<p>Promising applications in the design of various biological systems hold critical implications as heralded in the rising field of synthetic biology. But, to achieve these goals, the ability to synthesize and screen in situ DNA constructs of any size or sequence rapidly, accurately and economically is crucial. Today, the process of DNA oligonucleotide synthesis has been automated but the overall development of gene and genome synthesis and error correction technology has far lagged behind that of gene and genome sequencing. What even lagged behind is the capability of screening a large population of information on a single cell, protein or gene level. Compartmentalization of single cells in water-in-oil emulsion droplets provides an opportunity to screen vast numbers of individual assays with quantitative readouts. However these single-emulsion droplets are incompatible with aqueous phase analysis and are not controllable through molecule transports. </p><p>This thesis presents the development of a multi-tool ensemble platform targeted at high-throughput gene synthesis, error correction and screening. An inkjet oligonucleotide synthesizer is constructed to synthesize oligonucleotides as sub-arrays onto patterned and functionalized thermoplastic microchips. The arrays are married to microfluidic wells that provide a chamber to for enzymatic amplification and assembly of the DNA from the microarrays into a larger construct. Harvested product is then amplified off-chip and error corrected using a mismatch endonuclease-based reaction. Bacterial cells baring individual synthetic gene variants are encapsulated as single cells into double-emulsion droplets where cell populations are enriched by up to 1000 times within several hours of proliferation. Permeation of Isopropyl-D-1-thiogalactopyranoside (IPTG) molecules from the external solution allows induction of target gene expression. The induced expression of the synthetic fluorescent proteins from at least ~100 bacteria per droplet generates clearly distinguishable fluorescent signals that enable droplets sorting through fluorescence-activated cell sorting (FACS) technique. The integration of oligo synthesis and gene assembly on the same microchip facilitates automation and miniaturization, which leads to cost reduction and increases in throughput. The capacity of double emulsion system (millions discrete compartments in 1ml solution) combined with high-throughput sorting by FACS provide the basis for screening complex gene libraries for different functionality and activity, significantly reducing the cost and turn-around time.</p> / Dissertation

<p>Promising applications in the design of various biological systems hold critical implications as heralded in the rising field of synthetic biology. But, to achieve these goals, the ability to synthesize in situ DNA constructs of any size or sequence rapidly, accurately and economically is crucial. Today, the process of DNA oligonucleotide synthesis has been automated but the overall development of gene and genome synthesis technology has far lagged behind that of gene and genome sequencing. This has meant that numerous ideas go unfulfilled due to scale, cost and impediments in the quality of DNA due to synthesis errors. </p><p>This thesis presents the development of a multi-tool ensemble platform targeted at gene synthesis. An inkjet oligonucleotide synthesizer is constructed to synthesize DNA microarrays onto silica functionalized cylic olefin copolymer substrates. The arrays are married to microfluidic wells that provide a chamber to for enzymatic amplification and assembly of the DNA from the microarrays into a larger construct. Harvested product is then amplified off-chip and error corrected using a mismatch endonuclease-based reaction. This platform has the potential to be particularly low-cost since it employs standard phosphoramidite reagents and parts that are cheaper than optical and electrochemical systems. Genes sized 160 bp to 993 bp were successfully harvested and, after error correction, achieved up to 94% of intended functionality.</p> / Dissertation

Cytochrome P450 3A4 is integral membrane protein residing in endoplasmic reticular membrane. In human the highest concentration cytochrome P450 3A4 is expressed in liver, where it plays a major role in metabolism of many drugs and xenobiotics. The main aim of the thesis was to evaluate the effect of gene optimization on heterologous expression of human cytochrome P450 3A4. At first expression constructs based on vectors pET22b were prepared. Then the efficiency of heterologous expression of optimized vs. natural gene sequence encoding truncated form of the human cytochrome P450 3A4 compared. The results show that the gen sequence optimized for E. coli strains K12 was expressed in significantly higher efficiency than the original human gene based on cDNA sequence. Another aim was to evaluate the suitability of pET22b based expression vectors for recombinant production of native (complete) form of human cytochrome P450 3A4. The amount of native form of the protein found in bacterial membrane was however substantially lower then that of the truncated form. Keywords: cytochrome P450 3A4, heterologous expression, pET22b, gene synthesis

Breakthroughs in molecular and synthetic biology through de novo gene synthesis are stimulating new vaccines, pharmaceutical applications, and functionalized biomaterials, and advancing the knowledge of the function of cells. This evolution in biological processing motivates the study of a class of manufacturing systems, defined here as genetic manufacturing systems, which produce a final product with a genetic construct. Genetic manufacturing systems rely on rare molecular events for success, resulting in waste and repeated work during the deoxyribonucleic acid (DNA) fabrication process. Inspection and real time monitoring strategies are possible as mitigation tools, but it is unclear if these techniques are cost efficient and value added for the successful creation of custom genetic constructs. This work investigates resource allocation strategies for DNA fabrication environments, with an emphasis on inspection allocation. The primary similarities and differences between traditional manufacturing systems and genetic manufacturing systems are described. A serial, multi-stage inspection allocation mathematical model is formulated for a genetic manufacturing system utilizing gene synthesis. Additionally, discrete event simulation is used to evaluate inspection strategies for a fragment synthesis process and multiple fragment assembly operation. Results from the mathematical model and discrete event simulation provide two approaches to determine the appropriate inspection strategies with respect to total cost or total flow time of the genetic manufacturing system. / Ph. D. / Breakthroughs in molecular and synthetic biology through <i>de novo</i> gene synthesis are stimulating new vaccines, pharmaceutical applications, and functionalized biomaterials, and advancing the knowledge of the function of cells. This evolution in biological processing motivates the study of a class of manufacturing systems, defined here as genetic manufacturing systems, which produce a final product with a genetic construct. Genetic manufacturing systems rely on rare molecular events for success, resulting in waste and repeated work during the deoxyribonucleic acid (DNA) fabrication process. Inspection and real time monitoring strategies are possible as mitigation tools, but it is unclear if these techniques are cost efficient and value added for the successful creation of custom genetic constructs. This work investigates resource allocation strategies for DNA fabrication environments, with an emphasis on inspection allocation. The primary similarities and differences between traditional manufacturing systems and genetic manufacturing systems are described. A serial, multi-stage inspection allocation mathematical model is formulated for a genetic manufacturing system utilizing gene synthesis. Additionally, discrete event simulation is used to evaluate inspection strategies for a fragment synthesis process and multiple fragment assembly operation. Results from the mathematical model and discrete event simulation provide two approaches to determine the appropriate inspection strategies with respect to total cost or total flow time of the genetic manufacturing system.

5 Abstract Cytochrome P450 1A1 (CYP1A1) is one of the major isoforms of the cytochrome P450 superfamily. It is primarily an extrahepatic enzyme which is responsible for oxidation of many polycyclic aromatic hydrocarbons and other xenobiotics. Besides of the role in detoxification metabolism CYP1A1 is the one most important isoform involved in activation of procarcinogens. The main aim of this project was preparation of two modifications of the rat CYP1A1 gene with codon optimization for expression in E. coli by gene synthesis. One was wild type (wt1A1) and the other was with modified N-terminal anchor (mod1A1) - for both modifications with or without His Tag at the C-end of CYP1A1. Furthermore, an aim was to compare their level of expression in different strains of E. coli and try to purify and assess enzymatic activity of the gene's products. From pre-prepared oligonucleotides 2 "syntons" (parts of gene) were synthetized and separately inserted into pUC19. After verified sequence of the "syntons" they were cleaved from pUC19 and inserted together into pET-22b. These vectors were prepared for transformation of 3 strains of E. coli (BL-21 (DE3) GOLD, RIL a RIPL). For production of proteins many conditions were tested: temperature (18, 22, 24, 27 a 37 řC), time of production (untill 48 hours), concentration...

Brinjal shoot and fruit borer (Leucinodes orbonalis Guenee) is a major limiting factor in commercial cultivation of eggplant in southeast Asia. Extensive use of pesticides as well as the conventional breeding methods have been ineffective in controlling the borer so there is a need for Integrated Pest Management (IPM) strategies for its control. Bacillus thuringiensis (Bt) is known to produce a variety of insecticidal crystal proteins toxic to lepidopteran, dipteran and coleopteran pests. The Cry2Aa protein has been found to be more toxic to brinjal shoot and fruit borer than Cry1Ab. My objective was to develop eggplant cultivars that express a codon-optimized cry2Aa gene, the sequence of which is based on that of an Indian isolate of Bt, with the eventual goal of producing fully resistant cultivars. The cry2Aa gene was modified for optimal expression in eggplant using the codon usage frequencies based on solanaceous sequences (eggplant, tomato and pepper). The GC content was increased from 34.3% in the native gene to 41.3% in the optimized gene, thus removing the AT-rich regions that are typical for Bt cry genes. Also, other mRNA destabilizing and hairpin forming structure sequences were removed. The gene was synthesized in four different parts with complementary restriction sites. A total of 152 oligonucleotides (oligos) was used to assemble the 1.9 kb gene using dual asymmetric (DA) and overlap extension (OE) PCR techniques. The individual parts were subsequently ligated using the complementary restriction sites and inserted into vector pCAMBIA 1302. Also, the transformation efficiency of 12 different eggplant cultivars was tested using plasmid pHB2892 to predict utility for transformation with the synthetic cry2Aa. / Master of Science

Avec l’apparition de plus en plus de souches de bactérie résistante aux antibiotiques, le développement de nouveaux antibiotiques est devenu une important problématique pour les agences de santé. C’est pour cela que la création de nouvelles plateformes pour accélérer la découverte de médicaments est devenu un besoin urgent. Dans les dernières décennies, la recherche était principalement orientée sur la modification de molécules préexistantes, la méta-analyse d’organismes produisant des molécules activent et l’analyse de librairies moléculaires pour trouver des molécules synthétiques activent, ce qui s’est avéré relativement inefficace. Notre but était donc de développer de nouvelles molécules avec des effets thérapeutiques de façon plus efficace à une fraction du prix et du temps comparé à ce qui se fait actuellement. Comme structure de base, nous avons utilisé des métabolites secondaires qui pouvaient altérer le fonctionnement des protéines ou l’interaction entre deux protéines. Pour générer ces molécules, j’ai concentré mes efforts sur les terpènes, une classe de métabolites secondaires qui possède un large éventail d’activités biologiques incluant des activités antibactériennes. Nous avons développé un système de chromosome artificiel de levure (YAC) qui permet à la fois l’assemblage directionnel et combinatoire de gènes qui permet la création de voies de biosynthèse artificielles. Comme preuve de concept, j’ai développé des YACs qui contiennent les gènes pour l’expression des enzymes impliquées dans la biosynthèse de la -carotène et de l’albaflavenone et produit ces molécules avec un haut rendement. Finalement, Des YACs produits à partir de librairies de gènes ont permis de créer une grande diversité de molécules. / With the appearance of more and more antibiotic resistant strains of bacteria, the development of new antibiotics becomes an issue of utmost importance for society. It is for that reason that new platforms and methodologies to accelerate the discovery of novel antibiotics are urgently needed. For the last decades, research was mainly oriented on modifying existing antibiotics, mining natural producers or screening for synthetic molecules from giant chemical libraries but these approaches did not manage to keep the pipelines filled with a sufficient number of novel antibiotics. Therefore, our goal was to develop a way to create and screen new molecules more efficiently at a fraction of the cost when compared to traditional approaches and within a short time frame. As chemical scaffolds we use natural product-like compounds that modulate the function of individual proteins or of protein-protein interactions. To generate these compounds, I focused first on the terpene scaffold class, a class containing molecules with a wide range of biological activities and includes compounds with antibacterial activities. We developed a yeast artificial chromosome (YAC) platform that allows both directional and combinatorial assembly of biosynthetic genes that can be used to create artificial biosynthetic pathways. As a proof of principle, YACs were successfully assembled containing genes coding for enzymes involved in the biosynthesis of both B-carotene and albaflavenone, and that allowed high yield production of these compounds. Finally, YACs encoding terpene gene libraries were also created and which produced a diversity of terpenoid molecules.