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.

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.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25172号 / 医博第5058号 / 新制||医||1071(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村川 泰裕, 教授 竹内 理, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Production of mutant biological molecules for understanding biological principles or as therapeutic agents has gained considerable interest recently. Synthetic genes are today being widely used for production of such molecules due to the substantial decrease in the costs associated with gene synthesis technology. Along one such line, we have engineered tRNA genes in order to dissect the effects of G:U base-pairs on the accuracy of the protein translation machinery. Our results provide greater detail into the thermodynamic interactions between tRNA molecules and an Elongation Factor protein (termed EF-Tu in bacteria and eEF1A in eukaryotes) and how these interactions influence the delivery of aminoacylated tRNAs to the ribosome. We anticipate that our studies not only shed light on the basic mechanisms of molecular machines but may also help us to develop therapeutic or novel proteins that contain unnatural amino acids. Further, the manipulation of the translation machinery holds promise for the development of new methods to understand the origins of life. Along another line, we have used the power of synthetic biology to experimentally validate an evolutionary model. We exploited the functional diversity contained within the EF-Tu/eEF1A gene family to experimentally validate the model of evolution termed ‘heterotachy’. Heterotachy refers to a switch in a site’s mutational rate class. For instance, a site in a protein sequence may be invariant across all bacterial homologs while that same site may be highly variable across eukaryotic homologs. Such patterns imply that the selective constraints acting on this site differs between bacteria and eukaryotes. Despite intense efforts and large interest in understanding these patterns, no studies have experimentally validated these concepts until now. In the present study, we analyzed EF-Tu/eEF1A gene family members between bacteria and eukaryotes to identify heterotachous patterns (also called Type-I functional divergence). We applied statistical tests to identify sites possibly responsible for biomolecular functional divergence between EF-Tu and eEF1A. We then synthesized protein variants in the laboratory to validate our computational predictions. The results demonstrate for the first time that the identification of heterotachous sites can be specifically implicated in functional divergence among homologous proteins. In total, this work supports an evolutionary synthetic biology paradigm that in one direction uses synthetic molecules to better understand the mechanisms and constraints governing biomolecular behavior while in another direction uses principles of molecular sequence evolution to generate novel biomolecules that have utility for industry and/or biomedicine.

The objective of the field of Synthetic Biology is to implement novel functionalities in a biological context or redesign existing biological systems. To achieve this, it employs tried and tested engineering principles, such as standardisation and the design-build-test cycle. A crucial part of this process is the convergence of modelling and experiment. The aim of this thesis is to improve the design principles employed by Synthetic Biology in the context of Gene Regulatory Networks (GRNs). Small Ribonucleic Acids (sRNAs), in particular, are focussed on as a mechanism for post-transcriptional expression regulation, as they present great potential as a tool to be harnessed in GRNs. Modelling sRNA regulation and its interaction with its associated chaperone Host-Factor of Bacteriophage Qβ (Hfq) is investigated. Inclusion of Hfq is found to be necessary in stochastic models, but not in deterministic models. Secondly, feedback is core to the thesis, as it presents a means to scale-up designed systems. A linear design framework for GRNs is then presented, focussing on Transcription Factor (TF) interactions. Such frameworks are powerful as they facilitate the design of feedback. The framework supplies a block diagram methodology for visualisation and analysis of the designed circuit. In this context, phase lead and lag controllers, well-known in the context of Control Engineering, are presented as genetic motifs. A design example, employing the genetic phase lag controller, is then presented, demonstrating how the developed framework can be used to design a genetic circuit. The framework is then extended to include sRNA regulation. Four GRNs, demonstrating the simplest forms of genetic feedback, are then modelled and implemented. The feedback occurs at three different levels: autoregulation, through an sRNA and through another TF. The models of these GRNs are inspired by the implemented biological topologies, focussing on steady state behaviour and various setups. Both deterministic and stochastic models are studied. Dynamic responses of the circuits are also briefly compared. Data is presented, showing good qualitative agreement between models and experiment. Both culture level data and cell population data is presented. The latter of these is particularly useful as the moments of the distributions can be calculated and compared to results from stochastic simulation. The fit of a deterministic model to data is attempted, which results in a suggested extension of the model. The conclusion summarises the thesis, stating that modelling and experiment are in good qualitative agreement. The required next step is to be able to predict behaviour quantitatively.

Synthetic biology is a relatively new discipline within the field of biotechnologies. In essence it is the artificial creation of microorganisms. Though similar in principle, it differs from genetic engineering because it creates an organism from scratch, rather than cutting and pasting DNA (deoxyribonucleic acid) between existing organisms. This study investigates the ethical aspects (both rational and theological) concerned with synthetic biology through the use of a literature analysis. The study starts by investigating and describing the origins, pioneers, science and uses of synthetic biology. Secondly, it describes and ethically assesses the rational arguments for and against synthetic biology by comparing its benefits and risks. Lastly, the study describes and ethically assesses synthetic biology within the Reformed tradition, mainly by using the creational perspective of Christian ethical evaluations (including concepts such as creatio ex nihilio; creatio continua and imago Dei) and secondary the re-creational and eschatological perspectives. The final conclusion reached shows that synthetic biology is acceptable from a Reformed theological-ethical perspective, because humans as the image of God can create, just as God constantly creates new things and created a new universe from nothing. The rational arguments state that the potential benefits of synthetic biology surpass the risks it poses. Hence, it supports the idea that synthetic biology can be used to fulfil God’s commandment to love one’s neighbour, by improving his circumstances and activating hope. Nevertheless, Christians should always stay vigilant about motives and possible uses when dealing with new technologies. How and for what synthetic biology is used should in the future be constantly reviewed. In this way Christian scientists can still inquire about their work: Does it glorify God? / MA (Ethics), North-West University, Potchefstroom Campus, 2015

Synthetic biology is a relatively new discipline within the field of biotechnologies. In essence it is the artificial creation of microorganisms. Though similar in principle, it differs from genetic engineering because it creates an organism from scratch, rather than cutting and pasting DNA (deoxyribonucleic acid) between existing organisms. This study investigates the ethical aspects (both rational and theological) concerned with synthetic biology through the use of a literature analysis. The study starts by investigating and describing the origins, pioneers, science and uses of synthetic biology. Secondly, it describes and ethically assesses the rational arguments for and against synthetic biology by comparing its benefits and risks. Lastly, the study describes and ethically assesses synthetic biology within the Reformed tradition, mainly by using the creational perspective of Christian ethical evaluations (including concepts such as creatio ex nihilio; creatio continua and imago Dei) and secondary the re-creational and eschatological perspectives. The final conclusion reached shows that synthetic biology is acceptable from a Reformed theological-ethical perspective, because humans as the image of God can create, just as God constantly creates new things and created a new universe from nothing. The rational arguments state that the potential benefits of synthetic biology surpass the risks it poses. Hence, it supports the idea that synthetic biology can be used to fulfil God’s commandment to love one’s neighbour, by improving his circumstances and activating hope. Nevertheless, Christians should always stay vigilant about motives and possible uses when dealing with new technologies. How and for what synthetic biology is used should in the future be constantly reviewed. In this way Christian scientists can still inquire about their work: Does it glorify God? / MA (Ethics), North-West University, Potchefstroom Campus, 2015

Bacteria use Two Component Systems (TCS) to sense and respond to changes in their external environment. TCS are used to navigate to nutrients or away from toxins (chemotaxis) and to adapt to changes in osmolarity (osomosensing). TCS are composed of a histidine protein kinase (HPK) which trans-autophosphorylates in response to environmental change, transferring the phosphoryl group to a cognate response regulator (RR). Phosphorylated RRs modulate an output response such as protein-protein interaction for chemotaxis, and transcription for osmosensing. RRs are composed of a conserved amino terminal REC domain, and where present a variable effector domain. CheY, the chemotaxis RR, contains only a REC domain, whilst OmpR, the osmosensing RR, also contains a DNA binding effector domain. Recently, TCS have been used in synthetic biology applications due to their modularity and conserved signalling mechanism. This thesis aimed to investigate whether it was possible to design a synthetic TCS composed of fused chemotaxis and osmosensing components. Synthetic RRs were designed, fusing the highly conserved REC domains of CheY and OmpR upstream of the OmpR effector domain. REC domains were fused across the α₄-β₅-α₅ region, a region which transmits REC domain phosphorylation into effector domain activation. Synthetic RRs were designed to undergo phosphotransfer to their fused REC domains from the chemotaxis HPK, CheA, activate the attached OmpR effector domain and bind promoter DNA. Four chimeric RRs were created, although only three were structurally viable; F2, F3 and F4. Each fusion bound CheA, and F3 and F4 bound CheA with a significantly higher affinity than CheY. The chimeric RRs could all be phosphorylated byCheA-P; F4 and F3 were phosphorylated to wild-type levels. DNA binding affinitywas investigated with fluorescence anisotropy, hosphorylated and unphosphorylated F3 could not bind promoter DNA. F2 bound promoter DNA regardless of phosphorylation state. These data indicate that phosphorylation of the F2 REC domain does not lead to activation of the effector domain. F2 is likely to be constitutively active suggesting a previously unknown role for OmpR α₅ as a mediator of effector domain activation. Furthermore, using a simple fusion approach to design RRs is not a viable method to create a synthetic TCS with a controllable output.

Synthetic biology views a complex biological system as an ensemble in the hierarchy of parts, devices, systems, and networks. The practice of using engineering rules such as decoupling and standardization to understand, predict, and re-build novel biological functions from model-driven designed genetic circuits is emphasized. It is one of the top ten technologies that could help solving the current and potential risks in human society. Cyanobacteria have been considered as a promising biological system in conducting oxygenic photosynthesis to convert solar energy into reducing power, which drives biochemical reactions to assimilate and generate chemicals for a specific purpose such as CO2 fixation, N2 fixation, bioremediation, or fuels production. The promoter is a key biological part to construct feedback loops in genetic circuits for a desired biological function. In this thesis, promoters that don't work in the cyanobacterium Synechocystis PCC 6803 in terms of promoter strength, and dynamic range of gene regulation are identified. Biological parts, such as ribosome binding sites, and reporter genes with and without protease tags were also characterized with the home-built broad-host-range BioBrick shuttle vector pPMQAK1. The strong L03 promoter, which can be tightly regulated in a wide dynamic range by the foreign Tet repressor, was created through an iterative promoter engineering cycle. The iteration cycle of DNA breathing dynamic simulations and quantification of a reporting signal at a single-cell level should guide through the engineering process of making promoters with intended regulatory properties. This thesis is an essential step in creating functional promoters and it could be applied to create more diverse promoters to realize the emphasized practices of synthetic biology to build synthetic cyanobacteria for direct fuel production and CO2 assimilation.