Systems biology is a rapidly growing field which seeks a refined quantitative understanding of organisms, particularly studying how molecular species such as metabolites, proteins and genes interact in cells to form the complex emerging behaviour exhibited by living systems. Synthetic biology is a related and emerging field which seeks to engineer new organisms for practical purposes. Both fields can benefit from formal languages for modelling, simulation and analysis. In systems biology there is however a trade-off in the landscape of existing formal languages: some are modular but may be difficult for some biologists to understand (e.g. process calculi) while others are more intuitive but monolithic (e.g. rule-based languages). The first major contribution of this thesis is to bridge this gap with a Language for Biochemical Systems (LBS). LBS is based on the modular Calculus of Biochemical Systems and adds e.g. parameterised modules with subtyping and a notion of nondeterminism for handling combinatorial explosion. LBS can also incorporate other rule-based languages such as Kappa, hence adding modularity to these. Modularity is important for a rational structuring of models but can also be exploited in analysis as is shown for the specific case of Petri net flows. On the synthetic biology side, none of the few existing dedicated languages allow for a high-level description of designs that can be automatically translated into DNA sequences for implementation in living cells. The second major contribution of this thesis is exactly such a language for Genetic Engineering of Cells (GEC). GEC exploits the recent advent of standard genetic parts (“biobricks”) and allows for the composition of such parts into genes in a modular and abstract manner using logical constraints. GEC programs can then be translated to DNA sequences using a constraint satisfaction engine based on a given database of genetic parts.

The ability to control expression of a gene of interest is an important tool of molecular biologists and genetic engineers. This allows the phenotype associated with the regulated gene or genetic pathway to be partially de-coupled from the genotype and expressed only under condition that lend to induction of the genetic control system employed. Such control is typically implemented through a repressor protein (Eg. TetR, LacI) which will repress transcription when bound to a promoter containing a binding site (operator) recognized specifically by that repressor. Many such repressors and their cognate promoters are well-defined and characterized in model genetic systems, such as Escherichia coli, and may function poorly in other bacterial species. A lack of genetic components that allow the controlled expression of heterologous genes in less well studied bacterial species may limit their bio-industrial potential and the sophistication of engineered phenotypes. The work presented here uses random mutagenesis and selection to isolate mutants of TetR that are inducible by increased culture temperature. Induction of protein expression by temperature change can have benefits over repressors that require small-molecule inducers in bio-industrial applications as reversal of induction and reuse of growth medium are possible. The host range of these, or any, repressor protein is limited by the host range in which its cognate promoter will function. To bypass this limitation and allow use of TetR in Francisella novicida, a method was developed by which TetR-responsive promoters that function in this host could be selected from random DNA sequence flanking the TetR binding site, tetO. Many unique TetR-repressible promoters that function in Francisella were recovered and tightly-regulated expression of both exogenous reporter genes and host virulence genes were demonstrated. This promoter selection technique was also applied to E. coli, which allowed comparison between Francisella-selected promoters and those selected in an E. coli host. Adaption of this process for production of promoters responsive to transcription factors other than TetR would simply require the use of a different operator sequence, suggesting diverse applications for this technique. This success in promoter engineering should enable advances in synthetic biology and genetic engineering in non-model bacterial species. / Graduate

Thesis advisor: Welkin E. Johnson / Thesis advisor: Peter G. Clote / Synthetic biology currently is a rapidly emerging discipline, where innovative and interdisciplinary work has led to promising results. Synthetic design of RNA requires novel methods to study and analyze known functional molecules, as well as to generate design candidates that have a high likelihood of being functional. This thesis is primarily focused on the development of novel algorithms for the design of synthetic RNAs. Previous strategies, such as RNAinverse, NUPACK-DESIGN, etc. use heuristic methods, such as adaptive walk, ensemble defect optimization (a form of simulated annealing), genetic algorithms, etc. to generate sequences that minimize specific measures (probability of the target structure, ensemble defect). In contrast, our approach is to generate a large number of sequences whose minimum free energy structure is identical to the target design structure, and subsequently filter with respect to different criteria in order to select the most promising candidates for biochemical validation. In addition, our software must be made accessible and user-friendly, thus allowing researchers from different backgrounds to use our software in their work. Therefore, the work presented in this thesis concerns three areas: Create a potent, versatile and user friendly RNA inverse folding algorithm suitable for the specific requirements of each project, implement tools to analyze the properties that differentiate known functional RNA structures, and use these methods for synthetic design of de-novo functional RNA molecules. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

A terapia gênica consiste em introduzir uma sequência de nucleotídeos, codificante ou não-codificante, que tenha a capacidade de interferir na progressão de uma doença por ativação, inativação ou modulação da expressão de um gene alvo. Uma das rotas de transferência gênica é a ex vivo. Essa rota consiste em realizar a modificação gênica ex vivo de um tipo celular do organismo afetado, seguida do transplante celular no indivíduo para a correção do fenótipo. Dentre os vários vetores virais utilizados para transferência gênica, o sistema lentiviral é considerado um dos mais eficazes, visto que é capaz de manter altos níveis de expressão a longo prazo. Além disso, dentre os vetores virais que se integram no genoma, esse é considerado o mais seguro, tendo apresentado apenas um caso com relatado de efeito colateral sem reação adversa. Uma nova tecnologia utilizada para padronizar sistemas biológicos é a biologia sintética. Essa tecnologia pode ser utilizada como ferramenta para produzir e/ou otimizar sistemas de expressão para produção de proteínas de interesse terapêutico. O objetivo desse trabalho é gerar um vetor lentiviral sintético, para terapia gênica ex vivo, para reposição enzimática. Foram geradas quatro linhagens celulares humanas com produção transiente das proteínas terapêuticas Prot_Ctr e Prot_Mut sob controle dos promotores CMV e HeF1a. Para padronização do ensaio de transfecção, foram geradas duas linhagens celulares humanas que expressão da proteína fluorescente verde (GFP) sob controle dos mesmos promotores citados acima. Como controle negativo das linhagens produtoras da proteína terapêutica, foi gerada uma linhagem como vetor plasmidial vazio pLV_GTW_Mock. O nível de expressão do mRNA relativo às proteínas terapêuticas variou de 3.581,95 ± 1.322 a 10.377,18 ± 2.562 URE, não havendo diferenças significativas entre as linhagens produzidas (p > 0,05). O nível de produção da proteínas terapêuticas variou de 30,98 UI/mL a 241,1 UI/mL. A linhagem com maior produção dessa proteína foi a 293T/HeF1a_Prot_Mut, e essa diferença é significativa (p < 0,05). A partir da transfecção dos plasmídeos auxiliares e plasmídeo portador do cDNA que codifica a Prot-Mut na linhagem celular 293-T foi obtido o vetor lentiviral com título de 1,68x107 pLV/mL. Em conclusão, é possível gerar um vetor lentiviral funcional portador da Prot_Mut para utilização em terapia gênica ex vivo. Espera-se que o produto desse projeto de pesquisa gere uma patente. Para evitar a quebra de novidade, atividade inventiva e suficiência descritiva, requisitos mínimos de patenteabilidade, os resultados foram apresentados sem mencionar a doença e o gene em estudo / Gene therapy involves introducing a nucleotide sequence, coding or non-coding that can to interfere with the progression of a disease by activating, inactivating or modulating the expression of a target gene. One of the gene transfer routes is the ex vivo. This route consists in producing a ex vivo gene modification of a cellular kind of the affected organismo, following the transplant of those cells to correct the fenotype. Among the various viral vectors used for gene transfer, the lentiviral system is considered one of the most effective, since it is able to maintain high levels of long-term expression. Between the viral vectors that integrate into the genome, the lentiviral system is considered the safest, and It has only filed a case with reported side effect without adverse reaction. A new technology used to standardize biological systems is the synthetic biology. This technology can be used as a tool to produce and/or optimize expression systems for production of proteins of therapeutic interest. The aim of this study is to generate a synthetic lentiviral vector for ex vivo gene therapy, for enzyme replacement therapy. It were generated four human cell lines with transient production of therapeutic proteins Prot_Ctr and Prot_Mut under the control of CMV and HeF1a promoters. To standardize the transfection assay were generated two human cell lines expressing green fluorescent protein (GFP) under control of the same promoters cited above. As a negative control of the producing cell lines, It was generated a human cell line with an empty plasmid vector pLV_GTW_Mock. The level of mRNA expression relative to the therapeutic proteins ranged from 3581,95 ± 1322 to 10377,18 ± 2562 REU, there were no significant differences among the produced cell lines ( p > 0.05 ). The production level of therapeutic proteins ranged from 30.98 IU/mL to 241.1 IU/mL. The cell line with the highest production of the therapeutic protein was 93T/HeF1a_Prot_Mut+, and this difference is significant (p < 0.05 ). From the transfection of the plasmid containing the cDNA encoding the Prot_Mut and packing plasmid It was obtained lentiviral vector with the titer 1,68x107 pLV/mL. In conclusion, it is possible to generate a functional lentiviral vector carrying the Prot_Mut for use in ex vivo gene therapy. It is expected that the product of this research project generates a patent. To avoid breaking novelty, inventive activity and descriptive sufficiency, minimum requirements for patentability, the results were presented without mentioning the disease and the gene under study

Mycoplasma hominis est un pathogène humain opportuniste responsable d’infections génitales et néo-natales. Modifier génétiquement cette bactérie est nécessaire afin de comprendre les mécanismes de virulence et d’infection de ce pathogène. Il n’existe à ce jour aucun outil moléculaire efficace permettant de manipuler le génome de M. hominis, limitant les recherches sur sa pathogénicité et son métabolisme particulier reposant sur l’arginine. De nouvelles technologies rassemblées sous le terme de Biologie de Synthèse (BS) ont récemment émergé, offrant des perspectives inédites pour l’étude des mycoplasmes en permettant de modifier leurs génomes à grande échelle et de produire des souches mutantes. Ces travaux menés au J. Craig Venter Institute (JCVI, USA) ont montré que le génome de mycoplasmes apparentés pouvait être cloné et manipulé dans la levure avant d’être transplanté dans une cellule receveuse. La levure sert d’hôte d’accueil temporaire pour modifier le génome de la bactérie. Cette approche novatrice ouvre de nombreuses perspectives dans le cadre du développement de la génomique fonctionnelle chez les mycoplasmes pour lesquels les outils génétiques efficaces sont peu nombreux. Le but de cette thèse a été d’adapter pour la première fois certains outils de BS à M. hominis dans le but de créer des mutants déficients pour une fonction donnée. Pour cela, le génome de la souche type de M. hominis PG21 (665 kb) a été cloné dans la levure Saccharomyces cerevisiae par « Transformation-Associated Recombination cloning » (TAR-cloning). Deux clones (B3-2 et B3-4) de levure possédant le génome complet de M. hominis ont été validés par analyse en PCR simplex, PCR multiplex et électrophorèse en champs pulsé (PFGE). Ces clones levures ont ensuite été propagés en milieu sélectif durant 180 générations (30 passages), afin d’évaluer la stabilité du génome bactérien dans son hôte. Cette expérience a montré que (i) si la taille du génome de M. hominis ne variait pas au cours des premiers passages, elle diminuait progressivement à partir du dixième passage (≈60 générations), et que (ii) les zones du génome enrichies en séquence répétées étaient préférentiellement perdues. En tenant compte de ces résultats, le génome de M. hominis a été modifié chez le clone B3-4 par la technique « Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 » (CRISPR/Cas9) lors de passages précoces. Des clones de S. cerevisiae possédant un génome de M. hominis PG21 complet délété du gène vaa, codant une protéine d’adhésion majeure, ont été ainsi produits. La dernière étape de cette approche consistait à transplanter le génome modifié dans une cellule receveuse de M. hominis ou de Mycoplasma arthritidis, espèce phylogénétiquement la plus proche de M. hominis. Aucun protocole de transformation de M. hominis n’étant disponible au début de nos travaux, cette étape constituait un verrou majeur dans la mise en place des outils de BS chez cette espèce. Ce verrou a été en partie levé puisqu’une méthode de transformation de M. hominis basée sur du polyéthylène glycol (PEG) et mettant en jeu le plasposon pMT85 (plasmide contenant un transposon conférant la résistance à la tétracycline) a été mise au point au laboratoire. Cette technique de transformation, développée pour la souche de référence M. hominis M132 (745 kb) reste encore peu efficace ; elle est néanmoins reproductible et a permis d’obtenir des mutants d’intérêt de M. hominis. Le transformant n°28-2 a, ainsi, été muté dans le gène Mhom132_2390, codant le précurseur de la protéine P75, une adhésine putative de M. hominis. Le séquençage des génomes complets d’autres transformants a révélé l’insertion de multiples copies du transposon et la présence d’évènements de duplication et d’inversion de larges fragments d’ADN dans au moins deux génomes de M. hominis. / Mycoplasma hominis is an opportunistic human pathogen responsible for genital and neonatal infections. Genetically modifying this bacterium is necessary to understand the virulence and infection mechanisms of this pathogen. There is currently no effective molecular tool to engineer the genome of this bacterium, limiting research on its pathogenicity and its peculiar metabolism based on arginine.New technologies have recently emerged in the field of Synthetic Biology (BS), offering new perspectives for the study of mycoplasmas by allowing large scale genome modifications and the production of mutant strains. Work at the J. Craig Venter Institute (JCVI, USA) has shown that the genome of related mycoplasmas can be cloned and manipulated in yeast before being transplanted into a recipient cell. The yeast serves as a temporary host to modify the genome of the bacterium. This innovative approach opens many perspectives in the development of functional genomics in mycoplasmas for which there are few effective genetic tools. The goal of this thesis was to adapt a number of BS tools to M. hominis for the first time, in order to create mutants deficient for a given function. To achieve this goal, the genome of the M. hominis type strain PG21 (665 kb) was cloned into the yeast Saccharomyces cerevisiae by Transformation-Associated Recombination cloning (TAR-cloning). Two yeast clones (B3-2 and B3-4) possessing the complete genome of M. hominis were validated by simplex PCR, multiplex PCR and Pulsed Field Gel Electrophoresis (PFGE) analyses. These yeast clones were then propagated in a selective medium for 180 generations (30 passages) to evaluate the stability of the bacterial genome in its host. This experiment showed that (i) the size of the genome of M. hominis did not change during the first passages, it decreased progressively from the tenth passage (≈60 generations), and (ii) the enriched genome areas in repeated sequence were preferentially lost. Thus, the genome of M. hominis was modified in the B3-4 clone at early passages using the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) technology. Yeast clones with a complete M. hominis PG21 genome with a deleted vaa gene, encoding a major adhesion protein, were produced using this approach. The final step of this approach was to transplant the modified genome into a recipient cell of M. hominis or Mycoplasma arthritidis, the species phylogenetically closest to M. hominis. As no M. hominis transformation protocol was available at the beginning of our work, this step constituted a major obstacle in the implementation of BS tools in this species. This barrier has been partially lifted since a method of transformation of M. hominis based on polyethylene glycol (PEG) and involving the plasposon pMT85 (plasmid carrying a transposon conferring resistance to tetracycline) has been developed in the laboratory. This transformation technique, developed for the reference strain M. hominis M132 (745 kb) still remains not very efficient; it is nevertheless reproducible and allowed to obtain M. hominis mutants of interest. The Mhom132_2390 gene, encoding the precursor of the P75 protein, a putative adhesin of M. hominis, was effectively mutated in transformant No. 28-2. Complete genome sequencing of other transformants revealed the insertion of multiple copies of the transposon and the presence of duplication and inversion of large DNA fragments within at least two M. hominis genomes.In conclusion, this data has opened the way for the development and transposition of existing genetic modification approaches to M. hominis, previously considered as a genetically intractable bacterium.

One goal of synthetic biology is to design and build genetic circuits in living cells for a range of applications with implications in health, materials, and sensing. Computational design methodologies allow for increased performance and reliability of these circuits. Major challenges that remain include increasing the scalability and robustness of engineered biological systems and streamlining and automating the synthetic biology workflow of “specify-design-build-test.” I summarize the advances in microfluidic technology, particularly microfluidic large scale integration, that can be used to address the challenges facing each step of the synthetic biology workflow for genetic circuits. Microfluidic technologies allow precise control over the flow of biological content within microscale devices, and thus may provide more reliable and scalable construction of synthetic biological systems. However, adoption of microfluidics for synthetic biology has been slow due to the expert knowledge and equipment needed to fabricate and control devices. I present an end-to-end workflow for a computer-aided-design (CAD) tool, Fluigi, for designing microfluidic devices and for integrating biological Boolean genetic circuits with microfluidics. The workflow starts with a ``netlist" input describing the connectivity of microfluidic device to be designed, and proceeds through placement, routing, and design rule checking in a process analogous to electronic computer aided design (CAD). The output is an image of the device for printing as a mask for photolithography or for computer numerical control (CNC) machining. I also introduced a second workflow to allocate biological circuits to microfluidic devices and to generate the valve control scheme to enable biological computation on the device. I used the CAD workflow to generate 15 designs including gradient generators, rotary pumps, and devices for housing biological circuits. I fabricated two designs, a gradient generator with CNC machining and a device for computing a biological XOR function with multilayer soft lithography, and verified their functions with dye. My efforts here show a first end-to-end demonstration of an extensible and foundational microfluidic CAD tool from design concept to fabricated device. This work provides a platform that when completed will automatically synthesize high level functional and performance specifications into fully realized microfluidic hardware, control software, and synthetic biological wetware.

Predictable, robust genetic parts including constitutive promoters are one of the defining attributes of synthetic biology. Ideally, candidate promoters should cover a broad range of expression strengths and yield homogeneous output, whilst also being orthogonal to endogenous regulatory pathways. However, such libraries are not always readily available in non-model organisms, such as the industrially relevant genus Geobacillus. A multitude of different approaches are available for the identification and de novo design of prokaryotic promoters, although it may be unclear which methodology is most practical in an industrial context. Endogenous promoters may be individually isolated from upstream of well-understood genes, or bioinformatically identified en masse. Alternatively, pre-existing promoters may be mutagenised, or mathematical abstraction can be used to model promoter strength and design de novo synthetic regulatory sequences. In this investigation, bioinformatic, mathematic and mutagenic approaches to promoter discovery were directly compared. Hundreds of previously uncharacterised putative promoters were bioinformatically identified from the core genome of four Geobacillus species, and a rational sampling method was used to select sequences for in vivo characterisation. A library of 95 promoters covered a 2-log range of expression strengths when characterised in vivo using fluorescent reporter proteins. Data derived from this experimental characterisation were used to train Artificial Neural Network, Partial Least Squares and Random Forest statistical models, which quantifiably inferred the relationship between DNA sequence and function. The resulting models showed limited predictive- but good descriptive-power. In particular, the models highlighted the importance of sequences upstream of the canonical -35 and -10 motifs for determining promoter function in Geobacillus. Additionally, two commonly used mutagenic techniques for promoter production, Saturation Mutagenesis of Flanking Regions and error-prone PCR, were applied. The resulting sequence libraries showed limited promoter activity, underlining the difficulty of deriving synthetic promoters in species where understanding of transcription regulation is limited. As such, bioinformatic identification and deep-characterisation of endogenous promoter elements was posited as the most practical approach for the derivation of promoter libraries in non-model organisms of industrial interest.

Biotechnology of microalgae is a fast-growing field and several species have become targets for transgenic manipulation. Microalgae provide low-cost and scalable production platforms for manufacturing recombinant proteins and other high value products. However, the exploitation of microalgae as expression systems is restricted by the low yield of recombinant proteins and the limited availability of tools for the genetic manipulation of commercially important species. This thesis explores transgene instability and gene autoregulation as causes for low recombinant protein accumulation in the chloroplast of Chlamydomonas reinhardtii and describes the isolation of a mutant phytoene desaturase (PDS) gene which confers resistance to the herbicide norflurazon for future use as a selection marker for the marine microalga Dunaliella tertiolecta. Recombination between short dispersed DNA repeats (SDR) found in the chloroplast genome of C. reinhardtii was identified as a cause of transgene instability. The genes coding for β-glucuronidase (GUS) and peridinin-chlorophyll binding protein (PCP) were inserted in the chloroplast genome next to the atpB 3' UTR by homologous recombination. Recombination of a 30bp SDR located within the 3' UTR of atpB was identified as the cause of transgene excision in the transplastomic lines. Such transgene instability was tackled by replacing the 3' UTR of atpB with the rbcL 3' UTR from D. tertiolecta. Using this 3'UTR sequence from a different species produced a photosynthetic strain and prevented excision of the transgene by SDR recombination in all transfomants. Very low levels of recombinant GUS and PCP accumulated in chloroplast transformants when using the psbD 5' regulatory region to drive their expression. To address low levels of accumulation caused by regulatory pathways that inhibit transgene expression, I have engineered the chloroplast genome of a non-photosynthetic atpB mutant of C. reinhardtii by replacing the endogenous psbD promoter and 5'UTR with the promoter and 5'UTR of psbA. The engineered strain was subsequently transformed with the wildtype atpB and two different reporter genes driven by the psbD regulatory regions: gusA and kat, which code for GUS and the fluorescent protein Katushka respectively. Analysis of the transformants showed that accumulation of recombinant proteins in our engineered strain was 10 to 20 fold higher than in the nonengineered cells. Most of the selectable markers used in plants and algae are inefficient in Dunaliella, which is naturally resistant to many of the antibiotics used for the selection of transformants. Norflurazon inhibits PDS, an essential enzyme for carotenoid biosynthesis. Using forward genetics I have isolated, sequenced and characterised mutant PDS alleles conferring norflurazon resistance in D. tertiolecta. Independent mutations in pds, leading to substitutions R265C, S472L, S472F and L502F, all result in high resistance to norflurazon but differ in sensitivity to other bleaching herbicides. By mapping the four amino acid substitutions on 3D models of D. tertiolecta PDS I determined that R265C, S472L, S472F and L502F, cluster together in proximity to a Rossman-like domain and to aminoacids F128 and V469, previously reported to confer norflurazon resistance. This suggests that the mode of action of norflurazon is by competition with flavin adenine dinucleotide (FAD) for its binding site. A unique aspect of the R265C substitution is its negative cross-resistance to diflufenican and beflutamid which could be advantageous for its use as a positive/negative selection marker for transformation.

Antimicrobial resistant microorganisms are predicted to pose an existential threat to humanity inside of the next 3 decades. Characterisation of novel acting antimicrobial small molecules from microorganisms has historically counteracted this evolutionary arms race, however the bountiful source of pharmaceutically relevant bioactive specialised metabolites discovered in the Golden era of drug discovery has long since dried up. The clinicians' arsenal of useful antimicrobials is diminishing, and a fresh perspective on specialised metabolite discovery is necessary. This call to action is being answered, in part, through advances in genome sequencing, bioinformatics predictions and the development of next generation synthetic biology tools aiming to translate the biological sciences into an engineering discipline. To expedite our route to new pharmaceutically relevant specialised metabolites using the synthetic biology toolbox several bottlenecks need to be addressed, and are tackled here in. Biosynthetic gene clusters (BGCs) represent blueprints to pharmaceuticals, however to date the vast wealth of knowledge about biosynthetic gene clusters is inconsistently reported and sporadically disseminated throughout the literature and databases. To bring the reporting of BGCs in line with engineering principles we designed and built a community supported standard, the Minimum Information about a Biosynthetic Gene cluster (MIBiG), for reporting BGCs in a consistent manner, and centralised this information in an easy to operate and open access repository for rapid retrieval of information, an essential resource for the bioengineer. Prioritisation represents the next bottleneck in specialised metabolite discovery. Bioinformatics tools have predicted a cache of thousands of BGCs within publicly available genome sequences, however high experimental attrition rates drastically slows characterisation of the corresponding specialised metabolite. We designed and built an Output Ordering and Prioritisation System (OOPS), to rank thousands of BGCs in parallel against molecular biology relevant parameters, pairing BGCs with appropriate heterologous expression hosts and facilitating a judicious choice of BGCs for characterisation to reduce experimental attrition. To fully realise the potential of synthetic biology in specialised metabolite discovery a genetically amenable heterologous host, capable of completing rapid design-build-test-learn cycles, is necessary. This cannot be achieved for the pharmaceutically important type II polyketides, as their biosynthetic machinery is largely restricted to Actinobacteria. Using MIBiG datasets, antiSMASH and BLASTP we identify 5 sets of soluble type II polyketide synthases (PKS) in Escherichia coli for the first time. We construct and test the robustness of a plug-and-play scaffold for bioproduction of aromatic polyketides using one PKS in E. coli, yielding anthraquinones, dianthrones and benzoisochromanequinones intermediates. Through bioprospecting for biological 'parts' to expand the chemical diversity of our plug-and-play scaffold we describe a new lineage of type II PKSs predominantly from non-Actinobacteria. The standards, softwares, and plug-and-play scaffold and biosynthetic 'parts' described here-in will act as an engine for rapid and automated bioproduction of existing, and novel, pharmaceutically relevant aromatic polyketides in E. coli using the synthetic biology toolbox.

abstract: DNA and RNA are generally regarded as one of the central molecules in molecular biology. Recent advancements in the field of DNA/RNA nanotechnology witnessed the success of usage of DNA/RNA as programmable molecules to construct nano-objects with predefined shapes and dynamic molecular machines for various functions. From the perspective of structural design with nucleic acid, there are basically two types of assembly method, DNA tile based assembly and DNA origami based assembly, used to construct infinite-sized crystal structures and finite-sized molecular structures. The assembled structure can be used for arrangement of other molecules or nanoparticles with the resolution of nanometers to create new type of materials. The dynamic nucleic acid machine is based on the DNA strand displacement, which allows two nucleic acid strands to hybridize with each other to displace one or more prehybridized strands in the process. Strand displacement reaction has been implemented to construct a variety of dynamic molecular systems, such as molecular computer, oscillators, in vivo devices for gene expression control. This thesis will focus on the computational design of structural and dynamic nucleic acid systems, particularly for new type of DNA structure design and high precision control of gene expression in vivo. Firstly, a new type of fundamental DNA structural motif, the layered-crossover motif, will be introduced. The layered-crossover allow non-parallel alignment of DNA helices with precisely controlled angle. By using the layered-crossover motif, the scaffold can go through the 3D framework DNA origami structures. The properties of precise angle control of the layered-crossover tiles can also be used to assemble 2D and 3D crystals. One the dynamic control part, a de-novo-designed riboregulator is developed that can recognize single nucleotide variation. The riboregulators can also be used to develop paper-based diagnostic devices. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2019