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

A Rigorous Approach to High-Resolution Entropy-Constrained Vector Quantization

The nonnegativity of relative entropy implies that the differential entropy of a random vector X with probability density function (pdf) f is upper-bounded by -E[log g(X)]for any arbitrary pdf g. Using this inequality with a cleverly chosen g, we derive a lower bound on the asymptotic excess rate of entropy-constrained vector quantization for d-dimensional sources and rth-power distortion, where the asymptotic excess rate is defined as the difference between the smallest output entropy of a vector quantizer satisfying the distortion constraint and the rate-distortion function in the limit as the distortion tends to zero. Specialized to the one-dimensional case, this lower bound coincides with the asymptotic excess rate achieved by a uniform quantizer, thereby recovering the result by Gish and Pierce that uniform quantizers are asymptotically optimal as the allowed distortion tends to zero. Furthermore, in the one-dimensional case, the derivation of the lower bound reveals a necessary condition for a sequence of quantizers to be asymptotically optimal. This condition implies that any sequence of asymptotically optimal almost-regular quantizers must converge to a uniform quantizer as the distortion tends to zero. While the obtained lower bound itself is not novel, to the best of our knowledge, we present the first rigorous derivation that follows the direct approach by Gish and Pierce without resorting to heuristic high-resolution approximations commonly found in the quantization literature. Furthermore, our derivation holds for all d-dimensional sources having finite differential entropy and whose integer part has finite entropy. In contrast to Gish and Pierce, we do not require additional constraints on the continuity or decay of the source pdf.This work has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement number 714161), from the 7th European Union Framework Programme under Grant 333680, from the Ministerio de Economía y Competitividad of Spain under Grants TEC2013-41718-R, RYC-2014-16332, IJCI-2015-27020, TEC2015-69648-REDC, and TEC2016-78434-C3-3-R (AEI/FEDER, EU), and from the Comunidad de Madrid under Grant S2103/ICE-2845. The material in this paper was presented in part at the 2016 IEEE International Symposium on Information Theory, Barcelona, Spain, July 2016

Generalized perfect codes for symmetric classical-quantum channels

We define a new family of codes for symmetric classical-quantum channels and establish their optimality. To this end, we extend the classical notion of generalized perfect and quasi-perfect codes to channels defined over some finite dimensional complex Hilbert output space. The resulting optimality conditions depend on the channel considered and on an auxiliary state defined on the output space of the channel. For certain N-qubit classical-quantum channels, we show that codes based on a generalization of Bell states are quasi-perfect and, therefore, they feature the smallest error probability among all codes of the same blocklength and cardinality.This work was supported in part by the European Research Council (ERC) under Grant 714161; in part by the Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación, the Spanish Government, under Grant RED2018-102668-T, Grant PID2019-104958RB-C41, and Grant PID2020-116683GB-C21; and in part by the Catalan Government, within the ERDF Program of Catalunya, under Grant 2017 SGR 578 AGAUR and Grant 001-P001644 QuantumCAT.Peer ReviewedPostprint (author's final draft

DNA assembly standards: Setting the low-level programming code for plant biotechnology

[EN] Synthetic Biology is defined as the application of engineering principles to biology. It aims to increase the speed, ease and predictability with which desirable changes and novel traits can be conferred to living cells. The initial steps in this process aim to simplify the encoding of new instructions in DNA by establishing low-level programming languages for biology. Together with advances in the laboratory that allow multiple DNA molecules to be efficiently assembled together into a desired order in a single step, this approach has simplified the design and assembly of multigene constructs and has even facilitated the automated construction of synthetic chromosomes. These advances and technologies are now being applied to plants, for which there are a growing number of software and wetware tools for the design, construction and delivery of DNA molecules and for the engineering of endogenous genes. Here we review the efforts of the past decade that have established synthetic biology workflows and tools for plants and discuss the constraints and bottlenecks of this emerging field.Marta Vazquez-Vilar is funded by Wageningen University & Research. Diego Orzaez is funded by the Spanish Ministry of Economy and Competitiveness [grant number BIO2016-78601-R]. Nicola Patron is funded by the UK Biotechnological and Biological Sciences Research Council (BBSRC) and Engineering and Physics Research Council (EPSRC)Synthetic Biology for Growth programme [OpenPlant Synthetic Biology Research Centre, grant number BB/L0I4130/1], and by the Earlham DNA Foundry [grant number BB/CCG1720/1].Vázquez-Vilar, M.; Orzáez Calatayud, DV.; Patron, N. (2018). DNA assembly standards: Setting the low-level programming code for plant biotechnology. Plant Science. 273:33-41. https://doi.org/10.1016/j.plantsci.2018.02.024S334127

CRISPR-Cas12a genome editing at the whole-plant level using two compatible RNA virus vectors

[EN] The use of viral vectors that can replicate and move systemically through the host plant to deliver bacterial CRISPR components enables genome editing at the whole-plant level and avoids the requirement for labor-intensive stable transformation. However, this approach usually relies on previously transformed plants that stably express a CRISPR-Cas nuclease. Here, we describe successful DNA-free genome editing of Nicotiana benthamiana using two compatible RNA virus vectors derived from tobacco etch virus (TEV; genus Potyvirus) and potato virus X (PVX; genus Potexvirus), which replicate in the same cells. The TEV and PVX vectors respectively express a Cas12a nuclease and the corresponding guide RNA. This novel two-virus vector system improves the toolbox for transformation-free virus-induced genome editing in plants and will advance efforts to breed more nutritious, resistant, and productive crops.This research was supported by grants BIO2017-83184-R and PID2019-108203RB-I00 from the Ministerio de Ciencia e Innovacion (Spain) through the Agencia Estatal de Investigacion (co-financed by the European Regional Development Fund) and H2020-760331 Newcotiana from the European Commission. M.U. andM.V.-V. are the recipients of fellowships FPU17/05503 from the Ministerio de Ciencia e Innovacion (Spain) and APOSTD/2020/096 from the Generalitat Valenciana (Spain), respectivelyUranga, M.; Vázquez-Vilar, M.; Orzáez Calatayud, DV.; Daròs, J. (2021). CRISPR-Cas12a genome editing at the whole-plant level using two compatible RNA virus vectors. The CRISPR Journal. 4(5):1-9. https://doi.org/10.1089/crispr.2021.0049194

Fenton and Photo-Fenton Nanocatalysts Revisited from the Perspective of Life Cycle Assessment

This study provides an overview of the environmental impacts associated with the production of different magnetic nanoparticles (NPs) based on magnetite (Fe3O4), with a potential use as heterogeneous Fenton or photo-Fenton catalysts in wastewater treatment applications. The tendency of Fe3O4 NPs to form aggregates in water makes necessary their decoration with stabilizing agents, in order to increase their catalytic activity. Different stabilizing agents were considered in this study: poly(acrylic acid) (PAA), polyethylenimine (PEI) and silica (SiO2), as well as the immobilization of the magnetite-based catalysts in a mesoporous silica matrix, SBA-15. In the case of photo-Fenton catalysts, combinations of magnetite NPs with semiconductors were evaluated, so that magnetic recovery of the nanomaterials is possible, thus allowing a safe discharge free of NPs. The results of this study suggest that magnetic nanoparticles coated with PEI or PAA were the most suitable option for their applications in heterogeneous Fenton processes, while ZnO-Fe3O4 NPs provided an interesting approach in photo-Fenton. This work showed the importance of identifying the relevance of nanoparticle production strategy in the environmental impacts associated with their useThis research was supported by two projects granted by Spanish Ministry of Science and Innovation: MODENA Project CTQ2016-79461-R and CLUSTERCAT Project MAT2015-67458-P, and Fundación Ramón Areces, Spain (Project CIVP18A3940). The authors belong to the Galician Competitive Research Groups ED431C-2017/22 and ED431C-2017/29 and CRETUS InstituteS

Mathematical analysis and optimal control of heavy metals phytoremediation techniques

In this work we optimize different issues related to phytoremediation techniques for heavy metals removal from shallow water, by means of a combination of mathematical modeling, optimal control of partial differential equations and numerical optimization. We introduce, analyze and solve a 2D mathematical system of nonlinear partial differential equations representing the concentrations of heavy metals, algae and nutrients in large waterbodies. Then, we formulate an optimal control problem related to the optimization of the phytoremediation process. In particular, we determine the minimal quantity of algae to be used in the heavy metals remediation process, and locate the optimal place for such algal mass. We also propose two different full algorithms for computing the numerical solution of the control problem and, finally, we present several numerical results for a realistic case.Ministerio de Economía y Competitividad | Ref. MTM2015-65570-

Design of universal multicoset sampling patterns for compressed sensing of multiband sparse signals

Many problems in digital communications involve wideband radio signals. As the most recent example, the impressive advances in Cognitive Radio systems make even more necessary the development of sampling schemes for wideband radio signals with spectral holes. This is equivalent to considering a sparse multiband signal in the framework of Compressive Sampling theory. Starting from previous results on multicoset sampling and recent advances in compressive sampling, we analyze the matrix involved in the corresponding reconstruction equation and define a new method for the design of universal multicoset codes, that is, codes guaranteeing perfect reconstruction of the sparse multiband signal

Saddlepoint Approximations for Noncoherent Single-Antenna Rayleigh Block-Fading Channels

Proceeding of: 2019 IEEE International Symposium on Information Theory (ISIT), 7-12 July 2019, Paris, FranceThis paper presents saddlepoint approximations of state-of-the-art converse and achievability bounds for noncoherent, single-antenna, Rayleigh block-fading channels. These approximations can be calculated efficiently and are shown to be accurate for SNR values as small as 0 dB, blocklengths of 168 channel uses or more, and when the channel's coherence interval is not smaller than two. It is demonstrated that the derived approximations recover both the normal approximation and the reliability function of the channel.A. Lancho, G. Vázquez-Vilar and T. Koch have received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (714161), the Spanish Ministerio de Economía y Competitividad (RYC-2014-16332, TEC2016-78434-C3-3-R (AEI/FEDER, EU) and IJCI2015-27020), the Spanish Ministerio de Educación, Cultura y Deporte (FPU014/01274), and the Comunidad de Madrid (S2103/ICE-2845). G. Durisi and J. Östman have been supported by the Swedish Research Council under Grants 2016-03293 and 2014-6066