DESIGN OF GENETIC ELEMENTS AND SOFTWARE TOOLS FOR PLANT SYNTHETIC BIOLOGY

Tesis por compendio[EN] Synthetic Biology is an emerging interdisciplinary field that aims to apply the engineering principles of modularity, abstraction and standardization to genetic engineering. The nascent branch of Synthetic Biology devoted to plants, Plant Synthetic Biology (PSB), offers new breeding possibilities for crops, potentially leading to enhanced resistance, higher yield, or increased nutritional quality. To this end, the molecular tools in the PSB toolbox need to be adapted accordingly, to become modular, standardized and more precise. Thus, the overall objective of this Thesis was to adapt, expand and refine DNA assembly tools for PSB to enable the incorporation of functional specifications to the description of standard genetic elements (phytobricks) and to facilitate the construction of increasingly complex and precise multigenic devices, including genome editing tools. The starting point of this Thesis was the modular DNA assembly method known as GoldenBraid (GB), based on type IIS restriction enzymes. To further optimize the GB construct-making process and to better catalog the phytobricks collection, a database and a set of software-tools were developed as described in Chapter 1. The final webbased software package, released as GB2.0, was made publicly available at www.gbcloning.upv.es. A detailed description of the functioning of GB2.0, exemplified with the building of a multigene construct for anthocyanin overproduction was also provided in Chapter 1. As the number and complexity of GB constructs increased, the next step forward consisted in the refinement of the standards with the incorporation of experimental information associated to each genetic element (described in Chapter 2). To this end, the GB package was reshaped into an improved version (GB3.0), which is a self-contained, fully traceable assembly system where the experimental data describing the functionality of each DNA element is displayed in the form of a standard datasheet. The utility of the technical specifications to anticipate the behavior of composite devices was exemplified with the combination of a chemical switch with a prototype of an anthocyanin overproduction module equivalent to the one described in Chapter 1, resulting in a dexamethasone-responsive anthocyanin device. Furthermore, Chapter 3 describes the adaptation and functional characterization of CRISPR/Cas9 genome engineering tools to the GB technology. The performance of the adapted tools for gene editing, transcriptional activation and repression was successfully validated by transient expression in N. benthamiana. Finally, Chapter 4 presents a practical implementation of GB technology for precision plant breeding. An intragenic construct comprising an intragenic selectable marker and a master regulator of the flavonoid biosynthesis was stably transformed in tomato resulting in fruits enhanced in flavonol content. All together, this Thesis shows the implementation of increasingly complex and precise genetic designs in plants using standard elements and modular tools following the principles of Synthetic Biology.[ES] La Biología Sintética es un campo emergente de carácter interdisciplinar que se fundamenta en la aplicación de los principios ingenieriles de modularidad, abstracción y estandarización a la ingeniería genética. Una nueva vertiente de la Biología Sintética aplicada a las plantas, la Biología Sintética Vegetal (BSV), ofrece nuevas posibilidades de mejora de cultivos que podrían llevar a una mejora de la resistencia, a una mayor productividad, o a un aumento de la calidad nutricional. Sin embargo, para alcanzar este fin las herramientas moleculares disponibles en estos momentos para BSV deben ser adaptadas para convertirse en modulares, estándares y más precisas. Por ello se planteó como objetivo general de esta Tesis adaptar, expandir y refinar las herramientas de ensamblaje de DNA de la BSV para permitir la incorporación de especificaciones funcionales en la descripción de elementos genéticos estándar (fitobricks) y facilitar la construcción de estructuras multigénicas cada vez más complejas y precisas, incluyendo herramientas de editado genético. El punto de partida de esta Tesis fue el método de ensamblaje modular de ADN GoldenBraid (GB) basado en enzimas de restricción tipo IIS. Para optimizar el proceso de ensamblaje y catalogar la colección de fitobricks generados se desarrollaron una base de datos y un conjunto de herramientas software, tal y como se describe en el Capítulo 1. El paquete final de software se presentó en formato web como GB2.0, haciéndolo accesible al público a través de www.gbcloning.upv.es. El Capítulo 1 también proporciona una descripción detallada del funcionamiento de GB2.0 ejemplificando su uso con el ensamblaje de una construcción multigénica para la producción de antocianinas. Con el aumento en número y complejidad de las construcciones GB, el siguiente paso necesario fue el refinamiento de los estándar con la incorporación de la información experimental asociada a cada elemento genético (se describe en el Capítulo 2). Para este fin, el paquete de software de GB se reformuló en una nueva versión (GB3.0), un sistema de ensamblaje auto-contenido y completamente trazable en el que los datos experimentales que describen la funcionalidad de cada elemento genético se muestran en forma de una hoja de datos estándar. La utilidad de las especificaciones técnicas para anticipar el comportamiento de dispositivos biológicos compuestos se ejemplificó con la combinación de un interruptor químico y un prototipo de un módulo de sobreproducción de antocianinas equivalente al descrito en el Capítulo 1, resultando en un dispositivo de producción de antocianinas con respuesta a dexametasona. Además, en el Capítulo 3 se describe la adaptación a la tecnología GB de las herramientas de ingeniería genética CRISPR/Cas9, así como su caracterización funcional. La funcionalidad de estas herramientas para editado génico y activación y represión transcripcional se validó con el sistema de expresión transitoria en N.benthamiana. Finalmente, el Capítulo 4 presenta una implementación práctica del uso de la tecnología GB para hacer mejora vegetal de manera precisa. La transformación estable en tomate de una construcción intragénica que comprendía un marcador de selección intragénico y un regulador de la biosíntesis de flavonoides resultó en frutos con un mayor contenido de flavonoles. En conjunto, esta Tesis muestra la implementación de diseños genéticos cada vez más complejos y precisos en plantas utilizando elementos estándar y herramientas modulares siguiendo los principios de la Biología Sintética.[CA] La Biologia Sintètica és un camp emergent de caràcter interdisciplinar que es fonamenta amb l'aplicació a la enginyeria genètica dels principis de modularitat, abstracció i estandarització. Una nova vessant de la Biologia Sintètica aplicada a les plantes, la Biologia Sintètica Vegetal (BSV), ofereix noves possibilitats de millora de cultius que podrien portar a una millora de la resistència, a una major productivitat, o a un augment de la qualitat nutricional. Tanmateix, per poder arribar a este fi les eines moleculars disponibles en estos moments per a la BSV han d'adaptar-se per convertir-se en modulars, estàndards i més precises. Per això es plantejà com objectiu general d'aquesta Tesi adaptar, expandir i refinar les eines d'ensamblatge d'ADN de la BSV per permetre la incorporació d'especificacions funcionals en la descripció d'elements genètics estàndards (fitobricks) i facilitar la construcció d'estructures multigèniques cada vegada més complexes i precises, incloent eines d'edidat genètic. El punt de partida d'aquesta Tesi fou el mètode d'ensamblatge d'ADN modular GoldenBraid (GB) basat en enzims de restricció tipo IIS. Per optimitzar el proces d'ensamblatge i catalogar la col.lecció de fitobricks generats es desenvolupà una base de dades i un conjunt d'eines software, tal i com es descriu al Capítol 1. El paquet final de software es presentà en format web com GB2.0, fent-se accessible al públic mitjançant la pàgina web www.gbcloning.upv.es. El Capítol 1 també proporciona una descripció detallada del funcionament de GB2.0, exemplificant el seu ús amb l'ensamblatge d'una construcció multigènica per a la producció d'antocians. Amb l'augment en nombre i complexitat de les construccions GB, el següent pas fou el refinament dels estàndards amb la incorporació de la informació experimental associada a cada element genètic (es descriu en el Capítol 2). Per a aquest fi, el paquet de software de GB es reformulà amb una nova versió anomenada GB3.0. Aquesta versió consisteix en un sistema d'ensamblatge auto-contingut i complemtament traçable on les dades experimentals que descriuen la funcionalitat de cada element genètic es mostren en forma de fulla de dades estàndard. La utilitat de les especificacions tècniques per anticipar el comportament de dispositius biològics compostos s'exemplificà amb la combinació de un interruptor químic i un prototip d'un mòdul de sobreproducció d'antocians equivalent al descrit al Capítol 1. Aquesta combinació va tindre com a resultat un dispositiu de producció d'antocians que respón a dexametasona. A més a més, al Capítol 3 es descriu l'adaptació a la tecnologia GB de les eines d'enginyeria genètica CRISPR/Cas9, així com la seua caracterització funcional. La funcionalitat d'aquestes eines per a l'editat gènic i activació i repressió transcripcional es validà amb el sistema d'expressió transitòria en N. benthamiana. Finalment, al Capítol 4 es presenta una implementació pràctica de l'ús de la tecnologia GB per fer millora vegetal de mode precís. La transformació estable en tomaca d'una construcció intragènica que comprén un marcador de selecció intragènic i un regulador de la biosíntesi de flavonoïdes resultà en plantes de tomaca amb un major contingut de flavonols en llur fruits. En conjunt, esta Tesi mostra la implementació de dissenys genètics cada vegada més complexos i precisos en plantes utilitzant elements estàndards i eines modulars seguint els principis de la Biologia Sintètica.Vázquez Vilar, M. (2016). DESIGN OF GENETIC ELEMENTS AND SOFTWARE TOOLS FOR PLANT SYNTHETIC BIOLOGY [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/68483TESISPremios Extraordinarios de tesis doctoralesCompendi

Understanding host-microbe interactions in maize kernel and sweetpotato leaf metagenomic profiles.

Functional and quantitative metagenomic profiling remains challenging and limits our understanding of host-microbe interactions. This body of work aims to mediate these challenges by using a novel quantitative reduced representation sequencing strategy (OmeSeq-qRRS), development of a fully automated software for quantitative metagenomic/microbiome profiling (Qmatey: quantitative metagenomic alignment and taxonomic identification using exact-matching) and implementing these tools for understanding plant-microbe-pathogen interactions in maize and sweetpotato. The next generation sequencing-based OmeSeq-qRRS leverages the strengths of shotgun whole genome sequencing and costs lower that the more affordable amplicon sequencing method. The novel FASTQ data compression/indexing and enhanced-multithreading of the MegaBLAST in Qmatey allows for computational speeds several thousand-folds faster than typical runs. Regardless of sample number, the analytical pipeline can be completed within days for genome-wide sequence data and provides broad-spectrum taxonomic profiling (virus to eukaryotes). As a proof of concept, these protocols and novel analytical pipelines were implemented to characterize the viruses within the leaf microbiome of a sweetpotato population that represents the global genetic diversity and the kernel microbiomes of genetically modified (GMO) and nonGMO maize hybrids. The metagenome profiles and high-density SNP data were integrated to identify host genetic factors (disease resistance and intracellular transport candidate genes) that underpin sweetpotato-virus interactions Additionally, microbial community dynamics were observed in the presence of pathogens, leading to the identification of multipartite interactions that modulate disease severity through co-infection and species competition. This study highlights a low-cost, quantitative and strain/species-level metagenomic profiling approach, new tools that complement the assay’s novel features and provide fast computation, and the potential for advancing functional metagenomic studies

Quantum Internet Protocol Stack: a Comprehensive Survey

Classical Internet evolved exceptionally during the last five decades, from a network comprising a few static nodes in the early days to a leviathan interconnecting billions of devices. This has been possible by the separation of concern principle, for which the network functionalities are organized as a stack of layers, each providing some communication functionalities through specific network protocols. In this survey, we aim at highlighting the impossibility of adapting the classical Internet protocol stack to the Quantum Internet, due to the marvels of quantum mechanics. Indeed, the design of the Quantum Internet requires a major paradigm shift of the whole protocol stack for harnessing the peculiarities of quantum entanglement and quantum information. In this context, we first overview the relevant literature about Quantum Internet protocol stack. Then, stemming from this, we sheds the light on the open problems and required efforts toward the design of an effective and complete Quantum Internet protocol stack. To the best of authors' knowledge, a survey of this type is the first of its own. What emerges from this analysis is that the Quantum Internet, though still in its infancy, is a disruptive technology whose design requires an inter-disciplinary effort at the border between quantum physics, computer and telecommunications engineering

Robustness of the projected squeezed state protocol

Projected squeezed (PS) states are multipartite entangled states generated by unitary spin squeezing, followed by a collective quantum measurement and post-selection. They can lead to an appreciable decrease in the state preparation time of the maximally entangled N-qubit Greenberger-Horne-Zeilinger (GHZ) state when compared to deterministic preparation by unitary transformations in physical systems where spin squeezing can be realized, such as ion, neutral atom, and superconducting qubits. Here we simulate the generation of PS states in non-ideal experimental conditions with relevant decoherence channels. By employing the Kraus operator method, and quantum trajectory method to reduce the computational complexity, we assess the quantum Fisher information and overlap fidelity with an ideal GHZ state. Our findings highlight PS states as useful metrological resources, demonstrating a robustness against environmental effects with increasing qubit number N.Comment: 19 pages, 12 figures, appendi

Design and analysis of dual-mode numerically controlled oscillators based controlled oscillator frequency modulation

In this paper, the design and analysis of dual-mode numerically controlled oscillators (NCO) based controlled oscillator frequency Modulation is implemented. Initially, input is given to the analog to digital (ADC) converter. This will change the input from analog to digital converter. After that, the pulse skipping mode (PSM) logic and proportional integral (PI) are applied to the converted data. After applying PSM logic, data is directly transferred to the connection block. The proportional and integral block will transfer the data will be decoded using the decoder. After decoding the values, it is saved using a modulo accumulator. After that, it is converted from one hot residue (OHR) to binary converter. The converted data is saved in the register. Now both data will pass through the gate driver circuit and output will be obtained finally. From simulation results, it can observe that the usage of metal oxide semiconductor field effect transistors (MOSFETs) and total nodes are very less in dual-mode NCO-based controlled oscillator frequency modulation

Connector tensor networks: a renormalization-type approach to quantum certification

As quantum technologies develop, we acquire control of an ever-growing number of quantum systems. Unfortunately, current tools to detect relevant quantum properties of quantum states, such as entanglement and Bell nonlocality, suffer from severe scalability issues and can only be computed for systems of a very modest size, of around $6$ sites. In order to address large many-body systems, we propose a renormalisation-type approach based on a class of local linear transformations, called connectors, which can be used to coarse-grain the system in a way that preserves the property under investigation. Repeated coarse-graining produces a system of manageable size, whose properties can then be explored by means of usual techniques for small systems. In case of a successful detection of the desired property, the method outputs a linear witness which admits an exact tensor network representation, composed of connectors. We demonstrate the power of our method by certifying using a normal desktop computer entanglement, Bell nonlocality and supra-quantum Bell nonlocality in systems with hundreds of sites

Bell nonlocality

Bell's 1964 theorem, which states that the predictions of quantum theory cannot be accounted for by any local theory, represents one of the most profound developments in the foundations of physics. In the last two decades, Bell's theorem has been a central theme of research from a variety of perspectives, mainly motivated by quantum information science, where the nonlocality of quantum theory underpins many of the advantages afforded by a quantum processing of information. The focus of this review is to a large extent oriented by these later developments. We review the main concepts and tools which have been developed to describe and study the nonlocality of quantum theory, and which have raised this topic to the status of a full sub-field of quantum information science.Comment: 65 pages, 7 figures. Final versio

Identification of functionally related enzymes by learning-to-rank methods

Enzyme sequences and structures are routinely used in the biological sciences as queries to search for functionally related enzymes in online databases. To this end, one usually departs from some notion of similarity, comparing two enzymes by looking for correspondences in their sequences, structures or surfaces. For a given query, the search operation results in a ranking of the enzymes in the database, from very similar to dissimilar enzymes, while information about the biological function of annotated database enzymes is ignored. In this work we show that rankings of that kind can be substantially improved by applying kernel-based learning algorithms. This approach enables the detection of statistical dependencies between similarities of the active cleft and the biological function of annotated enzymes. This is in contrast to search-based approaches, which do not take annotated training data into account. Similarity measures based on the active cleft are known to outperform sequence-based or structure-based measures under certain conditions. We consider the Enzyme Commission (EC) classification hierarchy for obtaining annotated enzymes during the training phase. The results of a set of sizeable experiments indicate a consistent and significant improvement for a set of similarity measures that exploit information about small cavities in the surface of enzymes