An Experimental Framework to Examine the Influence of Promoter Architecture and Genomic Context on Gene Expression

Transcription is a fundamental process of gene expression. Information stored in DNA is transcribed into different types of mobile RNA, which play a role in various essential processes of the cell, e.g. translation. However, cells do not need all the information stored in their DNA at the same time. Therefore, the process of transcription gets regulated by a plethora of mechanisms. One frequently discussed but poorly understood mechanism of transcription regulation is DNA supercoiling [Travers and Muskhelishvili, 2005]. Whereby, the process of transcription itself affects the DNA-topology up- and downstream of the transcription machinery as described in the twin supercoiling domain model [Liu and Wang, 1987]. This phenomenon is called Transcription Coupled DNA Supercoiling (TCDS). It has also been shown that genes react individually to changes in DNA supercoiling and that there is a selection pressure on adapting to the DNA supercoiling levels emitted by neighbouring gene expression [Sobetzko, 2016]. The system in which promoters react to changes in DNA supercoiling is as diverse as there are promoters; notably, some promoters seem not to respond to DNA supercoiling at all. Thus, this raises the question as to which elements within different promoter types cause them to respond to TCDS so differently. In this thesis, I built a pipeline to investigate the effects of TCDS and DNA supercoiling on promoters. Firstly, I created a plasmid toolbox, which allows modular assembly of transcription units. The central feature of this toolbox is the flexibility to test different arrangements of multiple transcription units. I achieved this by adapting the well established Modular Cloning (MoClo) standard [Weber et al., 2011] and build my toolbox around it. I thus created a system that works on both its own and is compatible with the existing standard MoClo protocol. In the second part of this thesis, I established an experimental pipeline using synthetic σ70-promoters to investigate the influence of DNA supercoiling on transcription. The experimental setup allowed precise changes in parts of the promoter and at the same time created a library of these promoters. Using this pipeline to investigate the spacer region of the promoter, I was able to confirm that the spacer influences the promoter strength. Further, I showed that the promoter spacer has only a limited effect on the supercoiling sensitivity of a promoter. I also showed that a 5‘-TGTG-3‘ motif in the spacer region could lower transcription by enhancing RNA-polymerase (RNAP)-binding. Moreover, the experimental setup also showed the constraints of using the DNA-relaxing drug novobiocin on a plasmid-based system. Hence, to further investigate the effects of TCDS on neighbouring transcription, I applied an optogenetically-controllable promoter to the previously established pipeline. Finally, I began to explore the possibility of integrating my experimental promoter setup into any genomic position. As such, a CRISPR/Cas9-based homologous re-combination system was developed further to make it modular and compatible with the Modular Cloning protocol. I could show the first features of this system to work

Inferring biological networks from genome-wide transcriptional and fitness data

In the last 15 years, the increased use of high throughput biology techniques such as genome-wide gene expression profiling, fitness profiling and protein interactomics has led to the generation of an extraordinary amount of data. The abundance of such diverse data has proven to be an essential foundation for understanding the complexities of molecular mechanisms and underlying pathways within a biological system. This thesis demonstrates the capabilities and applications of using biological networks to extrapolate biological information from the wealth of data available in the yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. This study marks the first time a mutual information based network inference approach has been applied to a set of specific genome-wide expression and fitness compendia. In particular, this work has generated hypotheses in S. pombe that have led to a deeper understanding of the relationship between ribosomal proteins and energy metabolism, a recently discovered pathway termed riboneogenesis. Experimental validation of this hypothesis has led to new theories on the role of energy metabolism enzymes in controlling ribosome biogenesis in S. pombe, including the novel finding that fructose-1, 6-bisphosphatase (FBP1) may have roles in both gluconeogenesis and riboneogenesis. This thesis also demonstrates how the use of multi-level data allows for comprehensive insight into nuclear functions of the S. pombe nonsense-mediated mRNA decay protein, UPF1. This study provides substantial evidence demonstrating the role of UPF1 in DNA replication. The applicability of fitness data in identifying targets of metal and metalloid toxicity in S. cerevisiae has also been investigated

Identification et regroupement de QTL influençant la pression artérielle en modules épistatiques et analyse de deux gènes candidats chez la souche Dahl Salt-Sensitive

L’hypertension essentielle étant un facteur majeur de morbidité, la compréhension de son l’étiologie est prépondérante. Ainsi, la découverte de nouvelles composantes ou mécanismes de régulation de la PA par l’identification de QTL et l’étude de leurs interactions s’avère une approche prometteuse. L’utilisation de souches congéniques de rats pour l’étude de l’hypertension est une stratégie payante puisqu’elle permet de masquer les effets de l’environnement, tout en gardant le caractère polygénique de la PA. Longtemps conçu comme un trait issu de l’accumulation des effets minimes des QTL, la PA est régulée par une architecture basée sur l’existence d’interactions épistatiques. L’analyse par paires de QTL individuels a permis d’établir une modularité dans l’organisation des QTL chez le rat Dahl Salt-sensitive en fonction de la présence ou de l’absence d’une interaction épistatique entre eux. Ainsi, deux modules épistatiques ont été établis; EM1 et EM2 où tous les QTL appartenant à EM1 sont épistatiques entre eux et agissent de façon additive avec les membres de EM2. Des hiérarchies dans la régulation peuvent alors être révélées si les QTL d’un même EM ont des effets opposés. L’identification de la nature moléculaire des candidats C18QTL4/Hdhd2 et C18QTL3/Tcof1, membres du EM1, et de l’interaction épistatique entre ces deux QTL, a permis, en plus, d’élucider une régulation séquentielle au sein du module. Hdhd2 pourrait agir en amont de Tcof1 et réguler ce dernier par une modification post-traductionnelle. Cette interaction est la première évidence expérimentale de la prédiction des relations entre QTL, phénomène établi par leur modularisation. Le dévoilement du fonctionnement de l’architecture génétique à la base du contrôle de la PA et la découverte des gènes responsables des QTL permettrait d’élargir les cibles thérapeutiques et donc de développer des traitements antihypertenseurs plus efficaces.Essential hypertension is a major risk factor for cardiovascular diseases. Understanding the etiology of this pathology is of the outmost importance. Thus, unraveling novel genetic components and mechanisms regulating blood pressure (BP) via QTL identification and QTL-QTL interaction analysis is a promising strategy. Congenic strains establishment is a common and fruitful means for achieving such goal. A quantitative trait, such as BP, has long been thought to result from the accumulation of infinitesimal effects exerted by multiple QTL. Nevertheless, BP is controlled by an epistasis-based architecture. Pair-wise comparisons of individual QTL based on the existence or lack-of epistatic interaction between them allowed us to establish a modularized organization of BP QTL in the Dahl Salt-sensitive model. Hence, two epistatic modules, namely EM1 and EM2 were constituted. In this fashion, any member of the EM1 is epistatic to all the other members of the same module and is additive to those of EM2. Regulatory hierarchies among BP with paradoxical effects can be revealed within each EM. The molecular identification of EM1 members, C18QTL4/Hdhd2 and C18QTL3/Tcof1, as well as the revelation of the molecular basis for their epistatic interaction enabled us to suggest a sequential regulation within this EM. Hdhd2 could act upstream of Tcof1 and regulate it by post-transcriptional modification. This interaction is the first experimental evidence derived from the predictive model of QTL modularization. The elucidation of the molecular mechanisms underlying the genetic architecture of BP, as well as the identification of the causal genes for QTL will lay the grounds for expanding therapeutic targets and for developing more efficient antihypertensive treatments

An Experimental Framework to Examine the Influence of Promoter Architecture and Genomic Context on Gene Expression

Transcription is a fundamental process of gene expression. Information stored in DNA is transcribed into different types of mobile RNA, which play a role in various essential processes of the cell, e.g. translation. However, cells do not need all the information stored in their DNA at the same time. Therefore, the process of transcription gets regulated by a plethora of mechanisms. One frequently discussed but poorly understood mechanism of transcription regulation is DNA supercoiling [Travers and Muskhelishvili, 2005]. Whereby, the process of transcription itself affects the DNA-topology up- and downstream of the transcription machinery as described in the twin supercoiling domain model [Liu and Wang, 1987]. This phenomenon is called Transcription Coupled DNA Supercoiling (TCDS). It has also been shown that genes react individually to changes in DNA supercoiling and that there is a selection pressure on adapting to the DNA supercoiling levels emitted by neighbouring gene expression [Sobetzko, 2016]. The system in which promoters react to changes in DNA supercoiling is as diverse as there are promoters; notably, some promoters seem not to respond to DNA supercoiling at all. Thus, this raises the question as to which elements within different promoter types cause them to respond to TCDS so differently. In this thesis, I built a pipeline to investigate the effects of TCDS and DNA supercoiling on promoters. Firstly, I created a plasmid toolbox, which allows modular assembly of transcription units. The central feature of this toolbox is the flexibility to test different arrangements of multiple transcription units. I achieved this by adapting the well established Modular Cloning (MoClo) standard [Weber et al., 2011] and build my toolbox around it. I thus created a system that works on both its own and is compatible with the existing standard MoClo protocol. In the second part of this thesis, I established an experimental pipeline using synthetic σ70-promoters to investigate the influence of DNA supercoiling on transcription. The experimental setup allowed precise changes in parts of the promoter and at the same time created a library of these promoters. Using this pipeline to investigate the spacer region of the promoter, I was able to confirm that the spacer influences the promoter strength. Further, I showed that the promoter spacer has only a limited effect on the supercoiling sensitivity of a promoter. I also showed that a 5‘-TGTG-3‘ motif in the spacer region could lower transcription by enhancing RNA-polymerase (RNAP)-binding. Moreover, the experimental setup also showed the constraints of using the DNA-relaxing drug novobiocin on a plasmid-based system. Hence, to further investigate the effects of TCDS on neighbouring transcription, I applied an optogenetically-controllable promoter to the previously established pipeline. Finally, I began to explore the possibility of integrating my experimental promoter setup into any genomic position. As such, a CRISPR/Cas9-based homologous re-combination system was developed further to make it modular and compatible with the Modular Cloning protocol. I could show the first features of this system to work

Towards an Aspect-Oriented Design and Modelling Framework for Synthetic Biology

Work on synthetic biology has largely used a component-based metaphor for system construction. While this paradigm has been successful for the construction of numerous systems, the incorporation of contextual design issues—either compositional, host or environmental—will be key to realising more complex applications. Here, we present a design framework that radically steps away from a purely parts-based paradigm by using aspect-oriented software engineering concepts. We believe that the notion of concerns is a powerful and biologically credible way of thinking about system synthesis. By adopting this approach, we can separate core concerns, which represent modular aims of the design, from cross-cutting concerns, which represent system-wide attributes. The explicit handling of cross-cutting concerns allows for contextual information to enter the design process in a modular way. As a proof-of-principle, we implemented the aspect-oriented approach in the Python tool, SynBioWeaver, which enables the combination, or weaving, of core and cross-cutting concerns. The power and flexibility of this framework is demonstrated through a number of examples covering the inclusion of part context, combining circuit designs in a context dependent manner, and the generation of rule, logic and reaction models from synthetic circuit designs

Systems level analysis of non-model organisms: a tool for understanding environmental stress

Omics techniques are changing the focus of ecotoxicology. In addition to challenges resulting from large amounts of data, there are further difficulties for non-model species: from lack of annotation to limited number of additional databases for molecular interactions and functions. In this thesis, I demonstrate the use of systems biology to relate molecular measurements to physiological parameters in non-model species in the context of environmental stress. Firstly, I make dynamical data-driven model of how gene expression changes in relation to the nerve conductance in earthworm Eisenia fetida exposed to single chemicals in the laboratory. The model reveals that gene expression changes might reflect the recovery from nerve damage. Using a similar approach, I use blue mussel Mytilus edulis sampled from their natural environment to model their annual cycle, integrating 1H-NMR metabolite levels with physiological and environmental parameters. I challenge this model created from data from a reference site to see site-effects for mussels sampled from an industrial harbour. Finally, I use systems biology to relate changing chemical concentrations and traditional toxicity assays in an effluent remediation system to stickleback gene expression and morphology. I demonstrate that data-driven systems biology can help the interpretation of complex problems. Supplementary data for this thesis can be found on the University of Birmingham eData repository at: https://doi.org/10.25500/edata.bham.0000065

Modularisation épistatique des loci à trait quantitatif associés à la pression artérielle et identification de gènes candidats pour l’hypertension

Problématique: L’hypertension artérielle essentielle, facteur de risque majeur dans le développement des maladies cardiovasculaires, est un trait multigénique complexe dont les connaissances sur le déterminisme génétique nécessitent d’être approfondies. De nombreux loci à trait quantitatif (QTLs); soit des gènes responsables de faire varier la pression artérielle (PA), ont été identifiés chez l’humain et le modèle animal. Cependant, le mystère plane encore sur la façon dont ces gènes fonctionnent ensemble pour réguler la PA. Hypothèse et objectif: Plutôt qu’une addition de QTLs ayant chacun une action infinitésimale sur la PA, une interaction épistatique entre les gènes serait responsable du phénotype hypertendu. Ainsi, l’étude de cette épistasie entre les gènes impliqués, directement ou indirectement, dans l’homéostasie de la PA nous permettrait d’explorer de nouvelles voies de régulation moléculaire en cause dans cette maladie. Méthodes: Via la réalisation de souches congéniques de rats, où un segment chromosomique provenant d’une souche receveuse hypertendue (Dahl Salt Sensitive, SS/Jr) est remplacé par son homologue provenant d’une souche donneuse normotendue (Lewis, LEW), des QTLs peuvent être mis en évidence. Dans ce contexte, la combinaison de QTLs via la création de doubles ou multiples congéniques constitue la première démonstration fonctionnelle des interactions intergéniques. Résultats: Vingt-sept combinaisons au total nous ont menés à l’appréciation d’une modularisation des QTLs. Ces derniers ont été catégorisés selon deux principaux modules épistatiques (EMs) où les QTLs appartenant à un même EM sont épistatiques entre eux et participent à une même voie régulatrice. Les EMs/cascades agissent alors en parallèle pour réguler la PA. Grâce à l’existence de QTLs ayant des effets opposés sur la PA, nous avons pu établir l’ordre hiérarchique entre trois paires de QTLs. Cependant, lorsque cette suite régulatrice ne peut être déterminée, d’autres approches sont nécessaires. Nos travaux nous ont mené à l’identification d’un QTL situé sur le chromosome 16 du rat (C16QTL), appartenant au EM1 et qui révélerait une nouvelle voie de l’homéostasie de la PA. Le gène retinoblastoma-associated protein 140 (Rap140)/family with sequence similarity 208 member A (Fam208a), présentant une mutation non synonyme entre SS/Jr et LEW est le gène candidat le plus plausible pour représenter C16QTL. Celui-ci code pour un facteur de transcription et semblerait influencer l’expression de Solute carrier family 7 (cationic amino acid transporter, y+ system) member 12 (Slc7a12), spécifiquement et significativement sous exprimé dans les reins de la souche congénique portant C16QTL par rapport à la souche SS/Jr. Rap140/Fam208a agirait comme un inhibiteur de la transcription de Slc7a12 menant à une diminution de la pression chez Lewis. Conclusions: L’architecture complexe de la régulation de la PA se dévoile mettant en scène de nouveaux acteurs, pour la plupart inconnus pour leur implication dans la PA. L’étude de la nouvelle voie de signalisation Rap140/Fam208a - Slc7a12 nous permettra d’approfondir nos connaissances quant à l’homéostasie de la pression artérielle et de l’hypertension chez SS/Jr. À long terme, de nouveaux traitements anti-hypertenseurs, ciblant plus d’une voie de régulation à la fois, pourraient voir le jour.Introduction: Essential hypertension, a major risk factor in the development of cardiovascular diseases, is a complex multigenic trait whose knowledge about genetic determinism require further study. Many quantitative trait loci (QTLs); genes responsible for varying blood pressure (BP), have been identified in humans and animal models. However, the mystery still lingers on how these genes work together to regulate the BP. Hypothesis and aim: Rather than an addition of QTLs, each with an infinitesimal effect on the BP, an epistatic interaction between genes would be responsible for the hypertensive phenotype. Thus, the study of this epistasis among genes involved directly or indirectly in the homeostasis of the BP allow us to explore new molecular regulatory pathways involved in this disease. Methods: Via the embodiment of rats congenic strains, where a chromosomal segment from a recipient hypertensive strain (Dahl Salt Sensitive, SS/Jr) is replaced by its homologue from a donor normotensive strain (Lewis, LEW); QTLs may be identified. In this context, the combination of QTLs by creating double or multiple congenics constitutes the first functional demonstration of intergenic interactions. Results: Twenty-seven combinations in total have led us to the assessment of a modularization of QTLs. These have been categorized according to two main epistatic modules (EMs) where QTLs belonging to the same EM are epistatic with each other and participate in the same regulatory pathway. The EMs / pathway then act in parallel to control the BP. Thanks to the existence of QTLs with opposite effects on BP, we were able to establish hierarchical order between three pairs of QTLs. However, when this regulatory sequence cannot be determined, other approaches are needed. Our work led us to the identification of a QTL on rat chromosome 16 (C16QTL) belonging to the EM1 revealing a new way of BP homeostasis. The retinoblastoma-associated protein gene 140 (Rap140) / family with sequence similarity 208 member A (Fam208a) having a nonsynonymous mutation between SS/Jr and LEW is the most plausible candidate gene to represent C16QTL. It encodes a transcription factor and appear to influence the expression of Solute carrier family 7 (cationic amino acid transport, y + system) member 12 (Slc7a12) specifically and significantly under expressed in the kidneys of the congenic strain carrying C16QTL compared to the strain SS/Jr. Rap140 / Fam208a acts as an inhibitor of Slc7a12 transcription leading to a BP decrease in the congenic strain. Conclusions: The complex architecture of the regulation of BP unveils featuring new players, mostly unknown to their involvement in the BP. The study of the new signaling pathway Rap140 / Fam208a - Slc7a12 will deepen our knowledge about the homeostasis of blood pressure and hypertension in SS/Jr. In the long term, new antihypertensive treatments, targeting more than one regulatory cascade at a time may emerge

Computational design of orthogonal microRNAs for synthetic biology

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.Upcoming applications of synthetic biology will require access to a wide array of robust genetic components (parts). The logic of a genetic system is encoded with regulatory elements such as pairs of transcription factors:promoters, miRNAs:target sites, or ribozymes:aptamers among others. Due to a relatively simple form and mode of operation of miRNAs, it is possible to design their synthetic variants. Out of all possible miRNA sequences the ones chosen should perform efficiently and should avoid cross-talk with both the host system circuits and within the imported synthetic ones. In this work, a computational method involving a series of heuristics is developed that can be used to design ensembles of such sequences depending on the host transcriptome. As an example, an ensemble of eight such miRNA sequences is produced using this method for use in a human host. Those have then been validated experimentally against the above-mentioned requirements by transfection into HEK 293 cells and flow cytometry measurements of fluorescent markers. The produced sequences are available for use from pENTR vectors of the Gateway cloning system. The required computations were facilitated by a modern cluster computing system—Kaichu—especially developed for this project, but fit for general purpose use and available under an open-source license.EPSR

Generalized disjunction decomposition for evolvable hardware

Evolvable hardware (EHW) refers to self-reconfiguration hardware design, where the configuration is under the control of an evolutionary algorithm (EA). One of the main difficulties in using EHW to solve real-world problems is scalability, which limits the size of the circuit that may be evolved. This paper outlines a new type of decomposition strategy for EHW, the “generalized disjunction decomposition” (GDD), which allows the evolution of large circuits. The proposed method has been extensively tested, not only with multipliers and parity bit problems traditionally used in the EHW community, but also with logic circuits taken from the Microelectronics Center of North Carolina (MCNC) benchmark library and randomly generated circuits. In order to achieve statistically relevant results, each analyzed logic circuit has been evolved 100 times, and the average of these results is presented and compared with other EHW techniques. This approach is necessary because of the probabilistic nature of EA; the same logic circuit may not be solved in the same way if tested several times. The proposed method has been examined in an extrinsic EHW system using the$(1 + lambda)$evolution strategy. The results obtained demonstrate that GDD significantly improves the evolution of logic circuits in terms of the number of generations, reduces computational time as it is able to reduce the required time for a single iteration of the EA, and enables the evolution of larger circuits never before evolved. In addition to the proposed method, a short overview of EHW systems together with the most recent applications in electrical circuit design is provided

Programming microbes to treat superbug infection

Superbug infection is one of the greatest public health threat with grave implications across all levels of society. Towards a new solution to combat infection by multi-drug resistant bacteria, this thesis presents an engineering framework and genetic tools applied to repurpose commensal bacteria into “micro-robots” for the treatment of superbug infection. Specifically, a prototype of designer probiotic was developed using the human commensal bacteria Escherichia coli. The engineered commensal was reprogrammed with user-specified functions to sense superbug, produced pathogen-specific killing molecules and released the killing molecules via a lytic mechanism. The engineered commensal was effective in suppressing ~99% of planktonic Pseudomonas and preventing ~ 90% of biofilm formation. To enhance the sensing capabilities of engineered commensal, genetic interfaces comprising orthogonal AND & OR logic devices were developed to mediate the integration and interpretation of binary input signals. Finally, AND, OR and NOT logic gates were networked to generate a myriad of cellular logic operations including half adder and half subtractor. The creation of half adder logic represents a significant advancement of engineering human commensal to be biological equivalent of microprocessor chips in programmable computer with the ability to process input signals into diversified actions. Importantly, this thesis provides exemplary case studies to the attenuation of cellular and genetic context dependent effects through principles elucidated herein, thereby advancing our capability to engineer commensal bacteria.Open Acces