<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

Evolutionary innovations often arise from complex genetic and ecological interactions, which can make it challenging to understand retrospectively how a novel trait arose. In a long-term experiment, Escherichia coli gained the ability (Cit⁺ ) to utilize abundant citrate in the growth medium after ~31,500 generations of evolution. Exploiting this previously untapped resource was highly beneficial: later Cit⁺ variants achieve a much higher population density in this environment. All Cit⁺ individuals share a mutation that activates aerobic expression of the citT citrate:C₄-dicarboxylate antiporter, but this mutation confers only an extremely weak Cit⁺ phenotype on its own. To determine which of the other >70 mutations in early Cit⁺ clones were needed to take full advantage of citrate, we developed a Recursive Genome-Wide Recombination and Sequencing (REGRES) method and performed genetic backcrosses to purge mutations not required for Cit⁺ from an evolved strain. We discovered a mutation that increased expression of the dctA C₄-dicarboxylate transporter greatly enhanced the Cit⁺ phenotype after it evolved, implicating the intracellular supply of succinate or other C₄-dicarboxylates to be a critical factor for the expression of the phenotype. The activity level of citrate synthase (CS), encoded by the gltA gene, was also found to be important for Cit⁺. A mutation to gltA (gltA1) occurred before the evolution of Cit⁺ and led to an increase in CS activity by diminishing allosteric inhibition by NADH. This mutation was found to be deleterious for high-level citrate utilization, a situation that was remedied shortly after the evolution of Cit⁺ by the evolution of compensatory mutations to gltA which decreased CS activity. We speculate that the gltA1 mutation may have been important to 'potentiate' the evolution of a weak Cit⁺ phenotype by increasing succinate production via an upregulated glyoxylate pathway but that as cells became able to import succinate by virtue of the dctA mutation that this pathway became maladaptive, prompting this evolutionary reversal. Overall, our characterization of this metabolic innovation highlights the degree to which interactions between alleles shape the evolution of complex traits and emphasizes the need for novel whole-genome methods to explore such relationships.

L'acétate nuit à la croissance de la bactérie E. coli. Malgré les recherches qui tentent d'en d'écrire les raisons, il nous est impossible actuellement de fournir le schéma explicatif complet de ce phénomène. Cette thèse propose de déterminer l'importance des différents mécanismes possibles de l'inhibition de la croissance par l'acétate. Pour cela, nous avons construit une collection de mutants des voies métaboliques de l'acétate que nous avons systématiquement caractérisés avec/sans l'ajout de 128 mM d'acétate au cours de la phase exponentielle de croissance. La voie Pta AckA semble contribuer à 20% à l'inhibition par l'acétate, probablement par l'action de l'acétyle-phosphate sur l'expression des gènes ou la régulation de l'activité enzymatique. Nous montrons que le mécanisme généralement invoqué, l'effet d ́ecouplant de l'acétate, ne joue pas de rôle dans l'inhibition. L'effet majeur est dû à un déséquilibre des anions de la cellule. Nous présentons également deux projets de biologie synthétique : l'un visant à quantifier le mercure dans un échantillon d'eau; et l'autre contrôlant le nombre de cellules vivantes par la lumière au sein d'une population bactérienne. / Acetate inhibits the growth of Escherichia coli on glucose. Despite many studies that have attempted to elucidate the underlying mechanisms, we are currently unable to provide a comprehensive explanation of this phenomenon. Here, we construct a series of isogenic mutants that inactivate specific parts of the metabolic pathways of acetate. By systematically measuring growth rate, as well as the fluxes of carbon metabolites entering and leaving the cell, we are able to propose an explanation for the growth inhibition by acetate. The Pta-AckA pathway contributes about 20% of the growth inhibition by acetate, probably through the action of acetyl-phosphate on gene expression or the regulation of enzyme activities. We also show that acetate does not function as a classical uncoupling agent. This mechanism was commonly assumed to account for the largest part of acetate inhibition. Our data support a model where the imbalance of cellular anions, caused by the massive influx of acetate into the cell, is the major determinant of growth inhibition by acetate. We also present two synthetic biology projects from the iGEM competition: one to quantify mercury in a water sample and the other controlling the number of living cells by light in a bacterial population.

No description available.

L'acétate nuit à la croissance de la bactérie E. coli. Malgré les recherches qui tentent d'en d'écrire les raisons, il nous est impossible actuellement de fournir le schéma explicatif complet de ce phénomène. Cette thèse propose de déterminer l'importance des différents mécanismes possibles de l'inhibition de la croissance par l'acétate. Pour cela, nous avons construit une collection de mutants des voies métaboliques de l'acétate que nous avons systématiquement caractérisés avec/sans l'ajout de 128 mM d'acétate au cours de la phase exponentielle de croissance. La voie Pta AckA semble contribuer à 20% à l'inhibition par l'acétate, probablement par l'action de l'acétyle-phosphate sur l'expression des gènes ou la régulation de l'activité enzymatique. Nous montrons que le mécanisme généralement invoqué, l'effet d ́ecouplant de l'acétate, ne joue pas de rôle dans l'inhibition. L'effet majeur est dû à un déséquilibre des anions de la cellule. Nous présentons également deux projets de biologie synthétique : l'un visant à quantifier le mercure dans un échantillon d'eau; et l'autre contrôlant le nombre de cellules vivantes par la lumière au sein d'une population bactérienne. / Acetate inhibits the growth of Escherichia coli on glucose. Despite many studies that have attempted to elucidate the underlying mechanisms, we are currently unable to provide a comprehensive explanation of this phenomenon. Here, we construct a series of isogenic mutants that inactivate specific parts of the metabolic pathways of acetate. By systematically measuring growth rate, as well as the fluxes of carbon metabolites entering and leaving the cell, we are able to propose an explanation for the growth inhibition by acetate. The Pta-AckA pathway contributes about 20% of the growth inhibition by acetate, probably through the action of acetyl-phosphate on gene expression or the regulation of enzyme activities. We also show that acetate does not function as a classical uncoupling agent. This mechanism was commonly assumed to account for the largest part of acetate inhibition. Our data support a model where the imbalance of cellular anions, caused by the massive influx of acetate into the cell, is the major determinant of growth inhibition by acetate. We also present two synthetic biology projects from the iGEM competition: one to quantify mercury in a water sample and the other controlling the number of living cells by light in a bacterial population.

El origen de la vida en la Tierra es una de las preguntas más difíciles presentadas a la ciencia. En los últimos 60 años, ha habido un progreso considerable en entender cómo moléculas relativamente sencillas, que son relevantes para la vida, pueden ser generadas espontáneamente o pueden llegar a la Tierra desde el espacio. Además, los análisis de la evolución de la historia de ácidos nucleicos, los cuales almacenan la información genética, apuntan a un ancestro común universal ya extinto. Los estudios del origen de la vida ofrecen muchas pistas que apuntan hacia un origen común, quizás no solo en el Tierra sino también en algún otro punto del sistema solar. Debido al largo tiempo transcurrido desde que la Tierra empezó a albergar vida, las pistas más antiguas de los primeros organismos se han perdido. Es muy poco probable encontrar exactamente cómo fue este primer organismo. Sin embargo, en los últimos años la biología sintética ha logrado progresar mucho en la modificación de biomoléculas, en particular, los ácidos nucleicos. Es posible que pronto podamos construir y comprender un sistema minimalista en el cual las moléculas puedan copiarse a sí mismas dentro de una célula rudimentaria. El estudio de un sistema así podría permitirnos develar el origen de los primeros organismos. / The origin of life on Earth is one of the most challenging questions in science. In the last 60 years, considerable progress has been made in understanding how simple molecules relevant to life can be generated spontaneously or are known to arrive to Earth from space. Additionally, analysis of the evolution history of nucleic acids, which are the repository of genetic information, points to a now extinct, universal common ancestor for all life on Earth. The studies of the origin of life offer many clues towards a common origin, perhaps not just on Earth but somewhere else in the solar system. However due to the length of time that the Earth has harbored life, the oldest clues of the first organisms are mostly gone. It is unlikely to find exactly what this first organism was like. Nevertheless, in the last few years, synthetic biology has made remarkable progress at modifying biomolecules, particularly nucleic acids. It is possible that soon we will be able to construct and understand a minimalistic system in which molecules can copy themselves in a protocell. The study of such systems could shed light into the origin of the first organisms.

No description available.

La biologie de synthèse (BS) est une discipline scientifique qui se propose d'être à la biologie ce que la chimie synthétique est à la chimie analytique. La BS adopte des approches de l'ingénierie et vise à élaborer des systèmes biologiques fonctionnels réalisant des tâches techniques. Elle peut donc être qualifiée de technoscience, au sens où la technique est pour elle un débouché de ses recherches mais également une condition de ses découvertes. La BS ne se laisse cependant pas réduire à sa dimension intentionnelle. Elle est une discipline complexe, tant quant à son épistémologie qu'à son ontologie. Son inscription dans la société n'est pas moins complexe : les technosciences mettent toujours en jeu un grand nombre de dimensions de notre existence collective. Les enjeux éthiques de la BS sont donc majeurs, mais les crispations autour des nouvelles technologies rendent les débats difficiles, les positions se radicalisant entre utopies technophiles et dystopies technophobes.L'objectif de cette étude est de clarifier le contexte éthique, sans le simplifier, et d'apporter des éléments d'analyse des problèmes éthiques de la BS par-delà le simplisme rhétorique et le futurisme,qui minent les débats autour de cette technoscience. Il s'agit donc de se confronter à la complexité de la BS, de sa définition à son épistémologie et son ontologie, en passant par ses dimensions sociales et par le statut des êtres qu'elle produit. Les théories de W.V.O. Quine permettent d'éclairer les aspects épistémologiques et leurs conséquences ontologiques ; la philosophie des processus et des relations de Gilbert Simondon permet quant à elle de décrire la complexité des modes d'existence des êtres biosynthétiques entre contraintes techniques et devenir biologique. / Synthetic biology (SB) is a scientific field that aims at being to biology what syntheticchemistry is to analytic chemistry. SB adopts engineering approaches in order to develop functionalbiological systems carrying out technical tasks. It can thus be described as a technoscience, in the sensethat technic is both an outlet for its research and a material condition for its discoveries.However, SB does not let itself be reduced to that intentional dimension. It is a complexdiscipline, considering both its epistemology and its ontology. How SB is inscribed in society is notless complex: technosciences always involve several dimensions of our collective existence. SB's ethicalissues are thus crucial, but tensions about new technologies make the debates difficult, the positionsbeing often split between technophilic utopias and technophobic dystopias.The objective of this study is to clarify the ethical context without simplifying it, and to giveelements of analysis of the ethical problems in SB beyond the rhetorical simplism and the futurismthat undermine the debates about SB. SB's complexity must thus be confronted, from its definition toits epistemology and ontology, and through its social dimensions as well as the status of the beings itproduces. The theories of W.V.O. Quine enable the understanding of the epistemological aspects andtheir ontological consequences; the process and relations philosophy of Gilbert Simondon enables thedescription of the modes of existence of biosynthetic beings caught between their technical constraintsand their biological development.

La polyprotéine Gag du VIH-1 qui contient quatre principaux domaines (Matrix (MA), capside (CA), nucléocapside (NC), et P6) est l’orchestrateur privilégié de l'assemblage du virus HIV-1, assemblage qui a lieu pendant la phase tardive de la réplication. Il est bien connu que Gag interagit avec les lipides de la membrane plasmique de la cellule hôte et s’auto-assemble sur le feuillet interne de cette dernière afin de générer de nouvelles particules virales. Le bourgeonnement de ces particules virales hors de la cellule hôte est décrit comme étant dépendant de la machinerie cellulaire ESCRT. Différentes études structurales, fonctionnelles ainsi que des simulations de dynamique gros grain ont montré que la liaison de Gag à la membrane est médiée par une interaction duale. Une spécifique de nature éléctrostatique, qui associe une région hautement basique (HBR) du domaine MA de Gag au lipide acide,phosphatidyl inositol biphosphate (PI(4,5)P2) du feuillet interne de la membrane plasmique. Une de type hydrophobe, qui consiste en l’insertion du myristate de Gag dans la membrane plasmique. Savoir si Gag reconnait spécifiquement des domaines lipidiques pré-existants de type « rafts » ou si, au contraire, Gag tri ses lipides et les réorganise latéralement afin d’optimiser sa multimérisation et son bourgeonnement est une question à la fois fondamentale et d’actualité en virologie.Durant ma thèse, j’ai vérifié l’existence de la seconde hypothèse en utilisant des membranes modèles contenant du PI (4,5) P2 marqué de façon fluorescente et différent mutants et produits de la protéine Gag non-myristoylée. Ces expériences ont montré de fortes affinités de ces protéines pour les membranes contenant du PI (4,5) P2. S’appuyant sur les propriétés d’auto-extinction de fluorescence du marqueur choisit et à l’aide des différents variants de la protéine Gag, j'ai pu montré que la multimérisation de Gag génère l’existence de nanodomaines contenant du PI (4, 5) P2 et du cholestérol, la sphingomyéline étant au contraire exclue de ces domaines. En marquant la protéine Gag par un autre fluorophore, j’ai pu montrer par microscopie optique sur des vésicules lipidiques géantes (GUVs) que la protéine Gag partitionnait préférablement dans des microdomaines lipidiques de type liquide désordonnés (Ld). Par la suite, j’ai testé la capacité de la protéine Gag d’induire la formation de vésicules sur des membranes modèles (Bicouches supportés et GUVs) contenant du PI(4,5) P2 et de la phosphatidyl sérine (PS). En utilisant une microbalance à cristal de quartz (QCM-D) et des techniques de microscopie de fluorescence, j’ai suivi l'auto-assemblage de Gag dans le temps et ai montré que la protéine Gag était suffisante pour générer une courbure de la membrane et libérer des vésicules lipidiques. Grâce à différents produits de maturation de cette protéine, j’ai montré que la présence des domaines MA et CA est suffisante pour produire ces vésicules.L’ensemble de ces résultats suggèrent que la liaison et la multimérisation de la protéine Gag ne se produit pas dans des domaines lipidiques préexistants de type « raft », mais, au contraire, que la liaison et multimérisation de la protéine Gag génère l’existence de domaines lipidiques enrichis en PI (4,5) P2 et en cholestérol. La générescence de ces domaines lipidiques pourrait participer à la courbure de la membrane plasmique nécessaire au bourgeonnement du virus. / Gag polyprotein of HIV-1 is made of four main domains Matrix (MA), Capsid (CA), Nucleocapsid (NC), and P6 and is the prime orchestrator of virus assembly that occurs during the late phase of replication. It is well known that Gag interacts with host cell lipids and self-assemble along the inner-leaflet of the plasma membrane in order to generate virus like particles (VLPs). Budding of these VLPs out of the living cell is described to be ESCRT dependent. Structural, functional and simulation based studies has shown that Gag membrane binding is mediated by a bipartite interaction. One specific electrostatic interaction, between the highly basic region (HBR) of its MA domain and the host cell acidic lipid phosphatidyl inositol bisphophate (PI(4,5)P2), plus a hydrophobic interaction through Gag’s myristate insertion in the plasma membrane. It is still an opened question whether Gag would specifically recognize pre-existing lipid domains such as rafts to optimize its multimerization or, on the contrary, would reorganize lipids during its multimerization. During my Ph.D. I explored the second hypothesis using purified myr(-) Gag protein and model membranes containing fluorescently labelled PI(4,5)P2.Bonding experiments have shown strong affinities of these purified proteins towards PI(4,5)P2 containing lipid bilayers. Using PI(4,5)P2 fluorescence self-quenching properties, I found that multimerization Gag generates PI(4,5)P2/Cholesterol enriched nanoclusters. On the opposite, sphingomyelin was excluded from these nanoclusters. In addition to this, using a fluorescently labelled myr(-) Gag, I have observed its preferable partitioning into lipid disordered (Ld) phases of giant unilamellar vesicles (GUVs). Further, possibility of whether HIV-1 Gag alone, as a minimal system, can induce the formation of vesicles on PI(4,5)P2/PS containing supported lipid bilayers (SLBs) & GUVs was tested. Using quartz crystal microbalance (QCM-D) and fluorescence microscopy techniques, I monitored the self-assembly of HIV-1 Gag with time and found that Gag was sufficient to generate membrane curvature and vesicle release. Moreover, using mutants of this protein, I found that having MA and CA domain is enough for Gag to produce vesicle like structures. Taken together, these results suggest that binding and multimerization of Gag protein does not occur in pre-existing lipid domains (such as “rafts”) but this multimerization is more likely to induce PI(4,5)P2/Cholesterol nanoclusters. This nanophase separation could locally play a role in the membrane curvature needed for the budding of the virus.