Genetic engineering has allowed the design and production of recombinant antibodies (rmAbs) in plants. Nowadays, rmAbs are used in the treatment of a wide range of pathologies such as infectious diseases, inflammatory diseases and cancer, making rmAbs an important group of biomolecules within the pharmaceutical and biotechnology industry. By the time this study was started, the immunoglobulin G (IgG) was the antibody isotype predominantly expressed in plants. In recent years Modular DNA cloning technology has facilitated antibody engineering, with the development and expression of new rmAbs formats. However, there is hardly any study where different antibody formats are produced and compared in terms of yield and neutralizing capacity. Therefore, the starting point of the first chapter of this thesis is a comparative study where five different formats of the same commercial rmAb (Infliximab) against the human cytokine Tumor Necrosis Factor (TNF-¿) were expressed and compared. The results obtained in Chapter 1 demonstrate that both the isotype and the structure of the chosen rmAb influence the yield and the neutralizing capacity of rmAb. The expression of new antibody formats not only refers to the antibody isotype or structure; the format also refers to the combination of antibody idiotypes, leading to the production of oligo or polyclonal antibodies. Therefore, the possibility of co-expressing different monoclonal antibodies simultaneously in plants (creating oligoclonal or polyclonal formats) was raised. In the second chapter of this thesis, the expression of three rmAbs against the Ebola virus glycoprotein was studied. The three rmAbs were transiently expressed in N. benthamiana individually, by establishing separated production lines; in parallel, all three rmAbs were also co-expressed simultaneously in the same production line. The results obtained in this chapter demonstrated that the individual expression of rmAbs is feasible. However, when all three rmAbs are co-expressed, a drastic decrease in the binding of the antibody to the antigen was observed due to chain shuffling, as each heavy chain (HC) can be bound to any light chain (LC) other than its cognate chain, giving rise to an antibody cocktail with lower activity. With the objective of developing a method that allows co-expression of several rmAb in a single production line, we next proposed to exploit the viral interference phenomenon (also known as superinfection exclusion, SE). The results shown in Chapter three demonstrate that the production of an oligoclonal cocktail composed of 36 rmAbs in plants was possible using a viral expression system showing SE. The data obtained in this chapter showed that the resulting oligoclonal cocktail was active and capable of neutralizing toxic activities of the venom of the snake Bothrops asper in vitro and in vivo, wich was used as a model for studying the efficacy of the oligoclonal antibodies produced. The results of this thesis confirm and support the use of plants as platforms for the expression of alternative formats of antibodies. / La ingeniería genética ha permitido el diseño y la producción de anticuerpos recombinantes (rmAbs) en plantas. Hoy en día, los rmAbs se utilizan en el tratamiento de un amplio rango de patologías como enfermedades infecciosas, enfermedades inflamatorias y cáncer, convirtiéndose en un importante grupo de biomoléculas dentro de la industria farmacéutica y biotecnológica. Hasta la fecha de este estudio, en plantas se ha producido mayoritariamente la inmunoglobulina del tipo G (IgG). Gracias al desarrollo de la ingeniería del ADN recombinante y de la ingeniería de anticuerpos, es posible diseñar y producir nuevos formatos de rmAbs. Sin embargo, apenas existen estudios comparativos donde se demuestre si el formato de anticuerpo elegido es el idóneo en términos de rendimiento y capacidad neutralizante. Por tanto, el punto de partida del primer Capítulo de esta tesis consistió en la realización de un estudio comparativo de la expresión en plantas de cinco formatos distintos de un mismo rmAb comercial (Infliximab) frente a la citoquina humana Tumor Necrosis Factor (TNF-¿). Los resultados obtenidos en el Capítulo 1 demuestran que tanto el isotipo como la estructura del rmAb elegido influye en los niveles de rendimiento y en la capacidad neutralizante del rmAb. La expresión de nuevos formatos de anticuerpos no solo afecta al isotipo o a la estructura de las regiones constantes, sino que también se puede incluir en este término la expresión conjunta de distintos idiotipos de anticuerpos recombinantes, dando lugar a anticuerpos policlonales u oligoclonales recombinantes. Por tanto en esta tesis se planteó la posibilidad de co-expresar simultáneamente distintos anticuerpos monoclonales en plantas formando un cóctel oligoclonal. En el segundo Capítulo de esta tesis se diseñaron tres rmAbs frente a la glicoproteína de la cubierta del virus del Ébola. Los tres rmAbs se expresaron transitoriamente en N. benthamiana de manera individual mediante el establecimiento de líneas paralelas de producción y también se co-expresaron los tres rmAbs simultáneamente en una misma línea de producción. Los resultados obtenidos en este Capítulo demostraron que la expresión de los rmAbs de manera individual es factible. Sin embargo, cuando se co-expresan los tres rmAbs se observa una drástica disminución en la unión del anticuerpo al antígeno debido al barajado de cadenas, fenómeno por el cual cada cadena pesada (HC) se puede unir con cualquier cadena ligera (LC) distinta de su acompañante, dando lugar a un anticuerpo con una baja actividad. Finalmente, con el objetivo de desarrollar un método que permita co-expresar en una misma línea de producción varios rmAbs de forma reproducible se propuso explotar el fenómeno de la exclusión viral, un característica propia de los virus de plantas. Los resultados mostrados en el Capítulo 3 demuestran que es posible la producción de un cóctel oligoclonal compuesto por 36 rmAbs en N. benthamiana aprovechando el fenómeno de la exclusión viral. Los datos obtenidos en este capítulo muestran que el cóctel oligoclonal producido de esta forma mantiene intactas las actividades de los anticuerpos individuales y es capaz de neutralizar las actividades tóxicas del veneno de la serpiente Bothrops asper en ensayos in vitro e in vivo. Los resultados de esta tesis confirman y avalan el uso de las plantas como plataformas de expresión de formatos alternativos de anticuerpos. / El desenvolupament de l'enginyeria genètica ha permès el disseny i la producció d'anticossos recombinants (rmAbs) en plantes. Hui en dia, els rmAbs s'utilitzen en el tractament d'un ampli rang de patologies com malalties infeccioses, malalties inflamatòries i càncer convertint-se en un important grup de biomolècules dins de les indústries farmacèutiques i biotecnològiques. Fins a la data, s'han expressat majoritàriament la immunoglobulina del tipus G. Gràcies al desenvolupament de l'enginyeria de l'ADN recombinant i l'enginyeria dels anticossos s'han desenvolupat i expressat formats alternatius de rmAbs. Tanmateix, hi ha molts pocs estudis comparatius on es demostra si el format de l'anticòs elegit influeix en el rendiment i en la capacitat neutralitzant. Per tant, el punt de partida del primer Capítol d'esta Tesi és la realització d'un estudi comparatiu on s'expressen cinc formats diferents d'un mateix anticòs comercial (Infliximab) front a la citocina humana Tumor Necrosis Factor (TNF-¿). Els resultats obtesos demostren que tant l'isotip com l'estructura del rmAb elegit influeix en el rendiment i en la capacitat neutralitzant del rmAb. L'expressió de nous formats d'anticossos no sols afecta a l'isotip o a l'estructura del rmAb sinó que també pot incloure's dins d'aquest concepte l'expressió individual i l'expressió conjunta de diferents rmAbs. Partint d'aquesta hipòtesi, es va plantejar la possibilitat de co-expressar diferents rmAbs (còctel oligoclonal) en plantes. En el segon Capítol d'esta tesi es dissenyaren tres rmAbs front a la glicoproteïna del virus de l'Ébola. Els tres rmAbs s'expressaren transitòriament en N. benthamiana de manera individual mitjançant l'establiment de línies paral·leles de producció i també es co-expressaren els tres rmAbs en la mateixa línia de producció. Els resultats obtesos en este Capítol demostraren que l'expressió dels rmAbs de manera individual és factible. Tanmateix, quan es co-expressaren els tres rmAbs s'observà una dràstica disminució en la unió de l'anticòs a l'antigen com a conseqüència del shuffling chain, pel qual la cadena pesada (HC) s'uneix amb qualsevol cadena lleugera (LC) diferent a la seua acompanyant, formant un anticòs amb una baixa capacitat d'unió a l'antigen. Amb l'objectiu de desenvolupar un mètode que permeta co-expressar, en una mateixa línia de producció, un còctel oligoclonal es proposà explotar el fenomen de l'exclusió viral. Els resultats obtesos en el Capítol 3 demostren que l'expressió d' un còctel oligoclonal format per 36 rmAbs en plantes és possible. Els resultats mostren que el nostre còctel oligoclonal es capaç de neutralitzar activitats tòxiques del verí de la serp Bothrops asper en assaigs in vitro i in vivo. Els resultat obtesos en aquesta Tesi confirmen i avalen l'ús de les plantes com plataformes d'expressió de formats alternatius d'anticossos. / Huet Trujillo, E. (2017). Nuevas metodologías para la producción de anticuerpos recombinantes en plantas [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90469

La biología sintética se define como la ingeniería de la biología: el (re)diseño y construcción de nuevas partes, dispositivos y sistemas biológicos para realizar nuevas funciones con fines útiles, que se basan en principios elucidados de la biología y la ingeniería. Para facilitar la construcción rápida, reproducible y predecible de estos sistemas biológicos a partir de conjuntos de componentes es necesario desarrollar nuevos métodos y herramientas. La tesis plantea la optimización multiobjetivo como el marco adecuado para tratar los problemas comunes que surgen en el diseño racional y el ajuste óptimo de los circuitos genéticos sintéticos. Utilizando un enfoque clásico de ingeniería de sistemas, la tesis se centra principalmente en: i) el modelado de circuitos genéticos sintéticos basado en los primeros principios, ii) la estimación de parámetros de modelos a partir de datos experimentales y iii) el ajuste basado en modelos para lograr el desempeño deseado de los circuitos. Se han utilizado dos circuitos genéticos sintéticos de diferente naturaleza y con diferentes objetivos y problemas: un circuito de realimentación de tipo 1 incoherente (I1-FFL) que exhibe la importante propiedad biológica de adaptación, y un circuito de detección de quorum sensing y realimentación (QS/Fb) que comprende dos bucles de realimentación entrelazados -uno intracelular y uno basado en la comunicación de célula a célula- diseñado para regular el nivel medio de expresión de una proteína de interés mientras se minimiza su varianza a través de la población de células. Ambos circuitos han sido analizados in silico e implementados in vivo. En ambos casos, se han desarrollado modelos de estos circuitos basado en primeros principios. Se presta especial atención a ilustrar cómo obtener modelos de orden reducido susceptibles de estimación de parámetros, pero manteniendo el significado biológico. La estimación de los parámetros del modelo a partir de los datos experimentales se considera en diferentes escenarios, tanto utilizando modelos determinísticos como estocásticos. Para el circuito I1-FFL se consideran modelos determinísticos. Aquí, la tesis plantea la utilización de modelos locales utilizando la optimización multiobjetivo para realizar la estimación de parámetros del modelo bajo escenarios con estructura de modelo incompleta. Para el circuito QS/Fb, una estructura controlada por realimentación, el problema tratado es la falta de excitabilidad de las señales. La tesis propone una metodología de estimación en dos etapas utilizando modelos estocásticos. La metodología permite utilizar datos de curso temporal promediados de la población y mediciones de distribución en estado estacionario para una sola célula. El ajuste de circuitos basado en modelos para lograr un desempeño deseado también se aborda mediante la optimización multiobjetivo. Para el circuito QS/Fb se realiza un análisis estocástico completo. La tesis aborda cómo tener en cuenta correctamente tanto el ruido intrínseco como el extrínseco, las dos principales fuentes de ruido en los circuitos genéticos. Se analiza el equilibrio entre ambas fuentes de ruido y el papel que desempeñan en el bucle de realimentación intracelular, y en la realimentación extracelular de toda la población. La principal conclusión es que la compleja interacción entre ambos canales de realimentación obliga al uso de la optimización multiobjetivo para el adecuado ajuste del circuito. En esta tesis además del uso adecuado de herramientas de optimización multiobjetivo, la principal preocupación es cómo derivar directrices para el ajuste in silico de parámetros de circuitos que puedan aplicarse de forma realista in vivo en un laboratorio estándar. Como alternativa al análisis de sensibilidad de parámetros clásico, la tesis propone el uso de técnicas de clustering a lo largo de los frentes de Pareto, relacionando el compr / La biologia sintètica es defineix com l'enginyeria de la biologia: el (re) disseny i construcció de noves parts, dispositius i sistemes biològics per a realitzar noves funcions útils que es basen a principis elucidats de la biologia i l'enginyeria. Per facilitar la construcció ràpida, reproduïble i predictible de aquests sistemes biològics a partir de conjunts de components és necessari desenvolupar nous mètodes i eines. La tesi planteja la optimització multiobjectiu com el marc adequat per a tractar els problemes comuns que apareixen en el disseny racional i l' ajust òptim dels circuits genètics sintètics. Utilitzant un enfocament clàssic d'enginyeria de sistemes, la tesi es centra principalment en: i) el modelatge de circuits genètics sintètics basat en primers principis, ii) l' estimació de paràmetres de models a partir de dades experimentals i iii) l' ajust basat en models per aconseguir el rendiment desitjat dels circuits. S'han utilitzat dos circuits genètics sintètics de diferent naturalesa i amb diferents objectius i problemes: un circuit de prealimentació de tipus 1 incoherent (I1-FFL) que exhibeix la important propietat biològica d'adaptació, i un circuit de quorum sensing i realimentació (QS/Fb) que comprèn dos bucles de realimentació entrellaçats -un intracel·lular i un basat en la comunicació de cèl·lula a cèl·lula- dis-senyat per regular el nivell mitjà d'expressió normal d'una proteïna d'interès mentre es minimitza la seua variació al llarg de la població de cèl·lules. Els dos circuits han estat analitzats in silico i implementats in vivo. En tots dos casos, s'han desenvolupat models basats en primers principis d'aquests circuits. Després es presta especial atenció a delinear com obtenir models d'ordre reduït susceptibles de estimació de paràmetres, però mantenint el significat biològic. L' estimació dels paràmetres del model a partir de les dades experimentals es considera en diferents escenaris, tant utilitzant models determinístics com estocàstics. Per al circuit I1-FFL es consideren models determinístics. La tesi planteja la utilització de models locals utilitzant la optimització multiobjectiu per realitzar l'estimació de parametres del model sota escenaris amb estructura de model incompleta (dinàmica no modelada). Per al circuit de QS/Fb, una estructura controlada per realimentació, el problema tractat és la manca d'excitabilitat dels senyals. La tesi proposa una metodologia de estimació en dues etapes utilitzant models estocàstics. La metodologia permet utilitzar dades de curs temporal promediats de la població i mesures de distribució en estat estacionari d'una sola una cèl·lula. L' ajust de circuits basat en models per aconseguir el rendiment desitjat dels circuits també s' aborda mitjançant la optimització multiobjectiu. Per al circuit QS/Fb, es fa un anàlisi estocàstic complet. La tesi aborda com tenir en compte correctament tant el soroll intrínsec com l' extrínsec, les dues principals fonts de soroll en els circuits genètics sintètics. S' analitza l'equilibri entre dues fonts de soroll i el paper que exerceixen en el bucle de realimentació intracel·lular, les i en la realimentació extracel·lular de tota la població. La principal conclusió es que la complexa interacció entre els dos canals de realimentació fa necessari l' ús de la optimització multiobjectiu per al adequat ajust del circuit. En aquesta tesi, a més de l'ús adequat d'eines d'optimització multiobjectiu, la principal preocupació és com derivar directives per al ajust in silico de paràmetres de circuits que puguin aplicar-se de forma realista en viu en un laboratori estàndard. Així, com a alternativa a l'anàlisi de sensibilitat de paràmetres clàssic, la tesi proposa l'ús de l' tècniques de l'agrupació al llarg dels fronts de Pareto, relacionant el compromís de dessempeny amb les regions en l'espai d'paràmetres. / Synthetic biology is defined as the engineering of biology: the deliberate (re)design and construction of novel biological and biologically based parts, devices and systems to perform new functions for useful purposes, that draws on principles elucidated from biology and engineering. Methods and tools are needed to facilitate fast, reproducible and predictable construction of biological systems from sets of biological components. This thesis raises multi-objective optimization as the proper framework to deal with common problems arising in rational design and optimal tuning of synthetic gene circuits. Using a classical systems engineering approach, the thesis mainly addresses: i) synthetic gene circuit modeling based on first principles, ii) model parameters estimation from experimental data and iii) model-based tuning to achieve desired circuit performance. Two gene synthetic circuits of different nature and with different goals and inherent problems have been used throughout the thesis: an Incoherent type 1 feedforward circuit (I1-FFL) that exhibits the important biological property of adaptation, and a Quorum sensing/Feedback circuit (QS/Fb) comprising two intertwined feedback loops -an intracellular one and a cell-to-cell communication-based one-- designed to regulate the mean expression level of a protein of interest while minimizing its variance across the population of cells. Both circuits have been analyzed in silico and implemented in vivo. In both cases, circuit modeling based on first principles has been carried out. Then, special attention is paid to illustrate how to obtain reduced order models amenable for parameters estimation yet keeping biological significance. Model parameters estimation from experimental data is considered in different scenarios, both using deterministic and stochastic models. For the I1-FFL circuit, deterministic models are considered. In this case, the thesis raises ensemble modeling using multi-objective optimization to perform model parameters estimation under scenarios with incomplete model structure (unmodeled dynamics). For the QS/Fb gene circuit, a feedback controlled structure, the lack of excitability of the signals is the problem addressed. The thesis proposes a two-stage estimation methodology using stochastic models. The methodology allows using population averaged time-course data and steady state distribution measurements at the single-cell level. Model-based circuit tuning to achieve desired circuit performance is also addressed using multi-objective optimization. First, for the QS/Fb feedback control circuit, a complete stochastic analysis is performed. Here, the thesis addresses how to correctly take into account both intrinsic and extrinsic noise, the two main sources of noise in gene synthetic circuits. The trade-off between both sources of noise, and the role played by in the intracellular single-cell feedback loop and the extracellular population-wide feedback is analyzed. The main conclusion being that the complex interplay between both feedback channels compel the use of multi-objective optimization for proper tuning of the circuit to achieve desired performance. Thus, the thesis wraps up all the previous results and uses them to address circuit tuning for desired performance. Here, besides the proper use of multi-objective optimization tools, the main concern is how to derive guidelines for circuit parameters tuning in silico that can realistically be applied in vivo in a standard laboratory. Thus, as an alternative to classical parameters sensitivity analysis, the thesis proposes the use of clustering techniques along the optimal Pareto fronts relating the performance trade-offs with regions in the circuits parameters space. / This work has been partially supported by the Spanish Government (CICYT DPI2014- 55276-C5-1) and the European Union (FEDER). The author was recipient of the grant Formación de Personal Investigador by the Universitat Politècnica de València, subprogram 1 (FPI/2013-3242). She was also recipient of the competitive grants for pre-doctoral stays Erasmus Student Placement-European Programme 2015, and FPI Mobility program 2016 of the Universitat Politècnica de València. She also received the competitive grant for a pre-doctoral stay Becas de movilidad para Jóvenes Profesores e Investigadores 2016, Programa de Becas Iberoamérica of the Santander Bank. / Boada Acosta, YF. (2018). A systems engineering approach to model, tune and test synthetic gene circuits [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/112725

Research on bacteria in their biofilm form is limited by the ability to artificially culture bacterial biofilms in a system that permits the visualization of individual cells. The experiments comprising this thesis research are on-going investigations of bacterial culture systems engineered to provide an environment that mimics biofilms while enabling real-time microscopy. Specifically, the microfluidic systems developed and assessed as part of this thesis permit the visualization of individual bacteria cells within consortia growing within a narrow space provided by a microfluidic device. This research demonstrates the versatility of these microfluidic systems across potentially high-throughput microbiological experiments utilizing genetically engineered Escherichia coli. Before demonstrating the efficacy of these systems, the development of the field of synthetic biology over the past half century is reviewed, focusing on synthetic genetic circuits and their applications (Chapter 2). The first and main microfluidic device explored in this research was developed to mimic the nutrient-deficient conditions within biofilms by forcing media to enter the culture area through a narrow, torturous channel. The microfluidic channel was thin enough (0.97 μm) to prevent the motility of 1-μm-wide E. coli cells, enabling visualization of individual cells. The bacteria cultured in the device contained either a simple Plux-driven quorum sensing receiver (Chapters 3 and 5) or a LacI- and TetR-driven genetic toggle switch (Chapter 4). Under the culture conditions, the quorum sensing reporter signal was detected even without addition of the signaling molecule (Chapter 3). The genetic toggle switch was stable when the system began in the high-LacI expression state, but after 5 days of culture, >5% of high-TetR expression cells began to consistently express the high-LacI state (Chapter 4). This system was also employed to track lineages of cells using real-time microscopy, which successfully characterized the inheritance of aberrant, enlarged cell phenotypes under stress (Chapter 5). Another microfluidic device, a droplet bioreactor, was also developed to culture small numbers of cells in an aqueous bubble suspended in oil (Chapter 6). Quorum sensing receiver cellswere cultured in this device, demonstrating that it is well suited for testing the effects of compounds on biofilms within water-in-oil droplets. / Ph. D. / Bacteria are the most abundant organisms globally, yet relatively little is understood about the basic biology of biofilms, one of the most common natural states of bacteria. Biofilms are ubiquitous consortia of individual microbial cells that send and received chemical signals from one another to carry out group behaviors such as quorum sensing. The impacts of biofilms range from the contamination of food processing equipment to antibiotic resistant bacterial infections. The vast majority of microbiological research has been conducted on bacteria in their planktonic state as individual cells cultured in a liquid medium. This form of culture does not permit the types of research that can help address the impacts of biofilms on human health and economic activities, never mind examine the biological mechanism of random gene and morphological expression within bacterial biofilm. This thesis presents research utilizing two microfluidic devices that will enable further large-scale studies to unravel the mechanisms that create biofilms as well as permit high-throughput testing of chemical compounds to control the growth and development of biofilms. Moreover, these devices permit the use of real-time microscopy to track cells and their growth over time. The first microfluidic device utilized in this research mimics the nutrient-limiting conditions of biofilms. This biofilm-mimicking device was used to culture a common research bacteria, Escherichia coli, with one of two engineered genetic circuits (reviewed in Chapter 2): a quorum sensing receiver (Chapters 3 and 5) or genetic toggle switch (Chapter 4). Both of these genetic circuits demonstrated stochasticity in their gene expression states under the culture conditions in the biofilm-mimicking device. The second microfluidic device successfully permitted the culture of small numbers of isolated cells within a small bubble of bacterial media suspended in oil (Chapter 6). Additionally, this device enabled the addition of chemical compounds to influence the growth and metabolism of the trapped cells. Collectively, these microfluidic devices provide the ability to effectively study both the mechanisms underlying random gene expression within biofilms as well as explore the chemical factors that can be used to control and mitigate biofilm formation and growth.

DNA provides an ideal substrate for nanoscale construction and programmable dynamic mechanisms. DNA mechanisms can be used to produce DNA motors which do mechanical work, e.g. transportation of a substrate along a track. I explore a method for control of a DNA mechanism ubiquitous in DNA motor designs, toehold-mediated strand displacement, by which one strand in a duplex can be swapped for another. My method uses a mismatch between a pair of nucleotides in the duplex, which is repaired by displacement. I find that displacement rate can be fine-tuned by adjusting the position of the mismatch in the duplex, enabling the design of complex kinetic behaviours. A bipedal motor [1, 2] is designed to walk along a single-stranded DNA track. Previously the motor has only taken a single step, due to a lack of designs to extend the single-stranded track. I present a novel design for track held under tension using a 3D DNA origami tightrope, and verify its assembly. The bipedal motor design is adapted and a method to specifically place motors on tightropes is demonstrated. Motor operation is investigated on truncated tracks and tightrope tracks by electrophoresis and spectrofluorometry. The motor does not accumulate appreciably at the track end; this is tentatively attributed to rearrangement of the motor between track sites without interaction with fuel. Tightrope origami can hold single-stranded DNA under pN tension. I use tightropes to study hybridization kinetics under tension and find dramatic, non-monotonic changes in hybridization rate constants and dissociation constants with tension in the range ∼0-15 pN. Extended tracks for a 'burnt-bridges' motor which destroys its track as it moves [3] are created on the inside of DNA nanotubes, which can be polymerised to create tracks up to a few mm in length, and on tiles which I attempt to join in a specific order. Crossing of the motor between tubes is verified, and microscopy experiments provide some evidence that track is being cleaved by the motor, a requirement for movement along the track. Tile based tracks are imaged by super-resolution DNA PAINT [4], providing proof-of-principle for track observation to infer motor movement.

Genetically modified organisms (GMOs) are confined physically in order to contain their spread in nature and to minimise chances of horizontal gene transfer. However, with the potential that GMOs hold as cheap, reliable and efficient micro-machines, their eventual uncontrolled release into the wider space is becoming more likely. Indeed, their application as environmental sensors is largely increasing. Nevertheless, the field of synthetic biology may also afford solutions to the problem. A major potential application of GMOs is the delivery of antigens to human and animal hosts, through the utilization of live, engineered microbes. Recombinant technology is promising for several reasons including their capacity to be less reactogenic, more potent, safer and genetically definable. Also, they have the potential to provide protection against multiple targets simultaneously, are relatively inexpensive and can be eradicated with antibiotics, as the need arises. Besides, delivery of vaccines to mucosal surfaces is more efficient. Mutant Salmonella expressing heterologous antigens have been shown to induce protection against a variety of pathogens. Nevertheless, limited containment systems are available that can be applicable for bacterial antigen carriers. This project aims to design safeguards for the bacterial antigen delivery systems that limit ORF translatability and self-inactivates/destructs upon exit from the host. In this work, double quadruplet codons were suppressed by orthogonal tRNAs, providing a barrier for gene translation in the recipient cells when antigen is horizontally transferred. Furthermore, three kill switches were designed that are activated by a decrease in temperature from 37 °C. First, Sau3AI endonuclease was activated by protein self-splicing at low temperature mediated by Mtu recA intein. The activation of the endonuclease led to three-fold logarithmic decrease in the number of viable cells within two hours of gene expression. Second, RNA-dependent activation of RNase 7 showed a reduction in the number of viable cells at low temperature of three logarithmic folds. RNase 7 was controlled by the cspA 5’UTR, which sequesters ribosome binding site at 37 °C and allows translation at low temperature. Third, CspA 5’UTR was shown to regulate expression of TEV protease at 37 °C and low temperature. This led to bacterial cellular inhibition within two hours of TEV induction and five-fold logarithmic reduction in the number of viable cells at low temperature. In addition, for the first time and contrary to previous studies, the TEV protease was shown to inhibit cellular growth. It was also shown that biofilm formation was drastically impaired by the TEV activity. The three killing switches and the quadruplet translation system are poised to function as robust safeguards for bacterial antigen delivery systems.

Das stäbchenförmige Bakterium Escherichia coli teilt sich in zwei gleich große Tochterzellen. Dies ist nur möglich, wenn sich die Zelle in der Mitte teilt. Bei E. coli wird die Zellteilung durch den Zusammenschluss der FtsZ-Proteine an der Membran zum Z-Ring eingeleitet. Topologische Regulierung des Z-Ringes erfolgt durch räumlich-zeitliche Oszillationen von Min-Proteinen zwischen den beiden Zellpolen. MinC, MinD und MinE binden an und lösen sich von der Membran unter Hydrolyse von ATP und in antagonistischer Art und Weise, was zu einer alternierenden Ansammlung von MinC und MinD an den Zellpolen führt. Gemittelt über die Zeit ergibt sich somit ein MinD-Verteilungsprofil, das maximale Konzentration an den Zellpolen und ein Minimum in der Zellmitte aufweist. MinC bindet an MinD und folgt somit seiner Verteilung. Der Zusammenschluss von FtsZ-Proteinen wird durch MinC unterbunden, und somit kann sich der Z-ring nur an einer Position herausbilden, die ein Minimum an MinC aufweist - der Zellmitte. Das Min-system wurde in der Vergangenheit auch mit einem in-vitro-Ansatz untersucht, indem Min-Proteine in künstliche, aufliegende Lipiddoppelschichten (supported lipid bilayers, SLB) rekonstitutiert wurden. Dabei bildeten die Min-Proteine kein oszillierendes Muster aus, sondern organisierten sich vielmehr in parallelen und propagierenden Wellen (Loose, 2008, Science, 320). In diesen in-vitro-Experimenten war das Membransubstrat wesentlich größer als die Wellenlänge der Min-Proteinwellen. In vivo hingegen ist die Länge der Zelle in der gleichen Größenordnung wie die charakteristische Länge des Oszillationsmusters der Min-Proteine. Daher war es das Ziel dieser Arbeit, den Einfluß einer beschränkten Fläche und geometrischer Formgebung der künstlichen Lipiddoppelschichten auf die Wellenpropagation der Min-Protein zu untersuchen. Flächige Beschränkung künstlicher Membranen erfolgte durch Mikrostrukturtechnologie. Deckglässchen wurden mit einer Goldschicht und mikroskopischen Aussparungen unterschiedlicher geometrischer Formen strukturiert. Funktionale SLBs bildeten sich nur auf Glasflächen ohne Goldbeschichtung aus. Nach der Rekonstitution der Min-Proteine, organisierten sich diese auf den Membranstücken in parallele Wellen. Dabei bestimmte die flächige Beschränkung der künstlichen Membranen die Ausbreitungsrichtung der Min-Proteinwellen. Min-Proteinwellen konnten entlang gekrümmter Membranstreifen, in Ring- und sogar in Slalomstrukturen geleitet werden. In geraden, länglichen Strukturen richteten sich die Wellen entlang der längsten Achse aus. Kopplung von Proteinwellen auf räumlich getrennten Membranstücken in Abhängigkeit des Abstandes und des sogenannten Molecular Crowdings in der wässrigen Lösung konnte ebenfalls beobachtet werden. Diese Kopplung ist ein Indiz für inhomogene Proteinverteilungen in der Lösung oberhalb der Membran. Desweiteren konnten Min-Proteinwellen auch in diversen dreidimensionalen künstlichen Membranen rekonstitituiert werden. Im Wildtyp von E. coli ähneln die Min-Proteindynamiken der einer Oszillation mit einer charakteristischen Länge von 5 µm. Auf SLBs, bilden Min-Proteine Wellen mit einer Wellenlänge aus, die ca. zehnmal größer ist als in vivo. Dieser Unterschied zwischen der in-vivo- und der in-vitro-Welt wurde untersucht und diskutiert. In vitro konnte die Wellenlänge um 50 % durch Erhöhung des Molecular Crowding in der Lösung sowie um 33 % durch Temperaturerhöhung verkleinert werden. Das oszillierende Muster könnte dahingegen eine Folge der Kompartimentierung sein. Erste Versuche, das Min-System in geschlossene Membrankompartimente zu rekonstitutieren, wurden getestet. / Escherichia coli, a rod-like bacterium, divides by binary fission. Cell division into two daughter cells of equal size requires that fission takes place at a midcell position. In E. coli, cell division is initiated by assembly of the FtsZ-proteins at the inner membrane to the Z-ring. Topological regulation of the Z-ring is achieved by spatiotemporal pole-to-pole oscillations of Min-proteins. MinC, MinD and MinE bind to and detach from - under hydrolysis of ATP - the membrane in an antagonistic manner leading to an alternating accumulation of MinC and MinD at the cell poles. Averaged over time, the distribution profile of MinD exhibits maximal concentration at the cell poles and a minimum at the cell center. MinC binds to MinD and thus follows its distribution. FtsZ assembly is inhibited by MinC and therefore the Z-ring can only form at a cell position low in MinC - at the cell center. In the past, the Min-system was also investigated in an in vitro approach by reconstitution of Min-proteins into a supported lipid bilayer (SLB). Here, Min-proteins did not self-organize into an oscillatory pattern but into parallel and propagating waves (Loose, 2008, Science, 320). In this in vitro assay, the membrane substrate was infinitely large compared to the wavelength. However, in vivo, the cell length is on the same order of magnitude as the respective length scale of the oscillatory pattern of Min-proteins. Therefore, we wished to investigate the effect of lateral confinement and geometric structuring of artificial lipid bilayers on the Min-protein wave propagation. Lateral confinement of artificial membranes was achieved by microfabrication technology. Glass slides were patterned by a gold coating with microscopic windows of different geometries, and functional SLBs were only formed on uncoated areas. Upon reconstitution, Min-proteins organized into parallel waves on the geometric membrane patches. Confinement of the artificial membranes determined the direction of propagation of Min-protein waves. Min-protein waves could be guided along curved membrane stripes, in rings and even along slalom-geometries. In elongated membrane structures, the protein waves always propagate along the longest axis. Coupling of protein waves across spatially separated membrane patches was observed, dependent on gap size and level of molecular crowding of the aqueous media above the bilayer. This indicates the existence of an inhomogeneous and dynamic protein gradient in the solution above the membrane. Furthermore, reconstitution of Min-protein waves in various three-dimensional artificial membranes was achieved. In wild-type E. coli, Min-protein dynamics resemble that of an oscillation with a characteristic length scale of 5 µm. On supported lipid bilayers, Min-proteins self-organize into waves with a wavelength approximately 10-fold larger than in vivo. These discrepancies between the in vivo and in vitro world were investigated and discussed. In vitro, the wavelength could be decreased by a factor of 50 % by increase of the molecular crowding in solution and by 33 % through temperature increase. The oscillatory pattern is thought to be a consequence of compartmentalization and first attempts to encapsulate the Min-system in closed bilayer compartments are presented.

As an inspiration from nature, polymeric vesicles can be formed from amphiphilic block-copolymers. These vesicles are called polymersomes and have applications in drug delivery and as nanoreactors. Within this thesis, photo cross-linked and pH sensitive polymersomes were synthesized, characterized and applied on cells as well as bionanoreactors. The stability due to the crosslinking yielded polymersomes which show a distinct and reproducible swelling upon repeated pH changes. If the non cross-linked vesicles were exposed to a plasma-cleaned surface, they formed a tethered singly and multiple bilayers. Upon studying these membranes, they turned out to harden upon crosslinking and showed a completely non-fluid behaviour. Additionaly, the polymersome-cell interactions were studied and yielded a high influence of the crosslinking conditions on cellular toxicity. If crosslinked for a long time in a phosphate-free enviroment, the polymersomes proved to be least toxic. Finally, an enzyme was incorporated into the polymersomes to create bionanoreactors. Due to the pH sensitivity and swelling, the vesicles created yielded a pH controlled nanoreactor with enzymatic activity and a swollen, e.g. acidic, state only.

There is an unmet medical need in the treatment of gout. This type of inflammatory arthritis can be efficiently alleviated by the enzyme uricase. This enzyme breaks down uric acid, the causative agent of gout, so it can be flushed from the body. In humans and the other great apes, uricase is a pseudogene and as such is inactive. Research on therapeutic uricases has focused on using enzymes from naturally occurring sources; however, these foreign proteins can be very antigenic and present a potentially life-threatening safety risk to patients. We address the challenges of developing a safer uricase therapeutic by exploiting evidence that, while inactive, the human pseudogene is expressed in the human body and may be recognized as self by the immune system. To develop a ﾓhuman-likeﾔ? uricase we apply the hybrid computational and experimental approach of Ancestral Sequence Reconstruction to search functional sequence space of uricase proteins to engineer an enzyme with high sequence identity to the human pseudogene, and possessing therapeutic levels of activity for the breakdown of uric acid. This dissertation describes the development and characterization of several uricase leads. The most active ancestral uricase possesses both enhanced in vitro and in vivo stability (in healthy rats) when assayed head-to-head Pegloticase, the only FDA approved uricase for the treatment of gout.

Formation of segments during vertebrate embryogenesis is regulated by a biological clock. Models and experimental data indicate that the core of this clock consists of a cell- autonomous single cell oscillator. This oscillator likely involves a genetic feedback loop of transcriptional repressors belonging to the hairy gene family. In zebrafish, three her genes, her1, hes6 and her7, have been identified as core oscillator components. The main purpose of this project was to study the molecular mechanism of the hairy gene negative feedback oscillator in single cells. To determine whether a single cell oscillator is part of the zebrafish segmentation clock, a cell dissociation protocol was established to track the expression of Her1 ex vivo. Upon dissociation, Her1 expression continued to oscillate for up to three cycles. The period of oscillations was significantly slower than that of the segmentation clock, but appears to speed up in the presence of serum. To test whether the hairy gene interactions are sufficient to generate oscillations in single cells, a protocol was established that uses synthetic biology principles to design, construct and characterize hairy gene networks in yeast. First a library of network parts, containing hairy genes, promoters and Her binding sites was generated and subsequently assembled into simple devices to test their functionality in yeast. The three core oscillator components, Her1, Hes6 and Her7, were characterized and optimized for expression in yeast. In the SWITCH-OFF assay, the Her1 protein, modified with a MigED yeast repressor domain, was found to function as a transcriptional repressor in yeast, while Hes6 with the same modification can not. The dissociation of segmentation clock cells provides the first direct evidence that single cell oscillators exist in zebrafish. In this system, oscillator dynamics can be studied without the interactions of higher level clock components. In parallel, establishing a yeast chassis for hairy gene networks provides a novel technique to directly test predicted oscillator mechanisms by constructing them ’bottom up’.

A fundamental property of living cells is the ability to sense and respond appropriately to changing environmental conditions. In budding yeast (Sacharomyces cerevisiae), changes in extracellular osmotic conditions are sensed by the HOG SAPK pathway, which orchestrates the cell adaptation program required to maximize cell survival upon stress. Although most of the HOG pathway components have been described, little was known about the dynamics of the response. The aim of this thesis was to analyze the dynamic behavior of the HOG pathway. By using a chemical inhibitor and extensive signal quantification we showed that the HOG pathway is controlled by high basal signaling counteracted by a negative feedback regulatory system. This property determines dynamic signaling in terms of faster response times and higher sensitivity to small variations in extracellular stimuli. This thesis also aimed to implement novel strategies for biological computation that allow increasing complexity of circuits. By engineering signaling pathways in yeast, we have shown that distribution of computation tasks among several wired cells reduces wiring constraints and allows scalability of circuit complexity. Moreover, reusability of cells permits implementation of multiple circuits. Overall, our results define novel dynamic properties of the HOG pathway and have been important to achieve a better view of signal transduction process though MAPK pathways. Moreover, we have developed and implemented novel strategies for biological computation that solved fundamental constrains in the field of synthetic biology. / Una propietat cel•lular fonamental és l’habilitat de detectar estímuls i respondre coherentment a un ambient dinàmic. En cèl•lules de llevat (Saccharomyces cerevisiae), els canvis en l’osmolaritat externa són detectats per la via de senyalització de HOG que organitza tot el programa d’adaptació cel•lular, indispensable per assegurar la supervivència cel•lular en estrès osmòtic. Tot i que la gran majoria dels components de la via de HOG han estat identificats, la dinàmica del procés de senyalització és encara força desconeguda. L’objectiu d’aquest projecte de tesis ha estat analitzar el comportament dinàmic de la via de HOG. Gràcies a la utilització d’un al•lel inhibible de la MAPK Hog1 i a la quantificació sistemàtica del procés de senyalització, hem pogut demostrar que en la via de HOG existeix una intensa senyal basal reprimida constantment per un feedback negatiu depenent de la MAPK Hog1. Aquesta tesi també té com a objectiu la implementació de noves estratègies de computació biològica que permetin un increment de la complexitat dels circuits. Gràcies a la bioenginyeria de les vies de senyalització de llevat, hem demostrat que la distribució de la computació en diferents cèl•lules connectades entre elles disminueix les limitacions de connexió i permet incrementar la complexitat dels circuits a un baix cost. En conjunt, els nostres resultats defineixen noves propietats dinàmiques de la via de HOG i han estat importants per tenir una visió global millorada del procés de senyalització per vies de MAPK. A més, hem dissenyat i implementat noves estratègies de computació biològica que han resolt problemes fonamentals del camp de la biologia sintètica.