While protein therapeutics are indispensable in the treatment of a variety of diseases, including cancer, rheumatoid arthritis, and diabetes, key limitations including short half-lives, high immunogenicity, protein instability, and centralized production complicate long-term use and on-demand production. Site-specific polymer conjugation provides a method for mitigating these challenges while minimizing negative impacts on protein activity. However, the location-dependent effects of polymer conjugation are not well understood. Cell-free protein synthesis provides direct access to the synthesis environment and rapid synthesis times, enabling rapid evaluation of multiple conjugation sites on a target protein. Here, work is presented towards developing cell-free protein synthesis as a platform for both design and on-demand production of next generation polymer-protein therapeutics, including (1) eliminating endotoxin contamination in cell-free reagents for simplified therapeutic preparation, (2) improving shelf-stability of cell-free reagents via lyophilization for on-demand production, (3) coupling coarse-grain simulation with high-throughput cell-free protein synthesis to enable rapid identification of optimal polymer conjugation sites, and (4) optimizing cell-free protein synthesis for production of therapeutic proteins

Un des objectifs de la biologie de synthèse est de concevoir et produire des organismes « à façon », pour des applications thérapeutiques et industrielles. Une des voies envisagées pour atteindre cet objectif repose sur des techniques de synthèse et de transplantation de génomes entiers, afin de créer des organismes mutants.Le but de cette thèse est de développer des outils de biologie de synthèse qui permettront de construire une cellule minimale et non pathogène, à partir de Mycoplasma pneumoniae. Cette bactérie est l'un des plus petits organismes vivants, avec une taille inférieure au micron et un génome de 816 kpb. Ce mycoplasme est l’un des plus étudiés, avec une collection de données génétiques et multi-« omiques » disponibles. Ces caractéristiques font de cette cellule naturellement « quasi minimale » un point de départ idéal pour la construction d’un châssis bactérien. Néanmoins, la manipulation génétique de ce mycoplasme est difficile, en raison du nombre restreint d'outils disponibles.Une approche récemment développée propose de contourner ces limitations en utilisant la levure Saccharomyces cerevisiae comme plateforme d’ingénierie du génome de M. pneumoniae. L’étape préliminaire à cette approche consiste à cloner le génome bactérien dans la levure. Pour ce faire, une cassette « éléments levure » est insérée dans le génome de M. pneumoniae, pour permettre son maintien comme chromosome artificiel. Les travaux menés au cours de cette thèse ont permis d’insérer cette cassette par le biais d’un transposon, et de cloner ce génome marqué dans la levure. La stabilité du génome cloné a ensuite été étudiée, mettant en évidence que le chromosome bactérien est maintenu durant une dizaine de passages. Nous avons ensuite développé une nouvelle stratégie d’insertion des « éléments levure » en utilisant le système CRISPR/Cas9 pour cloner et éditer simultanément un génome de mycoplasme chez la levure : le CReasPy-Cloning. Cette méthode a été utilisée pour supprimer trois loci différents contenant des gènes impliqués dans la virulence : MPN372 (toxine CARDS), MPN142 (protéine de cytoadhérence) et MPN400 (protéine bloquant les IgG). Elle a ensuite été utilisée pour en cibler deux puis trois en une seule étape.Une fois le clonage et l’ingénierie du génome bactérien réalisés dans la levure, il est nécessaire de pouvoir transférer le chromosome modifié dans une cellule receveuse, afin de produire une cellule mutante. Ce processus nommé transplantation de génome n’étant pas décrit pour M. pneumoniae, une part importante de cette thèse a été dédiée au développement de cet outil. Nous avons utilisé la transformation de plasmides comme mécanisme modèle pour étudier le processus d’entrée de l’ADN dans M. pneumoniae et tester l’utilisation du polyéthylène glycol, le réactif clé de la transplantation. Bien qu’ayant réussi à mettre au point un protocole de transformation de plasmides, nous n’avons pas réussi pour l’instant à réaliser la transplantation de génomes.En parallèle, nous avons développé une stratégie alternative d’édition de génome qui ne dépend pas de la transplantation. Cette approche, nommée « Genomic Transfer - Recombinase-Mediated Cassette Exchange » (GT-RMCE), consiste à capturer dans un vecteur une section du génome bactérien édité présent dans la levure. Ce vecteur est transformé dans M. pneumoniae, et grâce au système Cre-lox la section éditée est introduite dans le génome. Ce mécanisme permet de réaliser des modifications de grande ampleur, et est actuellement utilisé pour introduire chez M. pneumoniae les délétions ΔMPN372, ΔMPN400 et ΔMPN372-ΔMPN400 produites par CReasPy-cloning. Nous avons également utilisé le GT-RMCE pour générer une souche de M. pneumoniae portant deux copies de l’opéron ribosomal S10.Au final, les outils d’ingénierie du génome de M. pneumoniae développés au cours de cette thèse permettent de réaliser un pas significatif vers la construction de nouveaux châssis bactériens. / A goal of synthetic biology is to create and produce “custom” organisms, for therapeutic and industrial applications. One of the contemplated approaches to achieve this goal is based on synthesis techniques and transplantation of whole genomes, in order to create mutant organisms.The aim of this thesis is to develop synthetic biology tools that will enable the construction of a minimal and non-pathogenic cell based on Mycoplasma pneumoniae. This bacterium is one of the smallest living organisms, with a size smaller than one micron and a genome of 816 kbp. This mycoplasma is one of the most studied, with a large set of genetic and multi- “omics” data available. These characteristics make this naturally “almost minimal” cell an ideal starting point for the construction of a bacterial chassis. Nevertheless, the genetic manipulation of this mycoplasma is difficult, due to the limited number of available tools.A recently developed approach offers the possibility to circumvent these limitations by using the yeast Saccharomyces cerevisiae as a genome engineering platform for M. pneumoniae. The preliminary step to this strategy is to clone the bacterial genome in yeast. To do so, a "yeast elements" cassette is inserted into the genome of M. pneumoniae, to allow its maintenance as an artificial chromosome. The work carried out during this thesis allowed us to insert this cassette through a transposon, and to clone this marked genome in yeast. Then, the stability of the cloned genome was studied, demonstrating that the bacterial chromosome is maintained during ten passages. We then developed a new strategy for the insertion of the "yeast elements", using the CRISPR/Cas9 system to simultaneously clone and edit a mycoplasma genome in yeast: the CReasPy-Cloning. This method was used to remove three different loci containing genes involved in virulence: MPN372 (CARDS toxin), MPN142 (cytoadherence protein) and MPN400 (IgG blocking protein). This method was also used to target two and then three different loci in one step.Once in-yeast cloning and bacterial genome engineering is achieved, it is necessary to transfer the modified chromosome into a recipient cell, to produce a mutant organism. This process, called genome transplantation, is not described for M. pneumoniae, so a significant part of this thesis was dedicated to the development of this tool. We used plasmid transformation as a model mechanism to study the process of DNA entry into M. pneumoniae and to test the use of polyethylene glycol, the key reagent for transplantation. Although we succeeded in developing a plasmid transformation protocol, we have not yet been able to perform genome transplantation.Concurrently, we have developed an alternative strategy for genome editing that does not depend on transplantation. This approach, named "Genomic Transfer - Recombinase-Mediated Cassette Exchange" (GT-RMCE), is used to capture in a vector a section of the edited bacterial genome borne by the yeast. This vector is then transformed into M. pneumoniae, and through to the Cre-lox system the edited section is introduced into the genome. This mechanism allows to carry out large-scale modifications, and is currently used to introduce into M. pneumoniae the ΔMPN372, ΔMPN400 and ΔMPN372-ΔMPN400 deletions produced by CReasPy-cloning. We also used the GT-RMCE to generate a strain of M. pneumoniae carrying two copies of the S10 ribosomal operon.Overall, the M. pneumoniae genome engineering tools developed during this thesis constitute a significant step towards the construction of new bacterial chassis.

Les chercheurs en biologie de synthèse programment l’ADN pour construire des systèmes biologiques capables de répondre à certaines conditions de manière prédéfinie. Cette capacité pourrait avoir un impact sur plusieurs domaines, de la médecine à la fermentation industrielle. Le traitement de signal par des circuits biologiques synthétiques est en train d’être démontré à large échelle, mais hélas la variété des signaux d’entrée capables de contrôler ces circuits est pour l’instant limitée. Ce manque de diversité est un obstacle majeur au développement de nouvelles applications car en général chaque application requiert une réponse à des signaux de nature particulière qui lui sont spécifiques. Cette thèse cherche à apporter des solutions au manque de signaux d’entrée appropriés contrôlant les circuits biologiques en développant deux nouvelles stratégies d’induction. La première stratégie vise à étendre la diversité chimique des signaux d’entrée. A l’inverse des approches existantes, qui reposent sur la modification des systèmes de détections naturels tels que les riboswitchs ou les facteurs de transcription allostériques, j’ai cherché ici à modifier directement des molécules préalablement non-détectables afin de les rendre détectables par les systèmes de détection actuels. Pour ce faire, la transformation chimique des molécules cibles est réalisée in situ grâce à l’expression de voies métaboliques synthétiques dans la cellule. Afin de pouvoir utiliser cette stratégie de manière systématique, j’ai employé la conception assistée par ordinateur et puisé dans l’ensemble des réactions biochimiques connues afin de prédire des voies de détections pour de nouvelles molécules. J’ai ensuite implémenté in vivo plusieurs prédictions qui ont permis à E. coli de détecter de nouveaux composés. Au-delà de l’intérêt de cette méthode en biotechnologie, cela montre que le métabolisme peut jouer un rôle dans le transfert d’information, en plus de son rôle dans le transfert de matière et d’énergie, ce qui soulève la question de l’utilisation potentielle de cette stratégie de détection par la nature. Un second axe présente une façon d’épargner l’utilisation d’inducteurs chimiques pour les programmes biologiques simples, et propose d’utiliser des inducteurs biologiques à la place. Lorsqu’une seule étape d’induction ou de répression de gènes est nécessaire, comme c’est le cas en fermentation industrielle, je propose de remplacer la coûteuse étape d’induction chimique par l’infection simultanée de toutes les cellules d’une population par des particules virales capables d’injecter en temps réel l’ensemble des informations nécessaires pour déclencher l’activité biologique recherchée. A des fins de fermentation, j’ai développé des particules virales modifiées qui reprogramment dynamiquement le métabolisme d’une large population de bactérie au moment opportun et les forcent à produire des molécules à haute valeur ajoutée. / Synthetic biologists program DNA with the aim of building biological systems that react under certain conditions in a predefined way. This ability could have impact in several fields, from medicine to industrial fermentation. While the scalability of synthetic biological circuits in terms of signal processing in now almost demonstrated, the variety of input signals for these circuits is limited. Because each application typically requires a circuit to react to case-specific molecules, the lack of input diversity is a major obstacle to the development of new applications. Two axis are developed over the course of this thesis to try to address input-related problems. The main axis consists in a new strategy aiming at systematically and immediately increasing the chemical diversity of inputs for synthetic circuits. Current approaches to expand the number of potential inputs focus on re-engineering sensing systems such as riboswitches or allosteric transcription factors to make them react to previously non-detectable molecules. On the contrary, here we developed a method to transform the non-detectable molecules themselves into molecules for which sensing systems already exist. These chemical transformations are realized in situ by expressing synthetic metabolic pathways in the cell. In order to systematize this strategy, we leveraged computer-aided design to predict ways of detecting new molecules by digging into all known biochemical reactions. We then implemented several predictions in vivo that successfully enabled E. coli to detect new chemicals. Aside from the interest of the method for biotechnological applications, this shows that in addition to transferring matter and energy, metabolism can also play a role in transferring information, raising the question of potential occurrences of this sensing strategy in nature. A second axis introduce a way to exempt simple programs from the need for a chemical input, and explore the use of a biological input instead. In situations where a single timely induction or repression of multiple genes is required, such as in industrial fermentation processes, we propose to replace expensive chemical induction by simultaneous infection of all the members of a growing population of cells with viral particles inputting in real-time all the necessary information for the task at hand. In the context of fermentation, we developed engineered viral particles that can dynamically reprogram the metabolism of a large population of bacteria at the optimal stage of growth and force them to produce value-added chemicals.

Le SELEX est une technique d’évolution moléculaire dirigée qui permet, après plusieurs tours de sélection, d’enrichir une banque d’acides nucléiques en séquences capable de se lier de manière spécifique à une cible. Le séquençage est utilisé pour identifier ces séquences que l’on nomme « aptamères ». Depuis l’arrivée récente du séquençage à haut débit (HD), il est possible d’analyser des millions de séquences. L’objectif de la thèse était de développer des méthodes pour traiter et analyser les données de séquençage HD afin de faciliter l’identification des meilleurs aptamères d’un SELEX. Au cours de cette thèse, un test robotisé de liaison sur cellules adhérentes vivantes a été mis au point pour mesurer l’affinité d’aptamères issus de SELEX ciblant des cellules (cell-SELEX). Puis, l’évolution de l’abondance des séquences d’un cell-SELEX a été analysée par séquençage HD. Ceci nous a permis de concevoir une nouvelle approche phylogénétique baptisée FREDROGRAM. Cette approche évolutive a permis d’identifier des mutants avec une meilleure affinité au sein d’une famille d’aptamères issu de ce cell-SELEX. Enfin, le séquençage HD de deux SELEX dirigés contre des protéines a contribué à mieux comprendre l’impact des paramètres de sélection sur la population de séquences et à identifier de nouveaux aptamères, notamment en réduisant le nombre de tours de SELEX. En conclusion, ces travaux montrent l’utilité du séquençage HD pour l’identification des meilleurs aptamères et suggèrent de nouvelles pratiques pour la conduite des SELEX futurs. / SELEX is a directed molecular evolution technic which allows, after several rounds of selection, enriching a library from random nucleic acids to sequences able to bind specifically a target. Sequencing technics are then used to identify these sequences called « aptamers ». Since the arrival of High-Throughput Sequencing (HTS), it is now possible to analyse millions of sequences. The aim of the thesis was to develop methods for the treatment and the analysis of HTS data, in order to facilitate the identification of the best aptamers inside a SELEX. During this thesis, a semi-automatic binding test on adherent living cells has been developed to measure the affinity of aptamers identified in SELEX directed against specific cells (cell-SELEX). Then, the evolution of the sequence enrichment during a cell-SELEX has been analysed by HTS. This analysis gave us the possibility to design a new phylogenetic approch named FREDROGRAM. This evolutive approch allowed to identify variants of an aptamer’s family with a better affinity. Finally, HTS of two SELEX directed against proteins has contributed to a better understanding of the impact of selection parameters on the library and to identified new aptamers, notably by reducing the number of SELEX rounds. To conclude, this work shows the importance of HTS in the identification of the best aptamers and suggests new protocols to monitor the next SELEX in a different manner.

<p>Synthetic biology reprograms organisms to perform non-native functions for beneficial reasons. An important practice in synthetic biology is the ability to edit DNA to change a base pair, disrupt a gene, or insert a new DNA sequence. DNA edits are commonly made with the help of homologous recombination, which inserts new DNA flanked by sequences homologous to the target region. To increase homologous recombination efficiency, a double stranded break is needed in the middle of the target sequence. Common methods to induce double stranded breaks use nucleases, enzymes that cleave ribonucleotides (DNA and RNA). The most common nucleases are restriction enzymes, which recognize a short, fixed, palindromic DNA sequence (4-8 base pairs). Because of the short and fixed nature of the recognition sites, restriction enzymes do not make good candidates to edit large chromosomal DNA. Alternatively, scientists have turned to programmable endonucleases which recognize user-defined DNA sequences, often times much larger than the recognition sites of restriction enzymes (15-25 base pairs). Programmable endonucleases such as CRISPR-based systems and prokaryotic Argonautes are found throughout the prokaryotic kingdom and may differ significantly in activity and specificity. To compare activity levels among endonuclease enzymes, activity assays are needed. These assays must clearly delineate dynamic activity levels of different endonucleases and work with a wide variety of enzymes. Ideally, the activity assay will also function as a positive selection screen, allowing modifications to the enzymes via directed evolution. Here, we develop an <i>in vivo</i> assay for programmable endonuclease activity that can also serve as a positive selection screen using two plasmids, a lethal plasmid to cause cell death and a rescue plasmid to rescue cell growth. The lethal plasmid houses the homing endonuclease, I-SceI, which causes a deadly double-stranded break at an 18 base pair sequence inserted into an engineered <i>E. coli</i> genome. The rescue plasmid encodes for a chosen endonuclease designed to target and cleave the lethal plasmid, thereby preventing cell death. With this, cell growth is directly linked to programmable endonuclease activity. Three endonucleases were tested, SpCas9, eSpCas9, and xCas9, displaying recovered growth of 49.3%, 26.1%, and 16.4% respectively. These values translate to kinetic enzymatic activity and are congruent with current literature findings as reported values find WT-SpCas9 to have the fastest kinetics cleaving around 95% of substrate within 15 seconds, followed closely by eSpCas9 cleaving 75% of substrate within 15 seconds and finally trailed by xCas9 cleaving 20% of substrate in about 30 seconds. The differences between each endonuclease’s activity is exacerbated in our <i>in vivo</i> system when compared to similar <i>in vitro</i> methods with much lower resolution. Therefore, slight differences in activity between endonucleases within the first few minutes in an <i>in vitro</i> assay may be a few percentages different whereas in our <i>in vivo</i> assay, these differences in activity result in a more amplified signal. With the ability to display the dynamic response of enzymes, this assay can be used to compare activity levels between endonucleases, give insight into their kinetics, and serve as a positive selection screen for use in directed evolution applications. </p>

<p>Stem cell technologies hold great promise in solving problems within fields such as drug development, regenerative medicine, and disease modeling. Stem cell engineering provides a mechanism that will help stem cells achieve this promise. Currently, many applications within tissue engineering are limited by a lack of ability to create accurate micro-physiological structures that recapitulate multicellular tissue patterns <i>in vivo</i>. Precise control of spatial and temporal signaling is desired to perform concurrent differentiation to multiple cell types intentionally. The OptoWnt construct, a novel optogenetic system activating the Wnt signaling pathway, achieves precise spatiotemporal regulation, in pursuit of greater control in stem cell differentiation. We utilize OptoWnt, to differentiate stem cells into cardiovascular cells: endothelial progenitor cells and cardiomyocytes, valuable cell types for designing microtissues. Endothelial cells comprise the luminal lining of blood and lymphatic vessels, providing the integral structure for distribution within the body, separating mobile and stationary tissues. Cardiomyocytes provide the force required to pump blood throughout the human body and are a highly desired cell type in regenerative medicine.</p> <p>In this project, we have applied an optogenetic induced signaling pathway, OptoWnt, to differentiate human pluripotent stem cells (hPSCs) into cardiovascular cells via light-induced activation of Wnt signaling pathway. In the analysis of these cells and comparison to previous small molecule approaches to cardiovascular cell differentiation, we demonstrate the robustness of the optogenetic approach and similar efficiency that it has with the small molecule approach. In short, we have further demonstrated the utility and potential of optogenetic induction of developmental pathways, via the OptoWnt construct.</p>

Biological cell populations, such as in tissues or microbial communities, are constantly subject to different sources of noise and variability. Despite this, multicellular systems are still able to function properly because cells coordinate with each other by communication. Using biological model systems to study this multiscalar process can be challenging because of their innate complexity. In this thesis, we address this challenge by building a synthetic multicellular system using bottom-up in vitro assembly approaches. Using this platform, we aim to study the effect of cell-to-cell communication to population variability in a minimal and simplified context. To achieve this, we require a synthetic cell population with (i) quantifiable gene expression dynamics, (ii) customizable population variability, and (iii) intercellular communication. Having these characteristics will allow us to test different initial configurations of population variability and monitor population gene expression dynamics with and without cell-to-cell communication. To generate these synthetic cell populations, reconstituted cell-free expression systems (CFES) are encapsulated into monodisperse-sized liposomes using double-emulsion microfluidics. Both transcription and translation levels are simultaneously monitored and quantified to develop models of cell-free gene expression dynamics and differentiate between bulk and encapsulated formats. Population variability was then incorporated by combining different batches of cells to create distinct subpopulations or by using a two-inlet double-emulsion microfluidic device to generate single populations with a large dispersion of encapsulated DNA template. Lastly, genetic circuits based on the quorum sensing system of Vibrio fischeri are used to implement diffusion-mediated intercellular signalling. Quorum sensing gene circuits in Escherichia coli extract-based CFES were tested in bulk and phase transfer-generated synthetic cells. Together with these experimental systems, corresponding models of synthetic cell populations that can account for population variability and secrete-and-sensing communication are developed using mixed-effects models and moment dynamics. Overall, this work leverages CFES and microfluidic technologies to reproducibly generate a simplified in vitro model of multicellular systems that can be easily monitored spatiotemporally to study multi-scalar processes.:Preface Chapter 1 Bottom-up multicellular systems Chapter 2 Building blocks: cell-free expression and liposomes Chapter 3 Gene expression dynamics in synthetic cell populations Chapter 4 Variability and communication in synthetic cell populations Chapter 5 Modeling variability & communication in synthetic cell populations Summary and outlook Appendices Bibliography / Biologische Zellpopulationen, z.B. in Geweben oder mikrobiellen Gemeinschaften, sind ständig verschiedenen Quellen von Rauschen und Variabilität ausgesetzt. Trotzdem sind multizelluläre Systeme in der Lage, ordnungsgemäß zu funktionieren, weil sich die Zellen durch Kommunikation miteinander abstimmen. Die Verwendung biologischer Modellsysteme zur Untersuchung dieses multiskalaren Prozesses kann aufgrund ihrer angeborenen Komplexität eine Herausforderung darstellen. In dieser Arbeit gehen wir diese Herausforderung an, indem wir ein synthetisches multizelluläres System mit Hilfe von Bottom-up-in vitro-Assembly-Ansätzen aufbauen. Mit Hilfe dieser Plattform wollen wir die Auswirkungen der Kommunikation von Zelle zu Zelle auf die Populationsvariabilität in einem minimalen und vereinfachten Kontext untersuchen. Um dies zu erreichen, benötigen wir eine synthetische Zellpopulation mit (i) quantifizierbarer Genexpressionsdynamik, (ii) anpassbarer Populationsvariabilität und (iii) interzellulärer Kommunikation. Mit diesen Eigenschaften können wir verschiedene Ausgangskonfigurationen der Populationsvariabilität testen und die Genexpressionsdynamik der Population mit und ohne Zell-zu-Zell-Kommunikation beobachten. Um diese synthetischen Zellpopulationen zu erzeugen, werden rekonstituierte zellfreie Expressionssysteme (CFES) mit Hilfe der Doppelemulsions-Mikrofluidik in monodisperse Liposomen eingekapselt. Sowohl die Transkriptions- als auch die Translationsraten werden gleichzeitig überwacht und quantifiziert, um Modelle für die Dynamik der zellfreien Genexpression zu entwickeln und zwischen Bulk- und verkapselten Formaten zu unterscheiden. Die Variabilität der Populationen wurde dann durch die Kombination verschiedener Zellchargen zur Bildung unterschiedlicher Subpopulationen oder durch die Verwendung einer mikrofluidischen Doppelemulsionsvorrichtung mit zwei Einlässen zur Erzeugung einzelner Populationen mit einer großen Streuung der eingekapselten DNA-Vorlage einbezogen. Schließlich werden genetische Schaltkreise auf der Grundlage des Quorum-Sensing-Systems von Vibrio fischeri verwendet, um diffusionsvermittelte interzelluläre Signalübertragung zu implementieren. Quorum-Sensing-Genkreisläufe in CFES auf der Basis von Escherichia coli-Extrakten wurden in synthetischen Zellen getestet, die durch Bulk- und Phasentransfer erzeugt wurden. Zusammen mit diesen experimentellen Systemen wurden entsprechende Modelle synthetischer Zellpopulationen entwickelt, die die Populationsvariabilität und die Sekretions- und Sensing-Kommunikation mit Hilfe von Mixed-Effects-Modellen und Momentendynamik berücksichtigen können. Insgesamt nutzt diese Arbeit CFES- und Mikrofluidik-Technologien, um reproduzierbar ein vereinfachtes in vitro-Modell multizellulärer Systeme zu erzeugen, das leicht raum-zeitlich überwacht werden kann, um multiskalare Prozesse zu untersuchen.:Preface Chapter 1 Bottom-up multicellular systems Chapter 2 Building blocks: cell-free expression and liposomes Chapter 3 Gene expression dynamics in synthetic cell populations Chapter 4 Variability and communication in synthetic cell populations Chapter 5 Modeling variability & communication in synthetic cell populations Summary and outlook Appendices Bibliography

Yes / Computational models are perceived as an attractive alternative to mathematical models (e.g., ordinary differential equations). These models incorporate a set of methods for specifying, modeling, testing, and simulating biological systems. In addition, they can be analyzed using algorithmic techniques (e.g., formal verification). This paper shows how formal verification is utilized in systems and synthetic biology through qualitative vs quantitative analysis. Here, we choose two well-known case studies: quorum sensing in P. aeruginosas and pulse generator. The paper reports verification analysis of two systems carried out using some model checking tools, integrated to the Infobiotics Workbench platform, where system models are based on stochastic P systems. / EPSRC

Flavonoid glycosyltransferases (GTs), enzymes integral to plant ecological responses and human pharmacology, necessitate rigorous structural elucidation to decipher their mechanistic function and substrate specificity, particularly given their role in the biotransformation of diverse pharmacological agents and natural products. This investigation delved into a comprehensive exploration of the flavonol 3-O GT from Citrus paradisi (Cp3GT), scrutinizing the impact of a c-terminal c-myc/6x histidine tag on its enzymatic activity and substrate specificity, and successfully achieving its purification to apparent homogeneity. This established a strong foundation for potential future crystallographic and other structure/function analyses. Through the strategic implementation of site-directed mutagenesis, a thrombin cleavage site was incorporated proximal to the tag, followed by cloning in Pichia pastoris, methanol-induced expression, and cobalt-affinity chromatography for initial purification stages. Notably, the recombinant tags did not exhibit a discernible influence on Cp3GT kinetics, substrate preference, pH optima, or metal interactions, maintaining its specificity towards flavonols at the 3-OH position and favoring glucosylation of quercetin and kaempferol. Subsequent purification steps, including MonoQ anion exchange and size-exclusion chromatography, yielded Cp3GT with ≥95% homogeneity. In silico molecular models of Cp3GT and its truncated variants, Cp3GTΔ80 and Cp3GTΔ10, were constructed using D-I-TASSER and COFACTOR to assess binding interactions with quercetin and kaempferol. Results indicated minimal interference of c-myc/6x-his tags with the native Cp3GT structure. This study not only lays a foundation for impending crystallographic studies, aiming to solidify the understanding of Cp3GT's stringent 3-O flavonol specificity, but also accentuates the potential of microbial expression platforms and plant metabolic engineering in producing beneficial compounds. To this end, a thorough review of four pivotal classes of plant secondary metabolites, flavonoids, alkaloids, betalains, and glucosinolates, was conducted. This will open avenues for further research and applications in biotechnological, medical, and agricultural domains.

No description available.