BioSystems 97 (2009) 146–153 Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/biosystems A novel generalized design methodology and realization of Boolean operations using DN

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 Dna Strand Displacement Based Circuits

DNA is the basic building block of any living organism. DNA is considered a popular candidate for future biological devices and circuits for solving genetic disorders and several other medical problems. With this objective in mind, this research aims at developing novel approaches for the design of DNA based circuits. There are many recent developments in the medical field such as the development of biological nanorobots, SMART drugs, and CRISPR-Cas9 technologies. There is a strong need for circuits that can work with these technologies and devices. DNA is considered a suitable candidate for designing such circuits because of the programmability of the DNA strands, small size, lightweight, known thermodynamics, higher parallelism, and exponentially reducing the cost of synthesizing techniques. The DNA strand displacement operation is useful in developing circuits with DNA strands. The circuit can be either a digital circuit, in which the logic high and logic low states of the DNA strand concentrations are considered as the signal, or it can be an analog circuit in which the concentration of the DNA strands itself will act as the signal. We developed novel approaches in this research for the design of digital, as well as analog circuits keeping in view of the number of DNA strands required for the circuit design. Towards this goal in the digital domain, we developed spatially localized DNA majority logic gates and an inverter logic gate that can be used with the existing seesaw based logic gates. The majority logic gates proposed in this research can considerably reduce the number of strands required in the design. The introduction of the logic inverter operation can translate the dual rail circuit architecture into a monorail architecture for the seesaw based logic circuits. It can also reduce the number of unique strands required for the design into approximately half. The reduction in the number of unique strands will consequently reduce the leakage reactions, circuit complexity, and cost associated with the DNA circuits. The real world biological inputs are analog in nature. If we can use those analog signals directly in the circuits, it can considerably reduce the resources required. Even though analog circuits are highly prone to noise, they are a perfect candidate for performing computations in the resource-limited environments, such as inside the cell. In the analog domain, we are developing a novel fuzzy inference engine using analog circuits such as the minimum gate, maximum gate, and fan-out gates. All the circuits discussed in this research were designed and tested in the Visual DSD software. The biological inputs are inherently fuzzy in nature, hence a fuzzy based system can play a vital role in future decision-making circuits. We hope that our research will be the first step towards realizing these larger goals. The ultimate aim of our research is to develop novel approaches for the design of circuits which can be used with the future biological devices to tackle many medical problems such as genetic disorders

Before Peer Production: Infrastructure Gaps and the Architecture of Openness in Synthetic Biology

Legal scholarship on intellectual property needs to be reoriented to consider how state action helps to generate the infrastructure of emerging fields in ways that prove conducive to their development. In this Article, I contribute to that reorientation through an in-depth analysis of one important emerging technology, synthetic biology. The ambition of synthetic biology is to make biology easier to engineer through standardization and associated technical processes. Early successes indicate the scientific promise of the field and help to explain why its advocates are concerned to see the field develop in an open and publicly beneficial manner. What openness might mean in the patent-dominated context of biotechnology remains unclear, however, and requires a reassessment of software’s “copyleft” concept that provided initial inspiration to the scientists and activists working on open synthetic biology. In this Article, I focus on the efforts of the BioBricks Foundation (BBF), the leading non-profit in synthetic biology, to promote the open development of the field. I explore the rationale behind the BBF’s decision to pursue a “public domain” strategy via a new legal agreement, the BioBrick™ Public Agreement

Engineering Systems of Anti-Repressors for Next-Generation Transcriptional Programming

The ability to control gene expression in more precise, complex, and robust ways is becoming increasingly relevant in biotechnology and medicine. Synthetic biology has sought to accomplish such higher-order gene regulation through the engineering of synthetic gene circuits, whereby a gene’s expression can be controlled via environmental, temporal, or cellular cues. A typical approach to gene regulation is through transcriptional control, using allosteric transcription factors (TFs). TFs are regulatory proteins that interact with operator DNA elements located in proximity to gene promoters to either compromise or activate transcription. For many TFs, including the ones discussed here, this interaction is modulated by binding to a small molecule ligand for which the TF evolved natural specificity and a related metabolism. This modulation can occur with two main phenotypes: a TF shows the repressor (X+) phenotype if its binding to the ligand causes it to dissociate from the DNA, allowing transcription, while a TF shows the anti-repressor (XA) phenotype if its binding to the ligand causes it to associate to the DNA, preventing transcription. While both functional phenotypes are vital components of regulatory gene networks, anti-repressors are quite rare in nature compared to repressors and thus must be engineered. We first developed a generalized workflow for engineering systems of anti-repressors from bacterial TFs in a family of transcription factors related to the ubiquitous lactose repressor (LacI), the LacI/GalR family. Using this workflow, which is based on a re-routing of the TF’s allosteric network, we engineered anti-repressors in the fructose repressor (anti-FruR – responsive to fructose-1,6-phosphate) and ribose repressor (anti-RbsR – responsive to D-ribose) scaffolds, to complement XA TFs engineered previously in the LacI scaffold (anti-LacI – responsive to IPTG). Engineered TFs were then conferred with alternate DNA binding. To demonstrate their utility in synthetic gene circuits, systems of engineered TFs were then deployed to construct transcriptional programs, achieving all of the NOT-oriented Boolean logical operations – NOT, NOR, NAND, and XNOR – in addition to BUFFER and AND. Notably, our gene circuits built using anti-repressors are far simpler in design and, therefore, exert decreased burden on the chassis cells compared to the state-of-the-art as anti-repressors represent compressed logical operations (gates). Further, we extended this workflow to engineer ligand specificity in addition to regulatory phenotype. Performing the engineering workflow with a fourth member of the LacI/GalR family, the galactose isorepressor (GalS – naturally responsive to D-fucose), we engineered IPTG-responsive repressor and anti-repressor GalS mutants in addition to a D-fucose responsive anti-GalS TF. These engineered TFs were then used to create BANDPASS and BANDSTOP biological signal processing filters, themselves compressed compared to the state-of-the-art, and open-loop control systems. These provided facile methods for dynamic turning ‘ON’ and ‘OFF’ of genes in continuous growth in real time. This presents a general advance in gene regulation, moving beyond simple inducible promoters. We then demonstrated the capabilities of our engineered TFs to function in combinatorial logic using a layered logic approach, which currently stands as the state-of-the art. Using our anti-repressors in layered logic had the advantage of reducing cellular metabolic burden, as we were able to create the fundamental NOT/NOR operations with fewer genetic parts. Additionally, we created more TFs to use in layered logic approaches to prevent cellular cross-talk and minimize the number of TFs necessary to create these gene circuits. Here we demonstrated the successful deployment of our XA-built NOR gate system to create the BUFFER, NOT, NOR, OR, AND, and NAND gates. The work presented here describes a workflow for engineering (i) allosteric phenotype, (ii) ligand selectivity, and (iii) DNA specificity in allosteric transcription factors. The products of the workflow themselves serve as vital tools for the construction of next-generation synthetic gene circuits and genetic regulatory devices. Further, from the products of the workflow presented here, certain design heuristics can be gleaned, which should better facilitate the design of allosteric TFs in the future, moving toward a semi-rational engineering approach. Additionally, the work presented here outlines a transcriptional programming structure and metrology which can be broadly adapted and scaled for future applications and expansion. Consequently, this thesis presents a means for advanced control of gene expression, with promise to have long-reaching implications in the future.Ph.D

Supporting complex workflows for data-intensive discovery reliably and efficiently

Scientific workflows have emerged as well-established pillars of large-scale computational science and appeared as torchbearers to formalize and structure a massive amount of complex heterogeneous data and accelerate scientific progress. Scientists of diverse domains can analyze their data by constructing scientific workflows as a useful paradigm to manage complex scientific computations. A workflow can analyze terabyte-scale datasets, contain numerous individual tasks, and coordinate between heterogeneous tasks with the help of scientific workflow management systems (SWfMSs). However, even for expert users, workflow creation is a complex task due to the dramatic growth of tools and data heterogeneity. Scientists are now more willing to publicly share scientific datasets and analysis pipelines in the interest of open science. As sharing of research data and resources increases in scientific communities, scientists can reuse existing workflows shared in several workflow repositories. Unfortunately, several challenges can prevent scientists from reusing those workflows, which hurts the purpose of the community-oriented knowledge base. In this thesis, we first identify the repositories that scientists use to share and reuse scientific workflows. Among several repositories, we find Galaxy repositories have numerous workflows, and Galaxy is the mostly used SWfMS. After selecting the Galaxy repositories, we attempt to explore the workflows and encounter several challenges in reusing them. We classify the reusability status (reusable/nonreusable). Based on the effort level, we further categorize the reusable workflows (reusable without modification, easily reusable, moderately difficult to reuse, and difficult to reuse). Upon failure, we record the associated challenges that prevent reusability. We also list the actions upon success. The challenges preventing reusability include tool upgrading, tool support unavailability, design flaws, incomplete workflows, failure to load a workflow, etc. We need to perform several actions to overcome the challenges. The actions include identifying proper input datasets, updating/upgrading tools, finding alternative tools support for obsolete tools, debugging to find the issue creating tools and connections and solving them, modifying tools connections, etc. Such challenges and our action list offer guidelines to future workflow composers to create better workflows with enhanced reusability. A SWfMS stores provenance data at different phases of a workflow life cycle, which can help workflow construction. This provenance data allows reproducibility and knowledge reuse in the scientific community. But, this provenance information is usually many times larger than the workflow and input data, and managing provenance data is growing in complexity with large-scale applications. In our second study, we document the challenges of provenance management and reuse in e-science, focusing primarily on scientific workflow approaches by exploring different SWfMSs and provenance management systems. We also investigate the ways to overcome the challenges. Creating a workflow is difficult but essential for data-intensive complex analysis, and the existing workflows have several challenges to be reused, so in our third study, we build a recommendation system to recommend tool(s) using machine learning approaches to help scientists create optimal, error-free, and efficient workflows by using existing reusable workflows in Galaxy workflow repositories. The findings from our studies and proposed techniques have the potential to simplify the data-intensive analysis, ensuring reliability and efficiency

Development and characterization of two new tools for plant genetic engineering: A CRISPR/Cas12a-based mutagenesis system and a PhiC31-based gene switch

Tesis por compendio[ES] La mejora genética vegetal tiene como objetivo la obtención de plantas con rasgos mejorados o características novedosas que podrían ayudar a superar los objetivos de sostenibilidad. Para este fin, la biotecnología vegetal necesita incorporar nuevas herramientas de ingeniería genética que combinen una mayor precisión con una mayor capacidad de mejora. Las herramientas de edición genética recientemente descubiertas basadas en la tecnología CRISPR/Cas9 han abierto el camino para modificar los genomas de las plantas con una precisión sin precedentes. Por otro lado, los nuevos enfoques de biología sintética basados en la modularidad y la estandarización de los elementos genéticos han permitido la construcción de dispositivos genéticos cada vez más complejos y refinados aplicados a la mejora genética vegetal. Con el objetivo final de expandir la caja de herramientas biotecnológicas para la mejora vegetal, esta tesis describe el desarrollo y la adaptación de dos nuevas herramientas: una nueva endonucleasa específica de sitio (SSN) y un interruptor genético modular para la regulación de la expresión transgénica. En una primera parte, esta tesis describe la adaptación de CRISPR/Cas12a para la expresión en plantas y compara la eficiencia de las variantes de Acidaminococcus (As) y Lachnospiraceae (Lb) Cas12a con Streptococcus pyogens Cas9 (SpCas9) descritos anteriormente en ocho loci de Nicotiana benthamiana usando expresión transitoria. LbCas12a mostró la actividad de mutagénesis promedio más alta en los loci analizados. Esta actividad también se confirmó en experimentos de transformación estable realizados en tres plantas modelo diferentes, a saber, N. benthamiana, Solanum lycopersicum y Arabidopsis thaliana. Para este último, los efectos mutagénicos colaterales fueron analizados en líneas segregantes sin la endonucleasa Cas12a, mediante secuenciación del genoma descartándose efectos indiscriminados. En conjunto, los resultados muestran que LbCas12a es una alternativa viable a SpCas9 para la edición genética en plantas. En una segunda parte, este trabajo describe un interruptor genético reversible destinado a controlar la expresión génica en plantas con mayor precisión que los sistemas inducibles tradicionales. Este interruptor, basado en el sistema de recombinación del fago PhiC31, fue construido como un dispositivo modular hecho de partes de ADN estándar y diseñado para controlar el estado transcripcional (encendido o apagado) de dos genes de interés mediante la inversión alternativa de un elemento regulador central de ADN. El estado del interruptor puede ser operado externa y reversiblemente por la acción de los actuadores de recombinación y su cinética, memoria y reversibilidad fueron ampliamente caracterizados en experimentos de transformación transitoria y estable en N. benthamiana. En conjunto, esta tesis muestra el diseño y la caracterización funcional de herramientas para la ingeniería del genómica y biología sintética de plantas que ahora ha sido completada con el sistema de edición genética CRISPR/Cas12a y un interruptor genético reversible y biestable basado en el sistema de recombinación del fago PhiC31.[CA] La millora genètica vegetal té com a objectiu l'obtenció de plantes amb trets millorats o característiques noves que podrien ajudar a superar els objectius de sostenibilitat. Amb aquesta finalitat, la biotecnologia vegetal necessita incorporar noves eines d'enginyeria genètica que combinen una major precisió amb una major capacitat de millora. Les eines d'edició genètica recentment descobertes basades en la tecnologia CRISPR/Cas9 han obert el camí per modificar els genomes de les plantes amb una precisió sense precedents. D'altra banda, els nous enfocaments de biologia sintètica basats en la modularitat i l'estandardització dels elements genètics han permès la construcció de dispositius genètics cada vegada més complexos i sofisticats aplicats a la millora genètica vegetal. Amb l'objectiu final d'expandir la caixa d'eines biotecnològiques per a la millora vegetal, aquesta tesi descriu el desenvolupament i l'adaptació de dues noves eines: una nova endonucleasa específica de lloc (SSN) i un interruptor genètic modular per a la regulació de l'expressió transgènica . En una primera part, aquesta tesi descriu l'adaptació de CRISPR/Cas12a per a l'expressió en plantes i compara l'eficiència de les variants de Acidaminococcus (As) i Lachnospiraceae (Lb) Cas12a amb la ben establida Streptococcus pyogens Cas9 (SpCas9), en vuit loci de Nicotiana benthamiana usant expressió transitòria. LbCas12a va mostrar l'activitat de mutagènesi mitjana més alta en els loci analitzats. Aquesta activitat també es va confirmar en experiments de transformació estable realitzats en tres plantes model diferents, a saber, N. benthamiana, Solanum lycopersicum i Arabidopsis thaliana. Per a aquest últim, els efectes mutagènics col·laterals van ser analitzats en línies segregants sense l'endonucleasa Cas12a, mitjançant seqüenciació completa del genoma i descartant efectes indiscriminats. En conjunt, els resultats mostren que LbCas12a és una alternativa viable a SpCas9 per a l'edició genètica en plantes. En una segona part, aquest treball descriu un interruptor genètic reversible destinat a controlar l'expressió gènica en plantes amb major precisió que els sistemes induïbles tradicionals. Aquest interruptor, basat en el sistema de recombinació del bacteriòfag PhiC31, va ser construït com un dispositiu modular fet de parts d'ADN estàndard i dissenyat per controlar l'estat transcripcional (encès o apagat) de dos gens d'interès mitjançant la inversió alternativa d'un element regulador central d'ADN. L'estat de l'interruptor pot ser operat externa i reversiblement per acció dels actuadors de recombinació i la seva cinètica, memòria i reversibilitat van ser àmpliament caracteritzats en experiments de transformació transitòria i estable en N. benthamiana. En conjunt, aquesta tesi mostra el disseny i la caracterització funcional d'eines per a l'enginyeria del genòmica i biologia sintètica de plantes que ara ha sigut completat amb el sistema d'edició genètica CRISPR/Cas12a i un interruptor genètic biestable i reversible basat en el sistema de recombinació del bacteriòfag PhiC31.[EN] Plant breeding aims to provide plants with improved traits or novel features that could help to overcome sustainability goals. To this end, plant biotechnology needs to incorporate new genetic engineering tools that combine increased precision with higher breeding power. The recently discovered genome editing tools based on CRISPR/Cas9 technology have opened the way to modify plant¿s genomes with unprecedented precision. On the other hand, new synthetic biology approaches based on modularity and standardization of genetic elements have enabled the construction of increasingly complex and refined genetic devices applied to plant breeding. With the ultimate goal of expanding the toolbox of plant breeding techniques, this thesis describes the development and adaptation to plant systems of two new breeding tools: a site-specific nuclease (SSNs), and a modular gene switch for the regulation of transgene expression. In a first part, this thesis describes the adoption of the SSN CRISPR/Cas12a for plant expression and compares the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described Streptococcus pyogens Cas9 (SpCas9) in eight Nicotiana benthamiana loci using transient expression experiments. LbCas12a showed highest average mutagenesis activity in the loci assayed. This activity was also confirmed in stable genome editing experiments performed in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, off-target effects in Cas12a-free segregating lines were discarded at genomic level by deep sequencing. Collectively, the results show that LbCas12a is a viable alternative to SpCas9 for plant genome engineering. In a second part, this work describes the engineering of a new reversible genetic switch aimed at controlling gene expression in plants with higher precision than traditional inducible systems. This switch, based on the bacteriophage PhiC31 recombination system, was built as a modular device made of standard DNA parts and designed to control the transcriptional state (on or off) of two genes of interest by alternative inversion of a central DNA regulatory element. The state of the switch can be externally and reversibly operated by the action of the recombination actuators and its kinetics, memory, and reversibility were extensively characterized in N. benthamiana using both transient expression and stable transgenics. Altogether, this thesis shows the design and functional characterization of refined tools for genome engineering and synthetic biology in plants that now has been expanded with the CRISPR/Cas12a gene editing system and the phage PhiC31-based toggle switch.Bernabé Orts, JM. (2019). Development and characterization of two new tools for plant genetic engineering: A CRISPR/Cas12a-based mutagenesis system and a PhiC31-based gene switch [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/133055TESISCompendi

Modelling tools and methodologies for rapid protocell prototyping

The field of unconventional computing considers the possibility of implementing computational devices using novel paradigms and materials to produce computers which may be more efficient, adaptable and robust than their silicon based counterparts. The integration of computation into the realms of chemistry and biology will allow the embedding of engineered logic into living systems and could produce truly ubiquitous computing devices. Recently, advances in synthetic biology have resulted in the modification of microorganism genomes to create computational behaviour in living cells, so called “cellular computing”. The cellular computing paradigm offers the possibility of intelligent bacterial agents which may respond and communicate with one another according to chemical signals received from the environment. However, the high levels of complexity when altering an organism which has been well adapted to certain environments over millions of years of evolution suggests an alternative approach in which chemical computational devices can be constructed completely from the bottom up, allowing the designer exquisite control and knowledge about the system being created. This thesis presents the development of a simulation and modelling framework to aid the study and design of bottom-up chemical computers, involving the encapsulation of computational re-actions within vesicles. The new “vesicle computing” paradigm is investigated using a sophisticated multi-scale simulation framework, developed from mesoscale, macroscale and executable biology techniques