic approach to synthesizing these GBL-type hormones. I have developed a highly modular plasmid-based fluorescence reporter assay to identify CSR/ligand pairs. While these assays can aid in Natural Product discovery, they also have broad applicability for building novel genetic circuitry for use in synthetic biology.</p>