Developments in the tools and methodologies of synthetic biology.

Synthetic biology is principally concerned with the rational design and engineering of biologically based parts, devices, or systems. However, biological systems are generally complex and unpredictable, and are therefore, intrinsically difficult to engineer. In order to address these fundamental challenges, synthetic biology is aiming to unify a body of knowledge from several foundational scientific fields, within the context of a set of engineering principles. This shift in perspective is enabling synthetic biologists to address complexity, such that robust biological systems can be designed, assembled, and tested as part of a biological design cycle. The design cycle takes a forward-design approach in which a biological system is specified, modeled, analyzed, assembled, and its functionality tested. At each stage of the design cycle, an expanding repertoire of tools is being developed. In this review, we highlight several of these tools in terms of their applications and benefits to the synthetic biology community

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

Synthetic biology advanced natural product discovery

A wide variety of bacteria, fungi and plants can produce bioactive secondary metabolites, which are often referred to as natural products. With the rapid development of DNA sequencing technology and bioinformatics, a large number of putative biosynthetic gene clusters have been reported. However, only a limited number of natural products have been discovered, as most biosynthetic gene clusters are not expressed or are expressed at extremely low levels under conventional laboratory conditions. With the rapid development of synthetic biology, advanced genome mining and engineering strategies have been reported and they provide new opportunities for discovery of natural products. This review discusses advances in recent years that can accelerate the design, build, test, and learn (DBTL) cycle of natural product discovery, and prospects trends and key challenges for future research directions

Capturing wheat phenotypes at the genome level

Recent technological advances in next-generation sequencing (NGS) technologies have dramatically reduced the cost of DNA sequencing, allowing species with large and complex genomes to be sequenced. Although bread wheat (Triticum aestivum L.) is one of the world’s most important food crops, efficient exploitation of molecular marker-assisted breeding approaches has lagged behind that achieved in other crop species, due to its large polyploid genome. However, an international public–private effort spanning 9 years reported over 65% draft genome of bread wheat in 2014, and finally, after more than a decade culminated in the release of a gold-standard, fully annotated reference wheat-genome assembly in 2018. Shortly thereafter, in 2020, the genome of assemblies of additional 15 global wheat accessions was released. As a result, wheat has now entered into the pan-genomic era, where basic resources can be efficiently exploited. Wheat genotyping with a few hundred markers has been replaced by genotyping arrays, capable of characterizing hundreds of wheat lines, using thousands of markers, providing fast, relatively inexpensive, and reliable data for exploitation in wheat breeding. These advances have opened up new opportunities for marker-assisted selection (MAS) and genomic selection (GS) in wheat. Herein, we review the advances and perspectives in wheat genetics and genomics, with a focus on key traits, including grain yield, yield-related traits, end-use quality, and resistance to biotic and abiotic stresses. We also focus on reported candidate genes cloned and linked to traits of interest. Furthermore, we report on the improvement in the aforementioned quantitative traits, through the use of (i) clustered regularly interspaced short-palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene-editing and (ii) positional cloning methods, and of genomic selection. Finally, we examine the utilization of genomics for the next-generation wheat breeding, providing a practical example of using in silico bioinformatics tools that are based on the wheat reference-genome sequence

Synthetic Genomics

The current advances in sequencing, data mining, DNA synthesis, cloning, in silico modeling, and genome editing have opened a new field of research known as Synthetic Genomics. The main goal of this emerging area is to engineer entire synthetic genomes from scratch using pre-designed building blocks obtained by chemical synthesis and rational design. This has opened the possibility to further improve our understanding of genome fundamentals by considering the effect of the whole biological system on biological function. Moreover, the construction of non-natural biological systems has allowed us to explore novel biological functions so far not discovered in nature. This book summarizes the current state of Synthetic Genomics, providing relevant examples in this emerging field

Applied Molecular Cloning: Present and Future for Aquaculture

With the grim picture of millions of people living in poverty and hunger, there is also an international alarm over future world food supply. This global concern of food scarcity has established the need to not only increase the production of traditional staples but also fisheries and aquaculture. Genetically, physiologically and phenotypically, fish are the most diverse group of livings. Similar to mammals, molecular biology is being extensively used in aquaculture, be it in disease management, or growth and reproduction enhancement. In this chapter we aim to discuss the molecular methodologies applied to uplift and attain sustainability in aqua farming

Characterizing a thermostable Cas9 for bacterial genome editing and silencing

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range

Mining and characterization of the candidate genes for distorted segregation in chromosome 4 of tomato

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em PlantasSolanum lycopersicum L. genetic variability was drastically diminished by successive genetic bottlenecks induced by the domestication process. The wild species of tomato, S. pimpinellifolium, is a small red-fruited plant native to Peru and is assumed as an ancestor species of the domesticated S. lycopersicum. One of the strategies to induce genetic variability to cultivated tomato is the development of introgression lines (ILs) containing a single segment of a donor wild genome in the genetic background of an elite tomato cultivar. In 2014, a genomic library of ILs that incorporates variability from the S. pimpinellifolium accession TO-937 in the genetic background of S. lycopersicum cultivar “Moneymaker” was developed. During the development of the IL collection, a region on the distal portion of chromosome 4 showed a segregation distortion (SD) favouring TO-937 alleles in detriment of “Moneymaker” alleles. Recently, the SD region was mapped to a 39Kb region of chromosome 4 containing seven gene annotations. The preliminary studies to assert gametic, post gametic and/or zygotic indicated that the SD was most probably caused by post-gametic or zygotic selection and it was a sex-independent phenomenon. The present study aims to characterize the genes included in the SD region and to propose a possible mechanism for the SD. Expression profile analysis by qRT-PCR and sequencing of genomic and transcriptomic sequences indicated a strong expression in the reproductive tissues of the two Heat-Shock Protein (HSP) genes contained in the SD region. Haplotyping of reciprocal and self-pollinating crosses between the SD haplotypes and “Moneymaker” gave new insights about the gametic and zygotic character of the SD. The analysis of natural sequence variations of the SD region revealed this region diverged in wild tomato accessions. Additionally, a reverse genetic approach was initated to assess if the HSPs are the cause of the SD using the GoldenBraid 3.0 standard assembly to create Agrobacterium-mediated transformation vectors, two CRISPR/Cas9 expression cassettes for the silencing of the HSP genes, and 3 expression cassettes.A variabilidade genética de Solanum lycopersicum L. foi drasticamente diminuída por sucessivos efeitos de gargalo genéticos induzidos pelo processo de domesticação. A espécie de tomate selvagem, S. pimpinellifolium, é uma pequena planta de frutos vermelhos nativas do Peru e é assumida como um ancestral do S. lycopersicum domesticado. Uma das estratégias para induzir variabilidade genética ao tomate cultivado é o desenvolvimento de linhas de introgressão (ILs - introgression lines) que contêm um único segmento do genoma selvagem no genoma de um cultivar de elite. Em 2014, foi desenvolvida uma biblioteca genómica de ILs que incorpora variabilidade da acessão TO-937 de S. pimpinellifolium no genoma do cultivar "Moneymaker" de S. lycopersicum. Durante o desenvolvimento da IL, uma região na porção distal do cromossoma 4 revelou uma segregação distorcida (SD) favorecendo os alelos TO-937 em detrimento dos alelos "Moneymaker". Recentemente, a região SD foi mapeada para uma região de 39Kb do cromossoma 4 contendo sete genes. Os estudos preliminares para afirmar o caracter gamético, pós-gamético e/ou zigótico indicaram que a SD provavelmente é causada pela seleção pósgamética ou zigótica e é um fenómeno independente do sexo. O presente estudo tem como objetivo a caracterização dos genes incluídos na região SD e um possível mecanismo de SD. A análise de perfil de expressão por qRT-PCR e sequenciação do genoma e transcriptoma indicou uma elevada expressão em tecidos reprodutores de dois genes contidos na região genómica da SD que codificam Heat-Shock Proteins (HSP). Haplotipagem de cruzamentos recíprocos e autocruzamentos entre os haplótipos de SD e "Moneymaker" revelou novas pistas acerca do caráter gamético e zigótico da SD. A análise da variação natural da região SD revelou uma significativa diversidade em acessões de tomate selvagem. Além disso, usando o sistema de clonagem GoldenBraid 3.0 para criar vetores de transformação mediada por Agrobacterium, duas cassetes de expressão CRISPR / Cas9 para o silenciamento dos genes HSP e 3 cassetes de expressão para foram desenvolvidas para futura aplicação

Application of CRISPR-Cas9 genome editing systems for improving oilseed rape (Brassica napus) disease resistance against Verticillium longisporum

Modern agriculture requires innovative techniques to advance established strategies in breeding. Traditional approaches have their limitations and are primarily based on the availability of a broad genetic spectrum and the selection of suitable genotypes to improve the desired characteristics. One of these characteristics is the improvement of resistance to biotic stresses or pathogens. In order to achieve this goal in crops with a narrow genetic base such as Brassica napus new strategies are required. An example for this is the disruption of plant factors that are required by the pathogen for a successful colonization of the host (susceptibility factors). Verticillium wilt is caused by the soil-borne and hemi-biotrophic fungus Verticillium longisporum, which is one of the most common fungal diseases in rapeseed cultivation. The disease leads to premature ripening and can cause considerable yield losses. The methods for disease management are very limited. Countermeasures are mainly based on soil hygiene or prevention strategies to reduce the number of spores in the soil. The genetic resources for resistance breeding are limited and no real resistance has been discovered so far. Susceptibility factors in combination with genome modification based on CRISPR/Cas9 can be used as a source of recessive resistance. CRISPR/Cas9 is the most promising system for targeted mutagenesis with the advantage of easy assembly and use as well as multiplexing. The knockout of target genes is possible by the induction of small InDels caused by non-homologous end joining (NHEJ) after a double-strand break in the target site. Another approach possible through this technology is the introduction of a repair template for homology directed repair (HDR), which allows the nucleotide sequence to be altered without disrupting the gene function. In this study we adapt several vector systems containing different nucleases and promoters for application in Brassica napus. After successful establishment of our expression cassettes, the focus relied on the knockout of the candidate genes BnCRT1a and BnHVA22c, which are involved in the V. longisporum -B. napus interaction. Loss-of-function genotypes for the genes BnCRT1a and BnHVA22c were generated and infected with V. longisporum. In addition to this NHEJ approach, miRNA binding sites of fungal miRNAs in the host genome are modified using the HDR approach without affecting the function for the genes BnAGO1 and BnTAO1. For this purpose, the viral genome of the Bean Yellow Dwarf Virus was adapted for use in B. napus hairy roots. In summary, all loss-of-function genotypes showed a greatly reduced expression of symptoms and growth inhibition. These results support our working hypothesis that CRT1a and HVA22c may induce resistance to V. longisporum in their mutated state. Following experiments focused on the function of these genes in plants. Unfortunately, no significant differences in the expression profile of marker genes for molecular and physiological processes between mutants and wild type were found. Only the ethylene marker gene ETR2 showed increased expression in knockout genotypes in the uninfected state, which is consistent with expression data from A. thaliana. The analysis of co-regulated genes, which play a possible role in protein folding / ER stress (for CRT1a) or vesicle transport / callose closure of plasmodesmata (for HVA22c), showed no difference between mutants and the wild type as well. The vector systems for the HDR-based approach were successfully implemented and viral replicons of the Bean Yellow Dwarf Virus could be detected within the cell. The miRNA binding sites in the genes BnAGO1 and BnTAO1, which are used by V. longisporum, were the target of our HDR approach. No positive HDR events could be identified that would be indicated by a sequence exchange. Overall, this work showed that the use of CRISPR/Cas in combination with susceptibility factors is a valuable strategy for generating recessive resistance to pathogens. In the future, this tool can be integrated into existing breeding methods, especially for crops with limited genetic resources