Genetic engineering of microbes has developed rapidly along with our ability to synthesize DNA de novo. Yet, even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. While technological advances have resulted in powerful techniques for in vitro and in vivo assembly of DNA, each suffers inherent disadvantages. Here, an ex vivo DNA cloning suite using crude cellular lysates derived from E. coli is demonstrated to amplify and assemble DNA containing small sequence homologies. Further, the advantages of an ex vivo approach are leveraged to rapidly optimize several parameters of the ex vivo DNA assembly methodology testing lysates from different engineered strains of E. coli, with various buffer components and using titrations of purified cloning enzymes. Finally, in order to complete the cloning suite, a vector expressing the Pyrococcus furiosis (Pfu) DNA polymerase was designed, constructed and expressed in E. coli to create a ‘functionalized lysate’ capable of ex vivo PCR. Not only do we demonstrate ex vivo cloning methodology as a complete cloning package capable of replacing the expensive cloning reagents currently required by synthetic biologists, but also establish ex vivo as an overarching approach for conducting molecular biology.

Orientador: Danielle Biscaro Pedrolli / Resumo: Os métodos de indução da expressão gênica disponíveis para linhagens bacterianas envolvem a adição de compostos indutores ao meio de cultura (por exemplo, isopropil β-D-1-tiogalactopiranosideo, xilose e arabinose), o que é indesejável para linhagens industriais, pois encarece o processo produtivo. Já a utilização da expressão constitutiva, alternativa à indução, pode ocasionar stress metabólico durante a fase lag de crescimento quando são utilizados promotores fortes. O objetivo do trabalho foi construir e padronizar um novo modelo de indução da expressão gênica para linhagens bacterianas industriais. O novo modelo de autoindução baseado no sistema de quorum-sensing bacteriano, permitindo que a célula se automonitore e induza a expressão gênica durante a fase exponencial de crescimento, eliminando assim não só a necessidade de adição de composto indutor como a necessidade de monitoramento da densidade celular pré-indução. Realizou-se amplificação e clonagem dos genes luxR e luxI, com e sem caudas de histidina, e suas respectivas sequências regulatórias de Aliivibrio fischeri, em plasmídeo contendo os genes responsáveis pela bioluminescência ou fluorescência com códons otimizados para Bacillus subtilis. Em seguida, foi realizada transformação e a integração do plasmídeo no cromossomo de B. subtilis. A funcionalidade do sistema foi avaliada em diferentes etapas de crescimento microbiano com o auxílio de um leitor de microplacas durante intervalos regulares. O sistema de autoind... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Current methods available for the autoinduction of gene expression in genetically engineered bacterial strains require addition of inducing compounds to the culture medium (e.g. Isopropyl β-D-1-thiogalactopyranoside, xylose, and arabinose), which is undesirable for industrial strains due to additional costs to the production process. Alternatively, constitutive gene expression is employed. However, the later can possibly cause metabolic stress during the lag growth phase if strong promoters are employed. The objective of this work was to construct and to standardize a new model for induction of gene expression in industrial bacterial strains. The new model is based on an autoinduction process triggered by the bacterial quorum-sensing system. It allows the cell to monitor itself and induce its own gene expression during the exponential growth phase, thereby eliminating both the need for an external inducing compound and the need for monitoring pre-inducing cell density. Bacterial cultures were grown in rich media, supplemented or not with antibiotics. Amplification and cloning of luxR and luxI genes, with and without histidine tags, and their respective regulatory sequences of Aliivibrio fischeri, were performed on a plasmid containing the genes responsible for bioluminescence or fluorescence with codons optimized for Bacillus subtilis. Next, transformation and integration of the plasmid into the B. subtilis chromosome were performed. The functionality of the system was evalua... (Complete abstract click electronic access below) / Doutor

Arsenic contamination of groundwater remains a serious health concern in many areas of the world. Developing countries such as Bangladesh and Nepal are particularly affected because access to high quality water infrastructure is low. Since the 1970s, most water in these countries is sourced from shallow tube wells installed to reduce the spread of diseases associated with poor water hygiene. In this goal they were successful, however by the mid 1990s it became apparent that many of these wells were contaminated by arsenic and that these countries’ rural poor were being slowly poisoned. No simple, cheap, and reliable test for arsenic exists, and efforts to mitigate arsenic contamination have been severely limited by this over the past two decades. Government backed well-testing efforts using commercially available field kits have many issues with reliability, safety, rigour, and transparency, and have lost their urgency over the past decade, while the expensive field test kits remain out of the reach of most ordinary people in these areas. Synthetic Biology offers the technology to develop a new class of biosensor by exploiting bacteria’s natural ability to sense and respond to levels of arsenic considerably lower than commercially available kits which are based on analytical chemistry. In order to reach this goal, we must first develop our understanding of the natural response to arsenic in our chosen host, B. subtilis. Although we have a reasonably good qualitative understanding of the operon responsible for arsenic sensing, very little quantitative analysis has been carried out, and a robust system for ratiometric characterisation has not been established in the bacteria. In this work, a robust platform for rapid ratiometric characterisation is established in B. subtilis. A rigorous mathematical model of the ars operon is developed and analysed before being verified experimentally. This new knowledge is then used to explore synthetic permutations to the natural system aimed at improving the sensor properties of the system. Finally, a biological architecture for an easily tunable biosensor with good characteristics is recommended.

Synthetic biology, which aims to enable the design and assembly of customized biological systems, holds great promise for delivering solutions to numerous modern day challenges in agriculture, sustainable energy production, and medicine. However, at its current stage, synthetic biology is not yet equipped with the necessary tools and understanding to reprogram the immensely complex molecular environment of the cell beyond simple proof of concept demonstrations. One current objective within synthetic biology is to create robust tools that can be used to manipulate biological systems in a predictable and reliable manner. While many transcription-based control devices have been reported, little consideration has been given to the eukaryotic protein translation apparatus as a target for engineering gene-regulatory tools. In this work, we explore the potential for reprogramming the protein synthesis machinery for cell engineering. We begin in Chapter 1 by reviewing canonical protein synthesis and survey the assortment of translation reprogramming mechanisms that exist in nature, focusing on the role of RNA in these processes. We then cover previous efforts to engineer the protein synthesis machinery and discuss their methodological approaches. Lastly, we examine potential opportunities for engineering protein synthesis that have not yet been explored. RNA’s prominent role in protein synthesis and its amenability to high-throughput in vitro selection approaches raises the possibility that the translation apparatus could be engineered through in vitro directed evolution of its RNA components. In Chapter 2, we develop an experimental framework for identifying mRNA sequence elements that reprogram protein synthesis, focusing on stop codon readthrough. By adapting a previously developed in vitro selection technology called mRNA display, we demonstrate that molecules of RNA derived from expansive libraries of random sequences can be enriched as a result of their translation reprogramming activity. We then analyze these stop codon readthrough signals and propose the use of these sequences for enhanced unnatural amino acid incorporation technologies. In Chapter 3, we apply this very same selection principle for the in vitro directed evolution of RNA sequences that stimulate -1 programmed ribosomal frameshifting. Then, using previously reported RNA aptamers, we rationally engineer RNA switches that regulate translation reading frame in response to small molecule inputs. To further optimize switch performance, an in vivo directed evolution platform was established. We explore the utility of these RNA switches, particularly their ability to regulate multi-protein stoichiometry, for performing cellular logic operations and controlling cell fate. A major focus of translation engineering has been the incorporation of unnatural amino acids for fluorescent labeling of proteins in living cells. The successful achievement of this goal will require small molecule fluorophores with desirable biological properties, as well as robust synthetic methods for their production. In Chapter 4, we present a scalable approach to oxazine and xanthene fluorophores that utilizes a general diaryl ether synthetic intermediate. Finally, in Chapter 5, we describe a photoactivatable oxazine fluorophore and demonstrate its utility as a live-cell imaging reagent with applicability to advanced microscopy techniques.

Biocatalysis represents an attractive route to the production of various compounds which are difficult or impossible to synthesise and isolate using traditional chemical synthesis. In particular, the production of chiral molecules is a function ideally suited to biocatalysis, due to the natural stereospecificity of enzymes. The synthesis of such chiral molecules is essential in the production of pharmaceuticals, additives for the food and drinks industry and the creation of specialist polymers. CYP105AS1, isolated from Amycolatopsis orientalis, is a cytochrome P450 enzyme which produces the inactive 6-epi-pravastatin of the blockbuster anti-cholesterol drug pravastatin. Previous directed evolution efforts have engineered this enzyme to produce a five-point mutant, known as P450prava, which partially reversed the stereospecificity of the enzyme to produce a majority pravastatin product mixture. This thesis details work to use structure-led engineering approaches to redesign the active site of P450prava to introduce stringent stereospecificity. A combinatorial approach of manual and computational rational design was pursued, leading to the creation of a novel T95F/V180M double mutant of P450prava. This double mutant was found to have successfully eliminated the unwanted 6-epi-pravastatin enantiomer from the product mix, leaving a pure pravastatin product. P450prava was also shown to bind and hydroxylate other statin substrate molecules, demonstrating its versatility in the production of drug metabolites and other high-value oxyfunctionalised molecules. This property, along with its proven tolerance of significant active site engineering efforts, demonstrates the viability of the P450prava as a platform for the creation of novel biocatalysts for the production of various hydroxylated products from diverse substrate molecules.

La faculté d’adaptation aux conditions environnementales des bactéries provient de lacomplexité de leur réseau de régulation génique, impliquant de nombreux régulateursspécifiques et la machinerie d’expression génique. Nous avons montré que le gène crl, codantun régulateur global d’Escherichia coli, est exprimé de façon transitoire lors de laphase exponentielle. Notre étude a permis d’identifier deux régulateurs responsables dece profil parmi une centaine testés. Ainsi, le complexe CRP-AMPc, réprime de façon indirectel’expression de crl, tandis que la nucléoprotéine Fis se fixe sur le promoteur de crlet active sa transcription directement. Le profil d’expression de crl étant similaire à celuide nombreux régulateurs globaux, nous nous sommes intéressés au rôle de la machinerieglobale d’expression des gènes et à son impact sur la croissance. Dans ce but, nous avonsconstruit un système nous permettant de contrôler la croissance d’E. coli en modulantl’expression des sous-unités _ et _’ de l’ARN polymérase et donc le niveau de transcriptiondans la cellule. Lorsque l’ARN polymérase est en faible concentration, le taux decroissance devient quasiment nul et les cellules filamentent. Ce contrôle de la croissanceest dose dépendant et a été mis en évidence autant à l’échelle de la population qu’à cellede la cellule unique. Nous avons enfin étudié par RNA-seq l’impact du niveau d’ARNpolymérase sur la transcription de l’ensemble du génome de cette souche. Cette étudemontre que toutes les classes fonctionnelles de gènes sont affectées par notre système, àl’exception des gènes qui codent les protéines ribosomales. / Bacteria can adapt to many different environmental conditions. This capacity of adaptationis conferred to the organism by a complex regulatory network, composed of specificregulators and the global gene expression machinery. We have studied the expression dynamicsof Crl, a global regulator of Escherichia coli, and observed a peak of transcriptionduring the exponential phase of growth. In order to identify potential regulators of crlexpression, we have measured the expression profile of crl in about one hundred differentmutant strains. This screen has revealed that CRP-cAMP represses indirectly the transcriptionof crl and the nucleoprotein Fis activates transcription of the crl promoter bybinding to the crl promoter region. We noted that the expression of most global regulatorsof E. coli have an expression profile similar to the one of Crl. We have thereforestudied the relationship between global gene expression machinery and cellular growth.We constructed a bacterium where the transcription of the two large subunits of RNApolymerase, _ et _’, is under external control. A small concentration of RNA polymeraseleads to a small growth rate of this engineered bacterium and the cells start to filament,whereas a high concentration of RNA polymerase produces phenotypically wild-type cells.We have characterized the control of growth rate by our system at the population level andin single cells. An analysis of the global transcription pattern of this strain by RNA-seqshows that the transcription of genes in all functional classes, with the possible exceptionof genes coding for ribosomal proteins, are almost equally affected by the modificationsof the intracellular concentration of RNA polymerase.

Le travail présenté s'articule autour de l'étude in silico des réseaux biologiques en abordant aussi bien les aspects d'intégration, de formalisation et de modélisation des réseaux et sous-réseaux biologiques. Dans ce contexte, les travaux ont porté dans un premier temps, sur le développement d'un outil d'intégration Cyclone à même d'assurer un accès et une exploitation simplifiés des données présentes dans la base de données BioCyc puis, dans un second temps, sur le développement d'un cadre de modélisation des graphes particulièrement adapté à l'étude de réseaux d'interactions hétérogènes, MIB (pour Modèle d'Interaction Biologique). Enfin, ces développements ont été mis à profit afin d'une part, de caractériser et d'étudier la présence et le mode de connexion de sous-réseaux ou motifs à l'intérieur de réseaux plus vastes et d'autre part, d'étudier et de modéliser la voie métabolique du galactose chez la levure Saccharomyces cerevisiae en tant que boucle de rétroaction impliquant régulation transcriptionnelle et interaction protéine-protéine.

Fibrin is the body's natural provisional matrix activated in response to vascular injury in which noncovalent knob:hole interactions between fibrin monomers lead to the assembly of fibrin for clot formation. In this study we aimed to exploit fibrin knob:hole affinity interactions with swelling, space filling microgels for the development of a potential bio-synthetic hybrid polymer system with hemostatic properties. Previous work has explored the inherent binding interactions of various fibrin knobs and their complementary polymerization holes, which have led to the development of fibrin knob peptide mimic (GPRPFPAC) with enhanced binding affinity for fibrin(ogen) holes. By coupling this enhanced fibrinogen binding peptide with a pNIPAm microgel system capable of being dynamically tuned and self-assembled, we hypothesized the specific and rapidly triggered formation of a bulk hydrogel in a wound environment (i.e. in the presence of fibrinogen). We found that at the peptide ligand density and concentrations of microgels used, that a rapid formation of a gel did not occur in the presence of fibrinogen alone. However with fibrinogen and thrombin, we found that fibrin network polymerization, structure, and viscoelastic properties were greatly altered in the presence of knob peptide-conjugated microgels.

<p>Interactions between nucleic acid substrates and the proteins and enzymes that bind and catalyze them are ubiquitous and essential for reading, writing, replicating, repairing, and regulating the genomic code by the proteomic machinery. In this dissertation, computational molecular engineering furthered the elucidation of spatial-temporal interactions of natural nucleic acid binding proteins and enzymes and the creation of synthetic counterparts with structure-function interactions at predictive proficiency. We examined spatial-temporal interactions to study how natural proteins can process signals and substrates. The signals, propagated by spatial interactions between genes and proteins, can encode and decode information in the temporal domain. Natural proteins evolved through facilitating signaling, limiting crosstalk, and overcoming noise locally and globally. Findings indicate that fidelity and speed of frequency signal transmission in cellular noise was coordinated by a critical frequency, beyond which interactions may degrade or fail. The substrates, bound to their corresponding proteins, present structural information that is precisely recognized and acted upon in the spatial domain. Natural proteins evolved by coordinating substrate features with their own. Findings highlight the importance of accurate structural modeling. We explored structure-function interactions to study how synthetic proteins can complex with substrates. These complexes, composed of nucleic acid containing substrates and amino acid containing enzymes, can recognize and catalyze information in the spatial and temporal domains. Natural proteins evolved by balancing stability, solubility, substrate affinity, specificity, and catalytic activity. Accurate computational modeling of mutants with desirable properties for nucleic acids while maintaining such balances extended molecular redesign approaches. Findings demonstrate that binding and catalyzing proteins redesigned by single-conformation and multiple-conformation approaches maintained this balance to function, often as well as or better than those found in nature. We enabled access to computational molecular engineering of these interactions through open-source practices. We examined the applications and issues of engineering nucleic acid binding proteins and enzymes for nanotechnology, therapeutics, and in the ethical, legal, and social dimensions. Findings suggest that these access and applications can make engineering biology more widely adopted, easier, more effective, and safer.</p> / Dissertation

<p>Driven by the development of new technologies and an ever expanding knowledge base of molecular and cellular function, Biology is rapidly gaining the potential to develop into a veritable engineering discipline - the so-called `era of synthetic biology' is upon us. Designing biological systems is advantageous because the engineer can leverage existing capacity for self-replication, elaborate chemistry, and dynamic information processing. On the other hand these functions are complex, highly intertwined, and in most cases, remain incompletely understood. Brazenly designing within these systems, despite large gaps in understanding, engenders understanding because the design process itself highlights gaps and discredits false assumptions. </p><p>Here we cover results from design projects that span several scales of complexity. First we describe the adaptation and experimental validation of protein functional assays on minute amounts of material. This work enables the application of cell-free protein expression tools in a high-throughput protein engineering pipeline, dramatically increasing turnaround time and reducing costs. The parts production pipeline can provide new building blocks for synthetic biology efforts with unprecedented speed. Tools to streamline the transition from the in vitro pipeline to conventional cloning were also developed. Next we detail an effort to expand the scope of a cysteine reactivity assay for generating information-rich datasets on protein stability and unfolding kinetics. We go on to demonstrate how the degree of site-specific local unfolding can also be determined by this method. This knowledge will be critical to understanding how proteins behave in the cellular context, particularly with regards to covalent modification reactions. Finally, we present results from an effort to engineer bacterial cell suicide in a population-dependent manner, and show how an underappreciated facet of plasmid physiology can produce complex oscillatory dynamics. This work is a prime example of engineering towards understanding.</p> / Dissertation