Les progrès récents de la microfluidique, la biologie synthétique, la microscopie automatisée rendent aujourd'hui possible le contrôle externe de l'expression des gènes en temps réel. Parmi les défis que devra relever le domaine du contrôle externe et temps-réel de l'expression des gènes, se trouve la possibilité de contrôler des réseaux de régulation génique aux dynamiques complexes et multi-stables et le contrôle de multiples gènes en parallèle. Pour faire avancer le domaine dans cette direction nous avons étudié la contrôlabilité d'un réseau bistable composé de deux gènes, appelé genetic toggle switch, ou bascule génétique, autour de son point d'équilibre instable sur de longues périodes. Dans ce document, nous présentons la mise en place d'une plateforme de contrôle externe de l'expression des gènes en cellule unique, ainsi que le développement d'un châssis cellulaire bactérien et d'une librairie de circuits de bascules génétiques à contrôler. Nous utilisons la plateforme pour diriger et maintenir notre système génétique dans sa région d'instabilité avec des techniques de stabilisation à la fois en boucle ouverte et en boucle fermée. Nous démontrons non seulement que les plateformes de contrôle in silico peuvent être utilisées pour contrôler un système génétique dans un état hors-équilibre, nous démontrons aussi la possibilité de maintenir une population de cellules dans leur région d'instabilité à l'aide de stimulations périodiques en boucle ouverte. Ces résultats suggèrent l'émergence de régimes de stabilité différents dans des réseaux de régulation génique lorsqu'ils sont soumis à des environnements fluctuants, et peuvent fournir de nouvelles perspectives dans l'étude de la prise de décision cellulaire. Nous présentons aussi une nouvelle approche pour l'analyse d'images de microscopie qui exploite l'information cachée dans plusieurs plans focaux autour du spécimen au lieu d'utiliser seulement un seul plan focal. L'objectif de cette méthode est d'identifier automatiquement les différentes parties d'une image à l'aide de techniques d'apprentissage-machine inspirées de l'imagerie hyperspectrale. La méthode facilite la segmentation de l'image et peut être facilement adaptée à différents organismes / Recent progresses in microfluidics, synthetic biology and microscopy automation now make it possible to control gene expression externally and in real time. Among the challenges facing the field of external real-time control of gene expression is the control of intricate, multistable gene regulation networks as well as the control of several target genes at the same time. To advance the domain in this direction we studied the controllability of a simple bistable two-genes network, the so-called genetic toggle switch, in the vicinity of its unstable equilibrium point for extended periods of time. Throughout this document, we present the development of a custom control platform for external control of gene expression at the single-cell level as well as a bacterial cellular chassis and a library of toggle switch genetic circuits for us to control. We use the platform to drive and maintain our genetic system in its region of unstability with both closed-loop and open-loop strategies. Not only do we demonstrate that in silico control platforms can control genetic systems in out-of-equilibrium states, we also notably maintain a population of cells in their unstable area with open-loop periodic stimulations. These results suggest the possible emergence of different regimes of stability in gene regulation networks submitted to fluctuating environments, and can potential insights in the study of cellular decision making. We also introduce a new approach for microscopy image analysis which exploits information hidden in several focal planes around the specimen instead of using only a single-plane image. The objective of this method is to automatically label different parts of an image with machine learning techniques inspired by hyperspectral imaging. The method is then shown to facilitate segmentation and be easily adaptable to various different organisms

This work deals with addressing the issues related to design of double toggle switch mechanisms with emphasis on structural, dimensional and dynamic aspects. Currently, almost all the issues related to electrical switches are dealt from electromagnetic point of view; the operating mechanism is hardly touched. It is observed that kinematic parameters influence electrical performance of switch significantly. Therefore, there is a need to develop methodologies for supporting exploration of diverse kinematic chains (KCs) for this purpose. Visual inspection is tedious and error prone even when a complete list of design criteria is available, hence, the work presented in the thesis contributes towards automated design of toggle switch mechanisms. In this context, in house modular kinematics data structure is found useful for using it as a tool in the design of toggle switch. Modular kinematics, typically used for kinematic analysis, works on the principle of finding the configuration of a mechanism using a given set of modules by a procedure called module sequence. This module sequence is used and interpreted in a number of ways for its effective use in various design stages. Structurally, a set of seven conditions must be satisfied by a KC to exhibit double toggle. These conditions are broadly classified into three categories: criteria for KC, function assignment criteria and criteria for stoppers. These three criteria are to be checked automatically by use of module sequence in the same order as mentioned. In the criteria for KC, one of the conditions is that, the KC should not have fractionated degrees of freedom (d.o.f.). Hence, detection of fractionation in a KC is inevitable. In literature, is was found that the algorithms for detection operate at their worst case complexity, O(n4), and some of them do not report joint fractionation. Thus, the algorithms are not only robust but also computationally expensive. Therefore, a frugal and comprehensive method O(n2) is implemented to detect fractionation using modular kinematics. Also, inherent structural pattern embedded in fractionated KCs is hardly studied in literature. It is found that the way body and joint fractionation is defined in fractionated KCs is inconsistent. So, fractionation is interpreted as symbolic partitioning of joints and links in the traditional body and joint fractionation types respectively. Based on the number of ways of partitioning, simple and multiple types of fractionation are recognized. Valid partitions are identified using the notion of fractionating and non-fractionating subchains. Relative locations of these subchains influence distribution of d.o.f. across the fractionated KC. Conventional representation of KCs as links and joints or graphs is difficult to comprehend this distribution. For this, a novel concept of fractionation graph is introduced that gives d.o.f. distribution information and the relative locations of the constituent subchains across the KC. Modular kinematics gives a constructive description of fractionated KCs. Characterization of fractionated KCs, based on presence of multiple separation links, is introduced as order of fractionation. Uniqueness for a given order of fractionation is also justified. After the criteria for KC, a KC is tested for feasibility for function assignment criteria. This requires recognition of active and passive subchains of the KC with respect to input and output pairs. For this, module sequence is characterized for recognition of the subchains. Based on these subchains, locations of stoppers are derived. Using this information, an algorithmic approach to assign functions (functions like spring, ground link, input link, etc.) to derive distinct driving mechanisms provided isomorphic elements (links and joints) of the KC are known beforehand, is introduced. The design parameters influencing dimensional synthesis have been identified as dimensions of links, spring anchor points and stopper locations. Sub-problems associated with each parameter are analyzed. It is found out that optimum location of stoppers for selecting operational range of motion is necessary by taking into account the considerations of timing of switch and impact velocity. Based on the analysis, an algorithmic way to design single toggle switch mechanisms is introduced. Timing for closing or opening of a switch is one of the critical measure that determines its performance. Timing should be as low as possible without exceeding the impact velocity at the instant contacts meet each other. Timing of a switch depends on the dimensions of the links, inertial parameters, spring stiffness etc. For a given timing for a mechanism, dynamic synthesis, in this thesis, deals with finding the inertial parameters of the links using Quinn's energy distribution method, modular kinematics, and Nelder and Mead's downhill simplex method for optimization. This thesis helps the designer to use modular kinematics as a potential automated tool to select a valid design to make the solution space more meaningful in the design of toggle switch mechanisms.

Research on bacteria in their biofilm form is limited by the ability to artificially culture bacterial biofilms in a system that permits the visualization of individual cells. The experiments comprising this thesis research are on-going investigations of bacterial culture systems engineered to provide an environment that mimics biofilms while enabling real-time microscopy. Specifically, the microfluidic systems developed and assessed as part of this thesis permit the visualization of individual bacteria cells within consortia growing within a narrow space provided by a microfluidic device. This research demonstrates the versatility of these microfluidic systems across potentially high-throughput microbiological experiments utilizing genetically engineered Escherichia coli. Before demonstrating the efficacy of these systems, the development of the field of synthetic biology over the past half century is reviewed, focusing on synthetic genetic circuits and their applications (Chapter 2). The first and main microfluidic device explored in this research was developed to mimic the nutrient-deficient conditions within biofilms by forcing media to enter the culture area through a narrow, torturous channel. The microfluidic channel was thin enough (0.97 μm) to prevent the motility of 1-μm-wide E. coli cells, enabling visualization of individual cells. The bacteria cultured in the device contained either a simple Plux-driven quorum sensing receiver (Chapters 3 and 5) or a LacI- and TetR-driven genetic toggle switch (Chapter 4). Under the culture conditions, the quorum sensing reporter signal was detected even without addition of the signaling molecule (Chapter 3). The genetic toggle switch was stable when the system began in the high-LacI expression state, but after 5 days of culture, >5% of high-TetR expression cells began to consistently express the high-LacI state (Chapter 4). This system was also employed to track lineages of cells using real-time microscopy, which successfully characterized the inheritance of aberrant, enlarged cell phenotypes under stress (Chapter 5). Another microfluidic device, a droplet bioreactor, was also developed to culture small numbers of cells in an aqueous bubble suspended in oil (Chapter 6). Quorum sensing receiver cellswere cultured in this device, demonstrating that it is well suited for testing the effects of compounds on biofilms within water-in-oil droplets. / Ph. D. / Bacteria are the most abundant organisms globally, yet relatively little is understood about the basic biology of biofilms, one of the most common natural states of bacteria. Biofilms are ubiquitous consortia of individual microbial cells that send and received chemical signals from one another to carry out group behaviors such as quorum sensing. The impacts of biofilms range from the contamination of food processing equipment to antibiotic resistant bacterial infections. The vast majority of microbiological research has been conducted on bacteria in their planktonic state as individual cells cultured in a liquid medium. This form of culture does not permit the types of research that can help address the impacts of biofilms on human health and economic activities, never mind examine the biological mechanism of random gene and morphological expression within bacterial biofilm. This thesis presents research utilizing two microfluidic devices that will enable further large-scale studies to unravel the mechanisms that create biofilms as well as permit high-throughput testing of chemical compounds to control the growth and development of biofilms. Moreover, these devices permit the use of real-time microscopy to track cells and their growth over time. The first microfluidic device utilized in this research mimics the nutrient-limiting conditions of biofilms. This biofilm-mimicking device was used to culture a common research bacteria, Escherichia coli, with one of two engineered genetic circuits (reviewed in Chapter 2): a quorum sensing receiver (Chapters 3 and 5) or genetic toggle switch (Chapter 4). Both of these genetic circuits demonstrated stochasticity in their gene expression states under the culture conditions in the biofilm-mimicking device. The second microfluidic device successfully permitted the culture of small numbers of isolated cells within a small bubble of bacterial media suspended in oil (Chapter 6). Additionally, this device enabled the addition of chemical compounds to influence the growth and metabolism of the trapped cells. Collectively, these microfluidic devices provide the ability to effectively study both the mechanisms underlying random gene expression within biofilms as well as explore the chemical factors that can be used to control and mitigate biofilm formation and growth.

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]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/133055 / Compendio

This dissertation is concerned with the potential multistability of protein concentrations in the cell that can arise in biochemical networks. That is, situations where one, or a family of, proteins may sit at one of two or more different steady state concentrations in otherwise identical cells, and in spite of them being in the same environment. Models of multisite protein phosphorylation have shown that this mechanism is able to exhibit unlimited multistability. Nevertheless, these models have not considered enzyme docking, the binding of the enzymes to one or more substrate docking sites, which are separate from the motif that is chemically modified. Enzyme docking is, however, increasingly being recognised as a method to achieve specificity in protein phosphorylation and dephosphorylation cycles. Most models in the literature for these systems are deterministic i.e. based on Ordinary Differential Equations, despite the fact that these are accurate only in the limit of large molecule numbers. For small molecule numbers, a discrete probabilistic, stochastic, approach is more suitable. However, when compared to the tools available in the deterministic framework, the tools available for stochastic analysis offer inadequate visualisation and intuition. We firstly try to bridge that gap, by developing three tools: a) a discrete `nullclines' construct applicable to stochastic systems - an analogue to the ODE nullcines, b) a stochastic tool based on a Weakly Chained Diagonally Dominant M-matrix formulation of the Chemical Master Equation and c) an algorithm that is able to construct non-reversible Markov chains with desired stationary probability distributions. We subsequently prove that, for multisite protein phosphorylation and similar models, in the deterministic domain, enzyme docking and the consequent substrate enzyme-sequestration must inevitably limit the extent of multistability, ultimately to one steady state. In contrast, bimodality can be obtained in the stochastic domain even in situations where bistability is not possible for large molecule numbers. We finally extend our results to cases where we have an autophosphorylating kinase, as for example is the case with $Ca^{2+}$/calmodulin-dependent protein kinase II (CaMKII), a key enzyme in synaptic plasticity.

X-Chromosom-Inaktivierung (XCI) ist der Mechanismus, den Säuger zur Dosiskompensierung zwischen weiblichen und männlichen Zellen verwenden. XCI wird ausgelöst durch die monoallelische Hochregulation der langen nicht-kodierenden RNA Xist von einem der zwei X-Chromosomen in weiblichen Zellen. Die Xist RNA vermittelt dann das Ausschalten der Gene auf diesem X-Chromosom. Das wirft einige interessante Fragen auf: Wie zählen Zellen ihre X-Chromosomen und stellen sicher, dass genau eines aktiv bleibt? Wie entscheiden sie, welches X-Chromosom aktiv bleibt und welches ausgeschaltet wird? Und wie erinnern sie sich an diese Entscheidung und behalten sie stabil bei durch alle weiteren Zellteilungen? Mithilfe eines stochastischen Modells zeigen wir, dass diese XCI Regulation prinzipiell durch nur zwei Regulatoren erklärt werden kann: Ein global (in trans) agierender XCI Aktivator und ein lokal (in cis) agierender XCI Repressor. Dieses Netzwerk aus nur zwei Regulatoren kann die Xist Expressionsmuster in verschiedenen Säugerspezies reproduzieren, von der Maus bis zum Mensch. Es sagt außerdem voraus, dass Zellen in der Lage sind, biallelische zu monoallelischer Xist Expression zu korrigieren, eine Vorhersage, für die wir tatsächlich experimentelle Belege finden. Mit einem mechanistischen Modell zeigen wir, dass das cis-Gedächtnis über den Xist Expressionszustand durch Antisense-Transkription zustande kommen könnte. Auf dieser Hypothese aufbauend untersucht der zweite Teil der Arbeit das Potential von Antisense-Transkription, ein lokales Gedächtnis über den Expressionszustand eines Gens zu generieren, genauer. Diese Analyse sagt vorher, dass Antisense-Repression den Expressionszustand eines Lokus tatsächlich für einige Tage stabil erhalten kann. / X-chromosome inactivation (XCI) is the mechanism for dosage compensation between the sexes in mammals. It is initiated through monoallelic upregulation of the long non-coding RNA Xist from one X chromosome, which mediates almost complete transcriptional silencing of this X chromosome. XCI regulation raises intriguing and thus far unanswered questions: How do cells count their X chromosomes and ensure that exactly one stays active? How do they make a mutually exclusive choice for one inactive X chromosome, and how do they then stably maintain this choice throughout subsequent cell divisions? Using stochastic modeling, we show that XCI onset only requires two regulators: A trans-acting Xist activator that ensures female specificity and a cis-acting Xist repressor that allows stable maintenance of alternative Xist expression states. This two-regulator network can recapitulate Xist expression patterns across different species and makes a novel prediction that is validated experimentally: Cells are able to revert biallelic Xist expression to monoallelic expression. With a mechanistic stochastic model we show that Xist's antisense transcript Tsix might be the cis-acting Xist repressor, uncovering the molecular mechanism behind the stabilization of the alternative Xist expression states. Building upon Tsix' possible functional role in stabilizing alternative Xist expression states on the active and inactive X chromosome, the second part of this thesis investigates the potential of antisense transcription to maintain a transient transcriptional memory. We find that mutual repression between a pair of antisense genes can allow the locus to remember the transcription state it has acquired due to a past signal for several days.

Electrical switches are ubiquitous. Performance requirement for a switch is stringent. The operating mechanism mostly decides the performance of an electromechanical switch. However, design of such mechanisms, which involve discontinuous motions, is not much addressed in literature. The present work proposes a systematic procedure to design and analyze toggle based switching mechanisms. The work defined the toggle phenomenon rigorously, and, based on the behaviour of the toggles, provided a classification scheme for the switch mechanisms. The existing switches fall in two major categories viz., single-toggle and double-toggle switches. The double toggle mechanism is more suitable for high power breaking as it can isolate the system’s behaviour from the operator’s behaviour. The kinematic and geometric attributes of the operating mechanism which affect the toggle sequence and timings have been identified. A systematic simulation based study has been performed to identify the influence of different kinematic and dynamic parameters on the functionality of a double toggle switching mechanism. The influence of the variable moment of inertia and mechanism singularities arising out of introduction of the four bar sub chain on the performance of the system have been studied in detail. It is observed that the performance of the double toggle systems is less susceptible, though not immune to the user behaviour; in extreme scenarios the switching performance could become erratic. The use of an additional spring in an existing system enhanced the system performance; but, connecting the main spring with the coupler link altered the system performance more dramatically. Thus it established that the influence of the kinematic configuration on the performance of a switching mechanism is more pronounced than the dynamic characteristics of a comparable system. For the ab initio design of double toggle switching mechanisms, necessary structural criteria for a mechanism to exhibit double toggle phenomenon have been identified and verified with various 2 d.o.f. systems. It is also established that any double toggle mechanism cannot be used directly as a switching mechanism; the link dimensions, link arrangements and the stopper locations have to be chosen properly. Towards that end, three necessary kinematic criteria for a switching mechanism are identified. A few mechanisms which satisfy all structural and kinematic criteria are identified; the switching and toggle behaviour of these mechanisms are examined through simulations using Pro/Mechanism. Finally, considering all the conditions a is constructed with consideration of mass and geometric shape of the links. Thus, it established that the proposed methodology can systematically generate novel, structurally distinct electrical switches.