The genomes of diverse organisms are predicted to contain 20 – 30% membrane protein encoding genes and more than half of all therapeutics target membrane proteins. However, only 2% of crystal structures deposited in the protein data bank represent integral membrane proteins. This reflects the difficulties in studying them using standard biochemical and crystallographic methods. The first problem frequently encountered when investigating membrane proteins is their low natural abundance, which is insufficient for biochemical and structural studies. The aim of my thesis was to provide a simple method to improve the production of recombinant proteins. One of the most commonly used methods to increase protein yields is codon optimization of the entire coding sequence. However, our data show that subtle synonymous codon substitutions in the 5’ region can be more efficient. This is consistent with the view that protein yields under normal conditions are more dependent on translation initiation than elongation. mRNA secondary structures around the 5’ region are in large part responsible for this effect although rare codons, as well as other factors, also contribute. We developed a PCR based method to optimize the 5’ region for increased protein production in Escherichia coli. For those proteins produced in sufficient quantities several additional hurdles remain before high quality crystals can be obtained. A second aim of my thesis work was to provide a simple method for topology mapping membrane proteins. A topology map provides information about the orientation of transmembrane regions and the location of protein domains in relation to the membrane, which can give information on structure-function relationships. To this end we explored the split-GFP system in which GFP is split between the 10th and 11th β-strands. This results in one large and one small fragment, both of which are non-fluorescent but can re-anneal and regain fluorescence if localized to the same cellular compartment. Fusing the 11th β-strand to the termini of a protein of interest and expressing it, followed by expression of the detector fragment in the cytosol, allows determination of the topology of inner membrane proteins. Using this strategy the topology of three model proteins was correctly determined. We believe that this system could be used to predict the topology of a large number of additional proteins, especially single-spanning inner membrane proteins in E. coli. The methods for efficient protein production and topology mapping engineered during my thesis work are simple and cost-efficient and may be very valuable in future studies of membrane proteins. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

Recombinant DNA technology has been commonly used in a number of fields to synthesize new products or generate products with a new pathway. Conventional cloning methods are expensive and require significant time and labor; λ-PCR, a new cloning method developed in the Senger lab, has a number of advantages compared to other cloning processes due to its employment of relatively inexpensive and widely available materials and time-efficiency. While the amount of lab work required for the cloning process is minimal, the importance of accurate primer design cannot be overstated. The target of this study was to create an effective procedure for λ-PCR primer design that ensures accurate cloning reactions. Additionally, synthetic ribosome binding sites (RBS) were included in the primer designs to test heterologous protein expression of the cyan fluorescent reporter with different RBS strengths. These RBS sequences were designed with an online tool, the RBS Calculator. A chimeric primer design procedure for λ-PCR was developed and shown to effectively create primers used for accurate cloning with λ-PCR; this method was used to design primers for CFP cloning in addition to two enzymes cloned in the Senger lab. A total of five strains of BL21(DE3) with pET28a + CFP were constructed, each with the same cyan fluorescent protein (CFP) reporter but different RBS sequences located directly upstream of the start codon of the CFP gene. Expression of the protein was measured using both whole-cell and cell-free systems to determine which system yields higher protein concentrations. A number of other factors were tested to optimize conditions for high protein expression, including: induction time, IPTG concentration, temperature, and media (for the cell-free experiments only). Additionally, expression for each synthetic RBS sequence was investigated to determine an accurate method for predicting protein translation. NUPACK and the Salis Lab RBS Calculator were both used to evaluate the effects of these different synthetic RBS sequences. The results of the plate reader experiments with the 5 CFP strains revealed a number of factors to be statistically significant when predicting protein expression, including: IPTG concentration, induction time, and in the cell-free experiments, type of media. The whole-cell system consistently produced higher amounts of protein than the cell-free system. Lastly, contrasts between the CFP strains showed each strain's performance did not match the predictions from the RBS Calculator. Consequently, a new method for improving protein expression with synthetic RBS sequences was developed using relationships between Gibbs free energy of the RBS-rRNA complex and expression levels obtained through experimentation. Additionally, secondary structure present at the RBS in the mRNA transcript was modeled with strain expression since these structures cause deviations in the relationship between Gibbs free energy of the mRNA-rRNA complex and CFP expression. / Master of Science / Recombinant DNA technology has been used to genetically enhance organisms to produce greater amounts of a product already made by the organism or to make an organism synthesize a new product. Genes are commonly modified in organisms using cloning practices which typically involves inserting a target gene into a plasmid and transforming the plasmid into the organism of interest. A new cloning process developed in the Senger lab, λ-PCR, improves the cloning process compared to other methods due to its use of relatively inexpensive materials and high efficiency. A primary goal of this study was to develop a procedure for λ-PCR primer design that allows for accurate use of the cloning method. Additionally, this study investigated the use of synthetic ribosome binding sites to control and improve expression of proteins cloned into an organism. Ribosome binding sites are sequences located upstream of the gene that increase the molecule’s affinity for the rRNA sequence on the ribosome, bind to the ribosome just upstream of the beginning of the gene, and initiate expression of the gene. Tools have been developed that create synthetic ribosome binding sites designed to produce specific amounts of protein. For example, the tools can increase or decrease expression of a gene depending on the application. These tools, the Salis Lab RBS Calculator and NUPACK, were used to design and evaluate the effects of the synthetic ribosome binding sites. Additionally, a new method was created to design synthetic ribosome binding sites since the methods used during the design process yielded inaccuracies. Each strain of E. coli contained the same gene, a cyan fluorescent protein (CFP), but had different RBS sequences located upstream of the gene. Expression of CFP was controlled via induction, meaning the addition of a particular molecule, IPTG in this system, triggered expression of CFP. Each of the CFP strains were tested with a variety of v conditions in order to find the conditions most suitable for protein expression; the variables tested include: induction time, IPTG (inducer) concentration, and temperature. Media was also tested for the cell-free systems, meaning the strains were grown overnight for 18 hours and lysed, a process where the cell membrane is broken in order to utilize the cell’s components for protein expression; the cell lysate was resuspended in new media for the experiments. ANOVA and multiple linear regression revealed IPTG concentration, induction time, and media to be significant factors impacting protein expression. This analysis also showed each CFP strain did not perform as the RBS Calculator predicted. Modeling each strain’s CFP expression using the RBS-rRNA binding strengths and secondary structures present in the RBS allowed for the creation of a new model for predicting and designing RBS sequences.

[ES] Esta Tesis ha sido dedicada al modelado multiescala considerando al anfitrión celular para el análisis y ajuste de circuitos genéticos sintéticos para bioproducción. Los objetivos principales fueron: 1. El desarrollo de un modelo que considere el anfitrión celular de tamaño reducido enfocado para simulación y análisis. 2. El desarrollo de herramientas de programación para el modelado y la simulación, orientada a la biología sintética. 3. La implementación de un modelo multiescala que considere las escalas relevantes para la bioproducción (biorreactor, célula y circuito sintético). 4. El análisis del controlador antitético considerando las interacciones célula-circuito, como ejemplo de aplicación de las herramientas desarrolladas. 5. El desarrollo y la validación experimental de leyes de control robusto para biorreactores continuos. El trabajo presentado en esta Tesis cubre las tres escalas del proceso de bioproducción. La primera escala es el biorreactor: esta escala considera la dinámica macroscópica del sustrato y la biomasa, y como estas dinámica se conecta con el estado interno de las células. La segunda escala es la célula anfitriona: esta escala considera la dinámica interna de la célula y la competencia por los recursos limitados compartidos para la expresión de proteínas. La tercera escala es el circuito genético sintético: esta escala considera la dinámica de expresión de los circuitos sintéticos exógenos y la carga que inducen en la célula anfitriona. Por último, como <<cuarta>> escala, parte de la Tesis se ha dedicado a desarrollar herramientas de software para el modelado y la simulación. Este documento se divide en siete capítulos. El Capítulo 1 es una introducción general al trabajo de la Tesis y su justificación; también presenta un mapa visual de la Tesis y enumera las principales contribuciones. El Capítulo 2 muestra el desarrollo del modelo del anfitrión celular (los Capítulos 4 y 5 hacen uso de este modelo para sus simulaciones). El Capítulo 3 presenta OneModel: una herramienta de software desarrollada en la Tesis que facilita el modelado y la simulación en biología sintética, en particular, facilita el uso del modelo del anfitrión celular. El Capítulo 4 utiliza el modelo del anfitrión celular para montar el modelo multiescala que considera el biorreactor y analiza el título, la productividad y el rendimiento en la expresión de una proteína exógena. El Capítulo 5 analiza un circuito más complejo, el recientemente propuesto y muy citado controlador biomolecular antitético, utilizando el modelo del anfitrión celular. El Capítulo 6 muestra el diseño de estrategias de control no lineal que permiten controlar la concentración de biomasa en un biorreactor continuo de forma robusta. El Capítulo 7 resume y presenta las principales conclusiones de la Tesis. En el Apéndice A se muestra el desarrollo teórico del modelo del anfitrión celular. Esta Tesis destaca la importancia de estudiar la carga celular en los sistemas biológicos, ya que estos efectos son muy notables y generan interacciones entre circuitos aparentemente independientes. La Tesis proporciona herramientas para modelar, simular y diseñar circuitos genéticos sintéticos teniendo en cuenta estos efectos de carga y permite el desarrollo de modelos que conecten estos fenómenos en los circuitos genéticos sintéticos, que van desde la dinámica intracelular de la expresión génica hasta la dinámica macroscópica de la población de células dentro del biorreactor. / [CA] Aquesta Tesi tracta del modelat multiescala considerant l'amfitrió ce\lgem ular per a l'anàlisi i ajust de circuits genètics sintètics per a bioproducció. Els objectius principals van ser: 1. El desenvolupament d'un model de grandària reduïda que considere l'amfitrió ce\lgem ular, enfocat al seu ús en simulació i anàlisi. 2. El desenvolupament d'eines de programari per al modelatge i la simulació, orientada a la biologia sintètica. 3. La implementació d'un model multiescala que considere les escales rellevants per a la bioproducció (bioreactor, cè\lgem ula i circuit sintètic). 4. L'anàlisi del controlador antitètic considerant les interacciones cè\lgem ula-circuit, com a exemple d'aplicació de les eines desenvolupades. 5. El desenvolupament i la validació experimental de lleis de control robust per a bioreactors continus. El treball presentat en aquesta Tesi cobreix les tres escales del procés de bioproducció. La primera escala és el bioreactor: aquesta escala considera la dinàmica macroscòpica del substrat i la biomassa, i com aquestes dinàmiques es connecten amb l'estat intern de les cè\lgem ules. La segona escala és la cè\lgem ula amfitriona: aquesta escala considera la dinàmica interna de la cè\lgem ula i la competència pels recursos limitats compartits per a l'expressió de proteïnes. La tercera escala és la del circuit genètic sintètic: aquesta escala considera la dinàmica d'expressió de circuits sintètics exógens i la càrrega que indueixen en la cè\lgem ula amfitriona. Finalment, com a <<quarta>> escala, part de la Tesi s'ha dedicat a desenvolupar eines de programari per al modelatge i la simulació. Aquest document es divideix en set capítols. El Capítol 1 és una introducció general al treball de la Tesi i la seua justificació; també presenta un mapa visual de la Tesi i enumera les principals contribucions. El Capítol 2 mostra el desenvolupament del model de l'amfitrió ce\lgem ular (els Capítols 4 i 5 fan ús d'aquest model per a les seues simulacions). El Capítol 3 presenta OneModel: una eina de programari desenvolupada en la Tesi que facilita el modelatge i la simulació en biologia sintètica, en particular, facilita l'ús del model de l'amfitrió ce\lgem ular. El Capítol 4 utilitza el model de l'amfitrió ce\lgem ular per a muntar el model multiescala que considera el bioreactor i analitza el títol, la productivitat i el rendiment en l'expressió d'una proteïna exògena. El Capítol 5 analitza un circuit més complex, el recentment proposat i molt citat controlador biomolecular antitètic, utilitzant el model de l'amfitrió ce\lgem ular. El Capítol 6 mostra el disseny d'estratègies de control no lineal que permeten controlar la concentració de biomassa en un bioreactor continu de manera robusta. El Capítol 7 resumeix i presenta les principals conclusions de la Tesi. En l'Apèndix A es mostra el desenvolupament teòric del model de l'amfitrió ce\lgem ular. Aquesta Tesi destaca la importància d'estudiar la càrrega ce\lgem ular en els sistemes biològics, ja que aquests efectes són molt notables i generen interaccions entre circuits aparentment independents. La Tesi proporciona eines per a modelar, simular i dissenyar circuits genètics sintètics tenint en compte aquests efectes de càrrega i permet el desenvolupament de models que connecten aquests fenòmens en els circuits genètics sintètics, que van des de la dinàmica intrace\lgem ular de l'expressió gènica fins a la dinàmica macroscòpica de la població de cè\lgem ules dins del bioreactor. / [EN] This Thesis was devoted to the multi-scale host-aware analysis and tuning of synthetic gene circuits for bioproduction. The main objectives were: 1. The development of a reduced-size host-aware model for simulation and analysis purposes. 2. The development of a software toolbox for modeling and simulation, oriented to synthetic biology. 3. The implementation of a multi-scale model that considers the scales relevant to bioproduction (bioreactor, cell, and synthetic circuit). 4. The host-aware analysis of the antithetic controller, as an example of the application of the developed tools. 5. The development and experimental validation of robust control laws for continuous bioreactors. The work presented in this Thesis covers the three scales of the bioproduction process. The first scale is the bioreactor: this scale considers the macroscopic substrate and biomass dynamics and how these dynamics connect to the internal state of the cells. The second scale is the host cell: this scale considers the internal dynamics of the cell and the competition for limited shared resources for protein expression. The third scale is the synthetic genetic circuit: this scale considers the dynamics of expressing exogenous synthetic circuits and the burden they induce on the host cell. Finally, as a <<fourth>> scale, part of the Thesis was devoted to developing software tools for modeling and simulation. This document is divided into seven chapters. Chapter 1 is an overall introduction to the Thesis work and its justification; it also presents a visual map of the Thesis and lists the main contributions. Chapter 2 shows the development of the host-aware model (Chapters 4 and 5 make use of this model for their simulations). Chapter 3 presents OneModel: a software tool developed in the Thesis that facilitates modeling and simulation for synthetic biology---in particular, it facilitates the use of the host-aware model---. Chapter 4 uses the host-aware model to assemble the multi-scale model considering the bioreactor and analyzes the titer, productivity (rate), and yield in expressing an exogenous protein. Chapter 5 analyzes a more complex circuit, the recently proposed and highly cited antithetic biomolecular controller, using the host-aware model. Chapter 6 shows the design of nonlinear control strategies that allow controlling the concentration of biomass in a continuous bioreactor in a robust way. Chapter 7 summarizes and presents the main conclusions of the Thesis. Appendix A shows the theoretical development of the host-aware model. This Thesis emphasizes the importance of studying cell burden in biological systems since these effects are very noticeable and generate interactions between seemingly unconnected circuits. The Thesis provides tools to model, simulate and design synthetic genetic circuits taking into account these burden effects and allowing the development of models that connect phenomena in synthetic genetic circuits, ranging from the intracelullar dynamics of gene expression to the macroscopic dynamics of the population of cells inside the bioreactor. / This research was funded by MCIN/AEI/10.13039/501100011033 grant number PID2020-117271RB-C21, and MINECO/AEI, EU grant number DPI2017-82896- C2-1-R. The author was recipient of the grant “Programa para la Formación de Personal Investigador (FPI) de la Universitat Politècnica de València — Subprograma 1 (PAID-01-2017)”. The author was also a grantee of the predoctoral stay “Ayudas para Movilidad de Estudiantes de Doctorado de la Universitat Politècnica de València 2019”. The Control Theory and Systems Biology Lab of the ETH Zürich is acknowledged for accepting the author in their facilities as predoctoral stay and their valuable collaboration sharing knowledge. / Santos Navarro, FN. (2022). Multi-Scale Host-Aware Modeling for Analysis and Tuning of Synthetic Gene Circuits for Bioproduction [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/183473 / TESIS / Premios Extraordinarios de tesis doctorales