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Using Natural Diversity of Quorum Sensing to Expand the Synthetic Biology Toolbox

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Currently in synthetic biology only the Las, Lux, and Rhl quorum sensing pathways have been adapted for broad engineering use. Quorum sensing allows a means of cell to cell communication in which a designated sender cell produces quorum sensing molecules

Currently in synthetic biology only the Las, Lux, and Rhl quorum sensing pathways have been adapted for broad engineering use. Quorum sensing allows a means of cell to cell communication in which a designated sender cell produces quorum sensing molecules that modify gene expression of a designated receiver cell. While useful, these three quorum sensing pathways exhibit a nontrivial level of crosstalk, hindering robust engineering and leading to unexpected effects in a given design. To address the lack of orthogonality among these three quorum sensing pathways, previous scientists have attempted to perform directed evolution on components of the quorum sensing pathway. While a powerful tool, directed evolution is limited by the subspace that is defined by the protein. For this reason, we take an evolutionary biology approach to identify new orthogonal quorum sensing networks and test these networks for cross-talk with currently-used networks. By charting characteristics of acyl homoserine lactone (AHL) molecules used across quorum sensing pathways in nature, we have identified favorable candidate pathways likely to display orthogonality. These include Aub, Bja, Bra, Cer, Esa, Las, Lux, Rhl, Rpa, and Sin, which we have begun constructing and testing. Our synthetic circuits express GFP in response to a quorum sensing molecule, allowing quantitative measurement of orthogonality between pairs. By determining orthogonal quorum sensing pairs, we hope to identify and adapt novel quorum sensing pathways for robust use in higher-order genetic circuits.

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2015-05

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Developing alternative genetic systems for structural DNA nanotechnology and Darwinian evolution

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A major goal of synthetic biology is to recapitulate emergent properties of life. Despite a significant body of work, a longstanding question that remains to be answered is how such a complex system arose? In this dissertation, synthetic nucleic acid

A major goal of synthetic biology is to recapitulate emergent properties of life. Despite a significant body of work, a longstanding question that remains to be answered is how such a complex system arose? In this dissertation, synthetic nucleic acid molecules with alternative sugar-phosphate backbones were investigated as potential ancestors of DNA and RNA. Threose nucleic acid (TNA) is capable of forming stable helical structures with complementary strands of itself and RNA. This provides a plausible mechanism for genetic information transfer between TNA and RNA. Therefore TNA has been proposed as a potential RNA progenitor. Using molecular evolution, functional sequences were isolated from a pool of random TNA molecules. This implicates a possible chemical framework capable of crosstalk between TNA and RNA. Further, this shows that heredity and evolution are not limited to the natural genetic system based on ribofuranosyl nucleic acids. Another alternative genetic system, glycerol nucleic acid (GNA) undergoes intrasystem pairing with superior thermalstability compared to that of DNA. Inspired by this property, I demonstrated a minimal nanostructure composed of both left- and right-handed mirro image GNA. This work suggested that GNA could be useful as promising orthogonal material in structural DNA nanotechnology.

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2011

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Development of an artificial genetic system capable of Darwinian evolution

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The principle of Darwinian evolution has been applied in the laboratory to nucleic acid molecules since 1990, and led to the emergence of in vitro evolution technique. The methodology of in vitro evolution surveys a large number of different molecules

The principle of Darwinian evolution has been applied in the laboratory to nucleic acid molecules since 1990, and led to the emergence of in vitro evolution technique. The methodology of in vitro evolution surveys a large number of different molecules simultaneously for a pre-defined chemical property, and enrich for molecules with the particular property. DNA and RNA sequences with versatile functions have been identified by in vitro selection experiments, but many basic questions remain to be answered about how these molecules achieve their functions. This dissertation first focuses on addressing a fundamental question regarding the molecular recognition properties of in vitro selected DNA sequences, namely whether negatively charged DNA sequences can be evolved to bind alkaline proteins with high specificity. We showed that DNA binders could be made, through carefully designed stringent in vitro selection, to discriminate different alkaline proteins. The focus of this dissertation is then shifted to in vitro evolution of an artificial genetic polymer called threose nucleic acid (TNA). TNA has been considered a potential RNA progenitor during early evolution of life on Earth. However, further experimental evidence to support TNA as a primordial genetic material is lacking. In this dissertation we demonstrated the capacity of TNA to form stable tertiary structure with specific ligand binding property, which suggests a possible role of TNA as a pre-RNA genetic polymer. Additionally, we discussed the challenges in in vitro evolution for TNA enzymes and developed the necessary methodology for future TNA enzyme evolution.

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2013

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RNA Aptamer-Based Systems for Pathogen Detection and Biomolecule Synthesis

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RNA aptamers adopt tertiary structures that enable them to bind to specific ligands. This capability has enabled aptamers to be used for a variety of diagnostic, therapeutic, and regulatory applications. This dissertation focuses on the use RNA aptamers in two

RNA aptamers adopt tertiary structures that enable them to bind to specific ligands. This capability has enabled aptamers to be used for a variety of diagnostic, therapeutic, and regulatory applications. This dissertation focuses on the use RNA aptamers in two biological applications: (1) nucleic acid diagnostic assays and (2) scaffolding of enzymatic pathways. First, sensors for detecting arbitrary target RNAs based the fluorogenic RNA aptamer Broccoli are designed and validated. Studies of three different sensor designs reveal that toehold-initiated Broccoli-based aptasensors provide the lowest signal leakage and highest signal intensity in absence and in presence of the target RNA, respectively. This toehold-initiated design is used for developing aptasensors targeting pathogens. Diagnostic assays for detecting pathogen nucleic acids are implemented by integrating Broccoli-based aptasensors with isothermal amplification methods. When coupling with recombinase polymerase amplification (RPA), aptasensors enable detection of synthetic valley fever DNA down to concentrations of 2 fM. Integration of Broccoli-based aptasensors with nucleic acid sequence-based amplification (NASBA) enables as few as 120 copies/mL of synthetic dengue RNA to be detected in reactions taking less than three hours. Moreover, the aptasensor-NASBA assay successfully detects dengue RNA in clinical samples. Second, RNA scaffolds containing peptide-binding RNA aptamers are employed for programming the synthesis of nonribosomal peptides (NRPs). Using the NRP enterobactin pathway as a model, RNA scaffolds are developed to direct the assembly of the enzymes entE, entB, and entF from E. coli, along with the aryl-carrier protein dhbB from B. subtilis. These scaffolds employ X-shaped RNA motifs from bacteriophage packaging motors, kissing loop interactions from HIV, and peptide-binding RNA aptamers to position peptide-modified NRP enzymes. The resulting RNA scaffolds functionalized with different aptamers are designed and evaluated for in vitro production of enterobactin. The best RNA scaffold provides a 418% increase in enterobactin production compared with the system in absence of the RNA scaffold. Moreover, the chimeric scaffold, with E. coli and B. subtilis enzymes, reaches approximately 56% of the activity of the wild-type enzyme assembly. The studies presented in this dissertation will be helpful for future development of nucleic acid-based assays and for controlling protein interaction for NRPs biosynthesis.

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2020