Subunits of highly fluorescent protein R-phycoerythrin as probes for cell imaging and single-molecule detection

R-phycoerythrin (R-PE) subunits and enzymatic digests were characterized by high-performance liquid chromatography (HPLC), capillary and gel electrophoresis, and HPLC-electrospray ionization mass spectrometry. Subunits were isolated from R-PE by HPLC and detected as single molecules by total internal reflection fluorescence microscopy (TIRFM). Favorable spectroscopic characteristics of R-PE subunits and digest peptides in the visible region of spectrum originate from phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores. High absorption coefficients and fluorescence (even under denaturing conditions), broad excitation and emission fluorescence spectra, and low molecular weights make these molecules suitable for fluorescence labeling of biomolecules and cells.;Fluorescent proteins were further formed both in vitro and in vivo by attachment of PEB to recombinant apo-subunits of R-PE and their genetic fusions to maltose binding protein (MBP). Apo-alpha and apo-beta R-PE subunits were cloned from red algae Polisiphonia boldii into bacterium Escherichia coli (E. coli). Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were formed after incubation of E. coli cells with PEB. Holo-subunits contained both PEB and urobilin (UB) chromophores. Fluorescence and differential interference contrast (DIC) microscopy localized holo-subunits at poles of E. coli cells. Proteins formed by attachment of PEB to MBP-subunit fusions were soluble, displayed high fluorescence, contained only PEB, and were located either throughout cells or at cell poles. As measured by flow cytometry, cells containing fluorescent holo-subunits or MBP-subunit fusions were up to ten times brighter than control cells. Proteins formed by attachment of PEB to R-PE apo-subunits can be used as florescence reporters of gene expression and protein localization in cells, and in flow cytometry.;Finally, a high-throughput method was demonstrated which recorded emission fluorescence spectra of individual E. coli cells containing fluorescent proteins. Upon excitation with a 488 nm argon-ion laser many bacterial cells were imaged by a 20x microscope objective while they moved through a capillary tube. Fluorescence was dispersed by a transmission diffraction grating, and an intensified charge-coupled device (ICCD) camera recorded simultaneously the zero order and the first order spectrum for each cell. Demonstrated method could have a higher throughput, better sensitivity, and better spectral resolution compared to spectral flow cytometry

Extensive translational regulation through the proliferative transition of Trypanosoma cruzi revealed by Multi-Omics

Trypanosoma cruzi is the etiological agent for Chagas disease, a neglected parasitic disease in Latin America. Gene transcription control governs the eukaryotic cell replication but is absent in trypanosomatids; thus, it must be replaced by posttranscriptional regulatory events. We investigated the entrance into the T. cruzi replicative cycle using ribosome profiling and proteomics on G1/S epimastigote cultures synchronized with hydroxyurea. We identified 1,784 translationally regulated genes (change > 2, false-discovery rate [FDR]  1.5, FDR < 0.05), respectively. A major translational remodeling accompanied by an extensive proteome change is found, while the transcriptome remains largely unperturbed at the replicative entrance of the cell cycle. The differentially expressed genes comprise specific cell cycle processes, confirming previous findings while revealing candidate cell cycle regulators that undergo previously unnoticed translational regulation. Clusters of genes showing a coordinated regulation at translation and protein abundance share related biological functions such as cytoskeleton organization and mitochondrial metabolism; thus, they may represent posttranscriptional regulons. The translatome and proteome of the coregulated clusters change in both coupled and uncoupled directions, suggesting that complex cross talk between the two processes is required to achieve adequate protein levels of different regulons. This is the first simultaneous assessment of the transcriptome, translatome, and proteome of trypanosomatids, which represent a paradigm for the absence of transcriptional control. The findings suggest that gene expression chronology along the T. cruzi cell cycle is controlled mainly by translatome and proteome changes coordinated using different mechanisms for specific gene groups

Cell size regulation of Escherichia coli at high osmolarity

The goal of the study of bacterial physiology, and in the study of biophysics more broadly, is to apply quantitative predictive models to the behaviour of complex living organisms. Just as is the case with thermodynamics, the challenge comes from the fact that the subject systems are far too intricate to be modelled from the bottom up using first principles. Instead, the focus is on the development of coarse-grained models which reduce the description of the system down to a small number of key state variables. As late as the mid 20th century the identity, and even existence, of these variables was an open question, because living cells are a non-equilibrium system. However, between the 1950s and 1970s a golden era of bacterial physiology would give rise to a series of robust quantitative models, that linked a number of important physiological parameters. Starting with the Copenhagen school in 1958, it would soon be discovered that cell size was connected to growth rate via a single exponential relation that was independent of the makeup of the media used to achieve it. Shortly after this key result, the work of Helmstetter and Cooper would reveal that the time taken to replicate the chromosome of E. coli was constant over a wide range of growth rates. This counter intuitive observation would lead to the realisation that in order to grow at doubling times shorter than their replication time, bacterial cells must overlap multiple rounds of replication simultaneously. Taking these findings and running with them, Donachie at the University of Edinburgh was able to develop a model in which cells accumulate a critical initiation mass in order to start new rounds of replication. Using previous data, he was able to show that this initiation mass remained constant across a wide range of growth conditions, and in doing so, was able to relate neatly the average cell size, growth rate, chromosome replication time and time to divide. Since its development Donachie’s constant initiation mass model has been tested under numerous different environmental conditions and found to hold. Although examples exist where the initiation mass can be shown to vary, the underlying relationship continues to stand up to scrutiny. Despite this extensive testing, one condition underrepresented in literature is that of growth at high osmolarity. Much like nutrient limitation, high osmolarity conditions have been shown to reduce the growth rate although the mechanism by which this happens remains poorly understood. To this end the objective of this doctoral thesis is to examine the balanced growth of E. coli cells in high osmolarity conditions, and determine how the cell physiology under such conditions fits within the paradigm of constant initiation mass, as described by Donachie. To achieve this, I characterise the bulk steady state growth rates of cells and show that in agreement with previous work they decrease significantly with increasing salt concentration. I go on to measure cell volume both using a custom built microscope I constructed, and a high throughput Coulter counter. By comparing the results from these independent measurement I am able to show that, in steady state, there is very little volume change that accompanies the significant decrease in growth rate at high osmolarity. Following the analysis of cell size, I proceed to measure how the DNA content of cells changes at high osmolarity, using flow cytometry. I argue that, in contrast to some recent findings, there is little change in cellular DNA content as the salt concentration is increased. Combining these results with measurements of the chromosome replication time from real time PCR reactions, I also demonstrate an increase in the cell cycle time with increasing osmolarity, in agreement with recent findings. Combining these results, I am able to show what effect high osmolarity has on the initiation mass. Additionally, I examine the results from a small number of experiments where the osmolarity was increased using soribtol instead of sodium chloride. I discuss if the nature of the osmolyte used changes the effect a high osmolarity condition has on cellular physiology, and suggest some interesting experiments going forward to further understand this relationship

Tools to study cytoadhesion of P. falciparum-infected erythrocytes under flow

Plasmodium falciparum infected erythrocytes are able to bind to a multitude of extracellular receptors through adhesins expressed on their membrane. The molecular interactions involved in parasite binding to these receptors are only partly understood, although the trait itself is well-known and used as an early indicator of severe malaria diagnosis and progress of infection. Binding to the microvasculature of blood vessels or the intervillous spaces of the placenta, ensures the survival of the parasite as it avoids splenic clearance in the later stages of maturation. In this thesis, the importance of sheer flow and cell morphology in binding dynamics of parasitized erythrocytes is further highlighted. While static adhesion assays would showcase similar amounts of cells adhering in different maturation stages, when observed in flow, this picture changes drastically. Trophozoite stage parasites seem to bind less frequently but more efficiently onto the simulated endothelium. In the last stage of maturation, schizont stage parasites alter the cell morphology to such an extent that adhesion is more likely but with less contact area and density of involved receptors. Changes in the membrane morphology between AA and AS erythrocytes also underline the significance of receptor presentation and accessibility influencing binding efficiency. The effects of a P. falciparum infection during primigravida are threatening to both the mother and the foetus, as infected erythrocytes sequester to the maternal side of the syncytiotrophoblast that lines the placenta. Despite the tremendous efforts in the field, the adhesive tropism of infected erythrocytes that leads to placental sequestration remains unsolved. In this thesis, I determined that measurements are not possible without the full length protein and interactions between the receptor and its erythrocyte-expressed ligand are not specific enough to distinguish from negative control experiments. Another form of cell adhesion investigated in this thesis is the formation of so-called rosettes, that form when an infected erythrocyte binds to non-infected erythrocytes. Rosette formation is considered either an indication of severe malaria or a symptom of progressed infection and is believed to propagate the severity of infection by obstructing smaller vasculature and normal blood flow. in this thesis, I developed a platform to study the position of rosettes within a channel in flow, in order to determine their margination tendance. In those experiments, I verified that regardless of haematocrit value and size of rosette, rosettes remain in flow and do not marginate towards the walls of the flow chamber

REGULATION OF MCM HELICASE LOADING DURING EARLY DIFFERENTIATION AND CELL CYCLE RE-ENTRY

Cells initiate DNA replication at thousands of DNA replication origins every cell cycle. Minichromosome maintenance complexes (MCM) unwind DNA to initiate replication in S phase. MCM loading onto DNA, called “origin licensing”, occurs in G1 phase. Multiple mechanisms restrict origin licensing to G1 phase to prevent aberrant MCM loading and genotoxic re-replication in S phase. For this reason, cells load 5-10 fold excess MCM in G1 as dormant origins to protect against replication stress in S phase. The origin licensing checkpoint ensures sufficient MCM loading before S phase entry. MCM loading occurs in G1, yet cells have a variety of G1 lengths. Stem cells have short G1s while differentiated cells have long G1 phases. Additionally, cells may exit the cell cycle to quiescence, a state without division. Cells re-entering the cell cycle from quiescence have longer G1s than actively proliferating cells. How cells accomplish the same MCM loading under these different G1 lengths is poorly understood. We used quantitative single cell flow cytometry and live cell imaging to measure MCM loading across varying G1 lengths. We found that stem cells with short G1s load MCM significantly faster than differentiated cells with long G1s. MCM loading rate decreases and G1 length increases during stem cell differentiation to all germ layers. The rapid licensing is due to increased Cdt1 protein in stem cells. Rapid origin licensing is intrinsic to pluripotency, as slowing MCM loading in stem cells promotes their differentiation. Cells re-entering into long G1 phases from quiescence are underlicensed with less loaded MCM at S phase entry than proliferating cells. The underlicensing causes replication stress sensitivity in S phase, and repeated rounds of quiescence and re-entry increase the replication stress sensitivity. A defective origin licensing checkpoint combined with a slow MCM loading rate causes the underlicensing during cell cycle re-entry, and extending the first G1 phase rescues the underlicensing. Thus, cell cycle re-entry is a higher risk cell cycle than active proliferation. Proper regulation of the rate and amount of MCM loading is critical across different G1 types to control differentiation, proliferation and genome stability.Doctor of Philosoph

Modification and Optimization of Conducting Polymer-Modified, Redox-Magnetohydrodynamics (R-MHD) Pumping for Enhanced and Sustained Microfluidics Applications

In this work, a novel microfluidic pumping approach, redox-magnetohydrodynamics (R-MHD) has improved by materials and device optimization to use in lab-on-a-chip applications. In R-MHD, magnetic flux (B) and ionic current density (j) interacts to generate body force (FB) in between active electrodes, according to the equation FB = j×B. This unique fluid pumping approach is scalable, tunable, generates flat flow profile, and does not require any channels or valves. Pumping performance, such as speed scales with the ionic current density (j) and duration depends on the total charge (Q). The ionic current density (j) results from the conversion of electronic current through redox reactions of a conducting polymer like PEDOT (poly-EDOT). The enhancement of j can be obtained by the modification of polymer morphology. Therefore, electropolymerization parameters such as solvent, monomer, electrolyte, and deposition method have been optimized to improve the electrochemical performance of PEDOT. Electrodeposited PEDOT film from propylene carbonate solvent and TBAPF6 electrolyte generated a maximum of 820 µm/s flow velocity and 210 s flow duration. This enhanced system used as an imaging cytometer by coupling with a light sheet confocal microscope. This microfluidic imaging platform was able to differentiate various leukocytes cells with ~ 5000 cell/s theoretical throughput and 0.6 µm image resolution. As, our existing microscope could not analyze the R-MHD velocity profile in height direction, astigmatism particle tracking velocimetry (APTV) was employed to analyze flow profiles in three dimensions. In a microfluidic setup, flow profile is dominated by stream wise component but with no significant contributions in y and z direction. Though we achieved significant improvement in fluidic speed, flow duration was still dependent upon the total charge. Therefore, an automated magnet switching device was built which synchronized the current and magnetic field to push fluid in single direction, for unlimited time

Checkpoint independence of most DNA replication origins in fission yeast

<p>Abstract</p> <p>Background</p> <p>In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, <it>Schizosaccharomyces pombe</it>, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of <it>rad3 </it>(which encodes the fission yeast homologue of ATR) or <it>cds1 </it>(which encodes the fission yeast homologue of Chk2).</p> <p>Results</p> <p>Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells.</p> <p>Conclusion</p> <p>The fact that ~97% of fission yeast replication origins – both early and late – are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks.</p

A systems engineering approach to model, tune and test synthetic gene circuits

La biología sintética se define como la ingeniería de la biología: el (re)diseño y construcción de nuevas partes, dispositivos y sistemas biológicos para realizar nuevas funciones con fines útiles, que se basan en principios elucidados de la biología y la ingeniería. Para facilitar la construcción rápida, reproducible y predecible de estos sistemas biológicos a partir de conjuntos de componentes es necesario desarrollar nuevos métodos y herramientas. La tesis plantea la optimización multiobjetivo como el marco adecuado para tratar los problemas comunes que surgen en el diseño racional y el ajuste óptimo de los circuitos genéticos sintéticos. Utilizando un enfoque clásico de ingeniería de sistemas, la tesis se centra principalmente en: i) el modelado de circuitos genéticos sintéticos basado en los primeros principios, ii) la estimación de parámetros de modelos a partir de datos experimentales y iii) el ajuste basado en modelos para lograr el desempeño deseado de los circuitos. Se han utilizado dos circuitos genéticos sintéticos de diferente naturaleza y con diferentes objetivos y problemas: un circuito de realimentación de tipo 1 incoherente (I1-FFL) que exhibe la importante propiedad biológica de adaptación, y un circuito de detección de quorum sensing y realimentación (QS/Fb) que comprende dos bucles de realimentación entrelazados -uno intracelular y uno basado en la comunicación de célula a célula- diseñado para regular el nivel medio de expresión de una proteína de interés mientras se minimiza su varianza a través de la población de células. Ambos circuitos han sido analizados in silico e implementados in vivo. En ambos casos, se han desarrollado modelos de estos circuitos basado en primeros principios. Se presta especial atención a ilustrar cómo obtener modelos de orden reducido susceptibles de estimación de parámetros, pero manteniendo el significado biológico. La estimación de los parámetros del modelo a partir de los datos experimentales se considera en diferentes escenarios, tanto utilizando modelos determinísticos como estocásticos. Para el circuito I1-FFL se consideran modelos determinísticos. Aquí, la tesis plantea la utilización de modelos locales utilizando la optimización multiobjetivo para realizar la estimación de parámetros del modelo bajo escenarios con estructura de modelo incompleta. Para el circuito QS/Fb, una estructura controlada por realimentación, el problema tratado es la falta de excitabilidad de las señales. La tesis propone una metodología de estimación en dos etapas utilizando modelos estocásticos. La metodología permite utilizar datos de curso temporal promediados de la población y mediciones de distribución en estado estacionario para una sola célula. El ajuste de circuitos basado en modelos para lograr un desempeño deseado también se aborda mediante la optimización multiobjetivo. Para el circuito QS/Fb se realiza un análisis estocástico completo. La tesis aborda cómo tener en cuenta correctamente tanto el ruido intrínseco como el extrínseco, las dos principales fuentes de ruido en los circuitos genéticos. Se analiza el equilibrio entre ambas fuentes de ruido y el papel que desempeñan en el bucle de realimentación intracelular, y en la realimentación extracelular de toda la población. La principal conclusión es que la compleja interacción entre ambos canales de realimentación obliga al uso de la optimización multiobjetivo para el adecuado ajuste del circuito. En esta tesis además del uso adecuado de herramientas de optimización multiobjetivo, la principal preocupación es cómo derivar directrices para el ajuste in silico de parámetros de circuitos que puedan aplicarse de forma realista in vivo en un laboratorio estándar. Como alternativa al análisis de sensibilidad de parámetros clásico, la tesis propone el uso de técnicas de clustering a lo largo de los frentes de Pareto, relacionando el comprLa biologia sintètica es defineix com l'enginyeria de la biologia: el (re) disseny i construcció de noves parts, dispositius i sistemes biològics per a realitzar noves funcions útils que es basen a principis elucidats de la biologia i l'enginyeria. Per facilitar la construcció ràpida, reproduïble i predictible de aquests sistemes biològics a partir de conjunts de components és necessari desenvolupar nous mètodes i eines. La tesi planteja la optimització multiobjectiu com el marc adequat per a tractar els problemes comuns que apareixen en el disseny racional i l' ajust òptim dels circuits genètics sintètics. Utilitzant un enfocament clàssic d'enginyeria de sistemes, la tesi es centra principalment en: i) el modelatge de circuits genètics sintètics basat en primers principis, ii) l' estimació de paràmetres de models a partir de dades experimentals i iii) l' ajust basat en models per aconseguir el rendiment desitjat dels circuits. S'han utilitzat dos circuits genètics sintètics de diferent naturalesa i amb diferents objectius i problemes: un circuit de prealimentació de tipus 1 incoherent (I1-FFL) que exhibeix la important propietat biològica d'adaptació, i un circuit de quorum sensing i realimentació (QS/Fb) que comprèn dos bucles de realimentació entrellaçats -un intracel·lular i un basat en la comunicació de cèl·lula a cèl·lula- dis-senyat per regular el nivell mitjà d'expressió normal d'una proteïna d'interès mentre es minimitza la seua variació al llarg de la població de cèl·lules. Els dos circuits han estat analitzats in silico i implementats in vivo. En tots dos casos, s'han desenvolupat models basats en primers principis d'aquests circuits. Després es presta especial atenció a delinear com obtenir models d'ordre reduït susceptibles de estimació de paràmetres, però mantenint el significat biològic. L' estimació dels paràmetres del model a partir de les dades experimentals es considera en diferents escenaris, tant utilitzant models determinístics com estocàstics. Per al circuit I1-FFL es consideren models determinístics. La tesi planteja la utilització de models locals utilitzant la optimització multiobjectiu per realitzar l'estimació de parametres del model sota escenaris amb estructura de model incompleta (dinàmica no modelada). Per al circuit de QS/Fb, una estructura controlada per realimentació, el problema tractat és la manca d'excitabilitat dels senyals. La tesi proposa una metodologia de estimació en dues etapes utilitzant models estocàstics. La metodologia permet utilitzar dades de curs temporal promediats de la població i mesures de distribució en estat estacionari d'una sola una cèl·lula. L' ajust de circuits basat en models per aconseguir el rendiment desitjat dels circuits també s' aborda mitjançant la optimització multiobjectiu. Per al circuit QS/Fb, es fa un anàlisi estocàstic complet. La tesi aborda com tenir en compte correctament tant el soroll intrínsec com l' extrínsec, les dues principals fonts de soroll en els circuits genètics sintètics. S' analitza l'equilibri entre dues fonts de soroll i el paper que exerceixen en el bucle de realimentació intracel·lular, les i en la realimentació extracel·lular de tota la població. La principal conclusió es que la complexa interacció entre els dos canals de realimentació fa necessari l' ús de la optimització multiobjectiu per al adequat ajust del circuit. En aquesta tesi, a més de l'ús adequat d'eines d'optimització multiobjectiu, la principal preocupació és com derivar directives per al ajust in silico de paràmetres de circuits que puguin aplicar-se de forma realista en viu en un laboratori estàndard. Així, com a alternativa a l'anàlisi de sensibilitat de paràmetres clàssic, la tesi proposa l'ús de l' tècniques de l'agrupació al llarg dels fronts de Pareto, relacionant el compromís de dessempeny amb les regions en l'espai d'paràmetres.Synthetic biology is defined as the engineering of biology: the deliberate (re)design and construction of novel biological and biologically based parts, devices and systems to perform new functions for useful purposes, that draws on principles elucidated from biology and engineering. Methods and tools are needed to facilitate fast, reproducible and predictable construction of biological systems from sets of biological components. This thesis raises multi-objective optimization as the proper framework to deal with common problems arising in rational design and optimal tuning of synthetic gene circuits. Using a classical systems engineering approach, the thesis mainly addresses: i) synthetic gene circuit modeling based on first principles, ii) model parameters estimation from experimental data and iii) model-based tuning to achieve desired circuit performance. Two gene synthetic circuits of different nature and with different goals and inherent problems have been used throughout the thesis: an Incoherent type 1 feedforward circuit (I1-FFL) that exhibits the important biological property of adaptation, and a Quorum sensing/Feedback circuit (QS/Fb) comprising two intertwined feedback loops -an intracellular one and a cell-to-cell communication-based one-- designed to regulate the mean expression level of a protein of interest while minimizing its variance across the population of cells. Both circuits have been analyzed in silico and implemented in vivo. In both cases, circuit modeling based on first principles has been carried out. Then, special attention is paid to illustrate how to obtain reduced order models amenable for parameters estimation yet keeping biological significance. Model parameters estimation from experimental data is considered in different scenarios, both using deterministic and stochastic models. For the I1-FFL circuit, deterministic models are considered. In this case, the thesis raises ensemble modeling using multi-objective optimization to perform model parameters estimation under scenarios with incomplete model structure (unmodeled dynamics). For the QS/Fb gene circuit, a feedback controlled structure, the lack of excitability of the signals is the problem addressed. The thesis proposes a two-stage estimation methodology using stochastic models. The methodology allows using population averaged time-course data and steady state distribution measurements at the single-cell level. Model-based circuit tuning to achieve desired circuit performance is also addressed using multi-objective optimization. First, for the QS/Fb feedback control circuit, a complete stochastic analysis is performed. Here, the thesis addresses how to correctly take into account both intrinsic and extrinsic noise, the two main sources of noise in gene synthetic circuits. The trade-off between both sources of noise, and the role played by in the intracellular single-cell feedback loop and the extracellular population-wide feedback is analyzed. The main conclusion being that the complex interplay between both feedback channels compel the use of multi-objective optimization for proper tuning of the circuit to achieve desired performance. Thus, the thesis wraps up all the previous results and uses them to address circuit tuning for desired performance. Here, besides the proper use of multi-objective optimization tools, the main concern is how to derive guidelines for circuit parameters tuning in silico that can realistically be applied in vivo in a standard laboratory. Thus, as an alternative to classical parameters sensitivity analysis, the thesis proposes the use of clustering techniques along the optimal Pareto fronts relating the performance trade-offs with regions in the circuits parameters space.This work has been partially supported by the Spanish Government (CICYT DPI2014- 55276-C5-1) and the European Union (FEDER). The author was recipient of the grant Formación de Personal Investigador by the Universitat Politècnica de València, subprogram 1 (FPI/2013-3242). She was also recipient of the competitive grants for pre-doctoral stays Erasmus Student Placement-European Programme 2015, and FPI Mobility program 2016 of the Universitat Politècnica de València. She also received the competitive grant for a pre-doctoral stay Becas de movilidad para Jóvenes Profesores e Investigadores 2016, Programa de Becas Iberoamérica of the Santander Bank.Boada Acosta, YF. (2018). A systems engineering approach to model, tune and test synthetic gene circuits [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/112725TESI