京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23818号 / 医科博第139号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 萩原 正敏, 教授 藤渕 航, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Biosensors are devices that use biological components to detect important analytes. Biosensing systems have various applications in areas such as medicine, environmental monitoring, and process control. Classical biosensors are often based on bacteria or purified enzymes that have limitations on efficiency or stability. I have developed several new biosensors to overcome these disadvantages. Two preliminary biosensors were first created based on the extremely strong and specific interaction between biotin and (strept)avidin. Both biosensors showed high sensitivity and reliability for measuring biotin with detection limits of 50-1000 pg/ml and 20-100 ng/ml, respectively. Following these, a new biosensor was developed by coupling a mobile, functionalized microsurface with cell-free expression approaches. This biosensor demonstrated a dynamic range of 1- 100 ng/ml. In addition, I also explored the possibility of combining these biosensing systems with engineered living cells. By leveraging the tools of synthetic biology, a genetic circuit was designed, constructed, and inserted into bacteria for enhanced biotin biosynthesis in vivo. Upon induction, a 17-fold increase in biotin production was measured in the engineered cells in comparison to wild type cells using the biosensors created herein. These new biosensors, particularly the mobile biosensing modality, form a building block for advanced biosensing and drug delivery systems due to enhancements in mobility and specificity. In the future, these biosensing and cellular production systems could impact a range of fields ranging from biomedicine to environmental monitoring. / Master of Science

In metabolic engineering of prokaryotes, combinatorial approaches have developed recently that induce random genetic perturbations to achieve a desired cell phenotype. A screening strategy follows the randomized genetic manipulations to select strain(s) with the more optimal phenotype of interest. This screening strategy is often divided into two categories: (i) a growth competition assay and (ii) selection by high-throughput screening. The growth competition assay involves culturing strains together. The strain with the highest growth rate will ultimately dominate the culture. This strategy is ideal for selecting strain with cellular fitness (e.g., solvent tolerance), but it does not work for selecting a strain that can over-produce a product (e.g., an amino acid). For the case of selecting highly productive phenotypes, high-throughput screening is used. This method analyzes strains individually and is costly and time-consuming. In this research, a synthetic genetic circuit was developed to select highly productive phenotypes using a growth competition assay rather than high-throughput screening. This novel system is called Feed-back Inhibition of Transcription for Growth Selection (FITSelect), and it uses a natural feedback inhibition mechanism in the L-arginine production pathway to select strains (transformed with a random genomic library) that can over-produce L-arginine in E. coli DH10B. With FITSelect, the cell can thrive in the growth competition assay when L-arginine is over-produced (i.e., growth is tied to L-arginine production). Cell death or reduced growth results if L-arginine is not over-produced by the cell. This system was created by including an L-arginine concentration responsive argF promoter to control a ccdB cell death gene in the FITSelect system. The effects of ccdB were modulated by the antidote ccdA gene under control of an L-tryptophan responsive trp promoter. Several insights and construction strategies were required to build a system that ties the growth rate of the cell to L-arginine concentrations. / Master of Science

Nuestro conocimiento de las interacciones moleculares nos ha conducido hoy hacia una perspectiva ingenieril, donde diseños e implementaciones de sistemas artificiales de regulación intentan proporcionar instrucciones fundamentales para la reprogramación celular. Nosotros aquí abordamos el diseño de redes de genes como una forma de profundizar en la comprensión de las regulaciones naturales. También abordamos el problema de la diseñabilidad dada una genoteca de elementos compatibles. Con este fin, aplicamos métodos heuríticos de optimización que implementan rutinas para resolver problemas inversos, así como herramientas de análisis matemático para estudiar la dinámica de la expresión genética. Debido a que la ingeniería de redes de transcripción se ha basado principalmente en el ensamblaje de unos pocos elementos regulatorios usando principios de diseño racional, desarrollamos un marco de diseño computacional para explotar este enfoque. Modelos asociados a genotecas fueron examinados para descubrir el espacio genotípico asociado a un cierto fenotipo. Además, desarrollamos un procedimiento completamente automatizado para diseñar moleculas de ARN no codificante con capacidad regulatoria, basándonos en un modelo fisicoquímico y aprovechando la regulación alostérica. Los circuitos de ARN resultantes implementaban un mecanismo de control post-transcripcional para la expresión de proteínas que podía ser combinado con elementos transcripcionales. También aplicamos los métodos heurísticos para analizar la diseñabilidad de rutas metabólicas. Ciertamente, los métodos de diseño computacional pueden al mismo tiempo aprender de los mecanismos naturales con el fin de explotar sus principios fundamentales. Así, los estudios de estos sistemas nos permiten profundizar en la ingeniería genética. De relevancia, el control integral y las regulaciones incoherentes son estrategias generales que los organismos emplean y que aquí analizamos. / Rodrigo Tarrega, G. (2011). Computational design and designability of gene regulatory networks [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/14179

The post-genomics era has put at the disposal of modern plant breeders an endless list of genetic building blocks for the design of new biotechnological crops. After a first wave of single-gene transgenic with controversial public acceptance, genomic information and technology is paving the way for increasingly complex designs based in multiple gene engineering. Those designs aiming at the production of inexpensive health-promoting compounds are most likely to be welcomed by consumers. In this project we plan to develop new multigene assembling tools. During this PhD, a standardized collection of interchangeable genetic parts (including promoters, CDS, P-DNAs, etc) and vectors will be developed. The collection, inspired in Synthetic Biology standards, will be made easy-to-assemble in an interchangeable, semi-idempotent and seamless fashion by the addition of flanking recognition sites of type IIS Restriction endonucleases. The construction of the collection will facilitate multigene engineering and will constitute a first step towards enabling Synthetic Biology in plants. / Sarrión Perdigones, MA. (2014). Design and development of modular DNA assembly tools for Multigene Engineering and Synthetic Biology in Plants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/35399

Plant hormones and their gene regulatory networks orchestrate a diverse array of metabolic and physiological changes crucial for growth, development, and environmental responses. Targeting the engineering of hormone signaling networks holds promise for enhancing plant health, crop productivity, and vigor. However, these networks are intricate, featuring negative feedback loops, extensive interconnections between pathways, pleiotropy, and overlapping gene expression. These complexities pose challenges in identifying candidate genes and parsing apart their isolated functions that could be strategically engineered to achieve desired plant phenotypes. Integration of comparative evolution, synthetic biology, and expression analysis facilitates the deconstruction of these networks. Through systems biology approaches data dimensionality can be reduced, enabling the attribution of specific phenotypes to associated genes. Here, I reviewed how the employment of these above-mentioned approaches can aid in the identification of candidate genes involved the regulation of growth and development within specific tissues, and how through synthetic biology we can explore the sequence-function space of candidate genes and their pathway modules. Candidate genes identified through this process can be evaluated through comparative evolutionary approaches, and efficiently tested in synthetic systems for engineering of their molecular functionalities in a high-throughput manner. Here, as a case study, I employ a systems biology approach to identify tissue-specific candidate genes within the auxin regulatory network in soybean shoot development. This method aims to minimize pleiotropy and off-target effects by utilizing expression analysis tissue-specificity score and principal component analysis. I primarily, focused on three pivotal components of the nuclear auxin signaling pathway: Aux/IAA transcriptional repressors, ARF transcription factors, and TIR1/AFB auxin receptors. These components collectively modulate auxin signaling, influencing various growth and environmental responses. I identified genes within the three pivotal components of auxin signaling involved in early shoot architecture development, which has advantages from weed suppression to yield in soybean cultivation. I used a yeast chassis to investigate the function of pleiotropic auxin receptors, which primarily regulate Aux/IAA levels and orchestrate transcriptional changes in response to auxin. I explored whether these receptors modulate auxin response in a concerted fashion, as they are generally not tissue specific. Here, I reported that auxin receptors interact in an epistatic manner to modulate auxin response. This case of study serves as a foundation in engineering plant genotype-phenotype via auxin signaling. / Doctor of Philosophy / Plant hormones are essential for controlling various processes that drive plant growth, development, and responses to the environment. Scientists are exploring ways to engineer the networks that regulate these hormones to improve plant health, boost crop yields, and enhance plant strength. However, these networks are complex, with many interacting parts, making it difficult to identify which genes to modify to achieve specific outcomes in plants. To tackle this challenge, researchers use a combination of approaches, including studying how these networks have evolved, analyzing large amounts of biological data, and using synthetic biology to test and refine their findings. By breaking down the complexity of these networks, they can link specific genes to particular plant traits. Once these genes–trait links are identified, they can be further tested and engineered to optimize plant characteristics. In this study, I focused on the auxin hormone, which plays a key role in numerous aspects of plant growth including soybean shoot development and Arabidopsis root development. I looked at three main components of the auxin regulatory network: Aux/IAA proteins (which act as repressors), ARF transcription factors (which control gene expression), and TIR1/AFB receptors (which detect auxin levels). These components work together to regulate how plants grow and respond to their environment. I identified key genes within these main auxin components that are important for early development of soybean shoots and minimizes off-target effects. This can help improve soybean farming by enhancing weed control and enhancing crop yields Using a synthetic biology yeast system, I studied the function of TIR1/AFB auxin receptors and how this family of receptors interact to perceive auxin and control the levels of Aux/IAA proteins, consequently controlling the plant's growth in response to auxin. I found that auxin receptors work in concert in a way that reduces their overall effect on the plants response to auxin. This research lays the groundwork for future efforts to engineer plant traits by modifying the auxin signaling pathway, which could lead to improved crop performance and resilience.

Yes / Marrying synthetic biology with synthetic chemistry provides a powerful approach toward natural product diversification, combining the best of both worlds: expediency and synthetic capability of biogenic pathways and chemical diversity enabled by organic synthesis. Biosynthetic pathway engineering can be employed to insert a chemically orthogonal tag into a complex natural scaffold affording the possibility of site-selective modification without employing protecting group strategies. Here we show that, by installing a sufficiently reactive handle (e.g., a C–Br bond) and developing compatible mild aqueous chemistries, synchronous biosynthesis of the tagged metabolite and its subsequent chemical modification in living culture can be achieved. This approach can potentially enable many new applications: for example, assay of directed evolution of enzymes catalyzing halo-metabolite biosynthesis in living cells or generating and following the fate of tagged metabolites and biomolecules in living systems. We report synthetic biological access to new-to-nature bromo-metabolites and the concomitant biorthogonal cross-coupling of halo-metabolites in living cultures. / European Research Council under the European Union’s Seventh Framework Programme (FP7/2007–2013/ERC consolidator grant GCGXC grant agreement no 614779) and ERAIB (Grant no. 031A338A) and H2020-MSCA-IF-2014 Grant no. 659399

Synthetic biology is a rapidly growing field that aims to treat cellular biological networks in an analogous way to electrical circuits. However, the field of plant synthetic biology has not grown at the same pace as bacterial and yeast synthetic biology, leaving a dearth of characterized tools for the community. Due to the need for tools for the synthetic plant biologist, I have endeavored to create a library of well-characterized TATA box variants in the cauliflower mosaic virus (CaMV) 35S promoter using the standardized assembly method Golden Braid 2.0. I introduced single nucleotide changes in the TATA box of the CaMV 35S promoter, a genetic part widely used in plant gene expression studies and agricultural biotechnology. Using a dual-luciferase reporter system, I quantified the transcriptional strength of the altered TATA box sequences and compared to the wild-type sequence, both in transient protoplast assays and stable transgenic Arabidopsis thaliana plants. The library of TATA-box modified CaMV 35S promoters with varying transcriptional strengths created here can provide the plant synthetic biology community with a series of modular Golden Braid-adapted genetic parts that can be used dependably and reproducibly by researchers to fine-tune gene expression levels in complex, yet predictable, synthetic genetic circuits.

Yes / This paper proposes a formal methodology to analyse bio-systems, in particular synthetic biology systems. An integrative analysis perspective combining different model checking approaches based on different property categories is provided. The methodology is applied to the synthetic pulse generator system and several verification experiments are carried out to demonstrate the use of our approach to formally analyse various aspects of synthetic biology systems. / EPSRC

This thesis shows advancements towards the development of engineered bacteria for sensing and responding to environmental pollutants by exploring the use of UDP-glycosyltransferases (UGTs) for their metabolism of toxins, along with the use of engineered tetracycline repressor protein (TetR) based transcriptional regulators as sensors for environmental toxins. The importance and applicability of UGTs as well as the adaptability of TetR systems for future developments are shown through a function-based review of UGTs, the development of high-throughput fluorescent UGT assay technique, and the creation of novel TetR transcription regulatory sequences. The assays effectively measured UGT71G1 activity based on the presence of reaction byproducts, leading to the identification of several new substrates including the toxin bisphenol A. Next, hybrid TetRs were assembled from complementary DNA-binding and ligand-binding domains of TetR homologs. The ability to interchange these domains while retaining their function multiplies the unique TetR systems available for use in cellular systems. In future, novel TetR and UGT71G1 systems may be developed to detect and respond to substrates like bisphenol A.