Matching Items (11)
Filtering by
- All Subjects: Synthetic Biology
- All Subjects: Metabolic Engineering
- Genre: Doctoral Dissertation
- Creators: Nielsen, David R
- Creators: Chaput, John C
- Creators: Green, Alexander
Description
The principle of Darwinian evolution has been applied in the laboratory to nucleic acid molecules since 1990, and led to the emergence of in vitro evolution technique. The methodology of in vitro evolution surveys a large number of different molecules simultaneously for a pre-defined chemical property, and enrich for molecules with the particular property. DNA and RNA sequences with versatile functions have been identified by in vitro selection experiments, but many basic questions remain to be answered about how these molecules achieve their functions. This dissertation first focuses on addressing a fundamental question regarding the molecular recognition properties of in vitro selected DNA sequences, namely whether negatively charged DNA sequences can be evolved to bind alkaline proteins with high specificity. We showed that DNA binders could be made, through carefully designed stringent in vitro selection, to discriminate different alkaline proteins. The focus of this dissertation is then shifted to in vitro evolution of an artificial genetic polymer called threose nucleic acid (TNA). TNA has been considered a potential RNA progenitor during early evolution of life on Earth. However, further experimental evidence to support TNA as a primordial genetic material is lacking. In this dissertation we demonstrated the capacity of TNA to form stable tertiary structure with specific ligand binding property, which suggests a possible role of TNA as a pre-RNA genetic polymer. Additionally, we discussed the challenges in in vitro evolution for TNA enzymes and developed the necessary methodology for future TNA enzyme evolution.
ContributorsYu, Hanyang (Author) / Chaput, John C (Thesis advisor) / Chen, Julian (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
Description
The production of monomer compounds for synthesizing plastics has to date been largely restricted to the petroleum-based chemical industry and sugar-based microbial fermentation, limiting its sustainability and economic feasibility. Cyanobacteria have, however, become attractive microbial factories to produce renewable fuels and chemicals directly from sunlight and CO2. To explore the feasibility of photosynthetic production of (S)- and (R)-3-hydroxybutyrate (3HB), building-block monomers for synthesizing the biodegradable plastics polyhydroxyalkanoates and precursors to fine chemicals, synthetic metabolic pathways have been constructed, characterized and optimized in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803). Both types of 3HB molecules were produced and readily secreted from Synechocystis cells without over-expression of transporters. Additional inactivation of the competing PHB biosynthesis pathway further promoted the 3HB production. Analysis of the intracellular acetyl-CoA and anion concentrations in the culture media indicated that the phosphate consumption during the photoautotrophic growth and the concomitant elevated acetyl-CoA pool acted as a key driving force for 3HB biosynthesis in Synechocystis. Fine-tuning of the gene expression levels via strategies, including tuning gene copy numbers, promoter engineering and ribosome binding site optimization, proved critical to mitigating metabolic bottlenecks and thus improving the 3HB production. One of the engineered Synechocystis strains, namely R168, was able to produce (R)-3HB to a cumulative titer of ~1600 mg/L, with a peak daily productivity of ~200 mg/L, using light and CO2 as the sole energy and carbon sources, respectively. Additionally, in order to establish a high-efficiency transformation protocol in cyanobacterium Synechocystis 6803, methyltransferase-encoding genes were cloned and expressed to pre-methylate the exogenous DNA before Synechocystis transformation. Eventually, the transformation efficiency was increased by two orders of magnitude in Synechocystis. This research has demonstrated the use of cyanobacteria as cell factories to produce 3HB directly from light and CO2, and developed new synthetic biology tools for cyanobacteria.
ContributorsWang, Bo (Author) / Meldrum, Deirdre R (Thesis advisor) / Zhang, Weiwen (Committee member) / Sandrin, Todd R. (Committee member) / Nielsen, David R (Committee member) / Arizona State University (Publisher)
Created2014
Description
Synthetic biology is constantly evolving as new ideas are incorporated into this increasingly flexible field. It incorporates the engineering of life with standard genetic parts and methods; new organisms with new genomes; expansion of life to include new components, capabilities, and chemistries; and even completely synthetic organisms that mimic life while being composed of non-living matter. We have introduced a new paradigm of synthetic biology that melds the methods of in vitro evolution with the goals and philosophy of synthetic biology. The Family B proteins represent the first de novo evolved natively folded proteins to be developed with increasingly powerful tools of molecular evolution. These proteins are folded and functional, composed of the 20 canonical amino acids, and in many ways resemble natural proteins. However, their evolutionary history is quite different from natural proteins, as it did not involve a cellular environment. In this study, we examine the properties of DX, one of the Family B proteins that have been evolutionarily optimized for folding stability. Described in chapter 2 is an investigation into the primitive catalytic properties of DX, which seems to have evolved a serendipitous ATPase activity in addition to its selected ATP binding activity. In chapters 3 and 4 we express the DX gene in E. coli cells and observe massive changes in cell morphology, biochemistry, and life cycle. Exposure to DX activates several defense systems in E. coli, including filamentation, cytoplasmic segregation, and reversion to a viable but non-culturable state. We examined these phenotypes in detail and present a model that accounts for how DX causes such a rearrangement of the cell.
ContributorsStomel, Joshua (Author) / Chaput, John C (Thesis advisor) / Korch, Shaleen (Committee member) / Roberson, Robert (Committee member) / Ghirlanda, Gionvanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
Advances in chemical synthesis have enabled new lines of research with unnatural genetic polymers whose modified bases or sugar-phosphate backbones have potential therapeutic and biotechnological applications. Maximizing the potential of these synthetic genetic systems requires inventing new molecular biology tools that can both generate and faithfully replicate unnatural polymers of significant length. Threose nucleic acid (TNA) has received significant attention as a complete replication system has been developed by engineering natural polymerases to broaden their substrate specificity. The system, however, suffers from a high mutational load reducing its utility. This thesis will cover the development of two new polymerases capable of transcribing and reverse transcribing TNA polymers with high efficiency and fidelity. The polymerases are identified using a new strategy wherein gain-of-function mutations are sampled in homologous protein architectures leading to subtle optimization of protein function. The new replication system has a fidelity that supports the propagation of genetic information enabling in vitro selection of functional TNA molecules. TNA aptamers to human alpha-thrombin are identified and demonstrated to have superior stability compared to DNA and RNA in biologically relevant conditions. This is the first demonstration that functional TNA molecules have potential in biotechnology and molecular medicine.
ContributorsDunn, Matthew Ryan (Author) / Chaput, John C (Thesis advisor) / LaBaer, Joshua (Committee member) / Lake, Douglas (Committee member) / Mangone, Marco (Committee member) / Arizona State University (Publisher)
Created2015
Description
The engineering of microbial cell factories capable of synthesizing industrially relevant chemical building blocks is an attractive alternative to conventional petrochemical-based production methods. This work focuses on the novel and enhanced biosynthesis of phenol, catechol, and muconic acid (MA). Although the complete biosynthesis from glucose has been previously demonstrated for all three compounds, established production routes suffer from notable inherent limitations. Here, multiple pathways to the same three products were engineered, each incorporating unique enzyme chemistries and/or stemming from different endogenous precursors. In the case of phenol, two novel pathways were constructed and comparatively evaluated, with titers reaching as high as 377 ± 14 mg/L at a glucose yield of 35.7 ± 0.8 mg/g. In the case of catechol, three novel pathways were engineered with titers reaching 100 ± 2 mg/L. Finally, in the case of MA, four novel pathways were engineered with maximal titers reaching 819 ± 44 mg/L at a glucose yield of 40.9 ± 2.2 mg/g. Furthermore, the unique flexibility with respect to engineering multiple pathways to the same product arises in part because these compounds are common intermediates in aromatic degradation pathways. Expanding on the novel pathway engineering efforts, a synthetic ‘metabolic funnel’ was subsequently constructed for phenol and MA, wherein multiple pathways were expressed in parallel to maximize carbon flux toward the final product. Using this novel ‘funneling’ strategy, maximal phenol and MA titers exceeding 0.5 and 3 g/L, respectively, were achieved, representing the highest achievable production metrics products reported to date.
ContributorsThompson, Brian (Author) / Nielsen, David R (Thesis advisor) / Nannenga, Brent (Committee member) / Green, Matthew (Committee member) / Wang, Xuan (Committee member) / Moon, Tae Seok (Committee member) / Arizona State University (Publisher)
Created2017
Description
Lignocellulose, the major structural component of plant biomass, represents arenewable substrate of enormous biotechnological value. Microbial production of
chemicals from lignocellulosic biomass is an attractive alternative to chemical synthesis.
However, to create industrially competitive strains to efficiently convert lignocellulose to
high-value chemicals, current challenges must be addressed. Redox constraints, allosteric
regulation, and transport-related limitations are important bottlenecks limiting the
commercial production of renewable chemicals from lignocellulose. Advances in
metabolic engineering techniques have enabled researchers to engineer microbial strains
that overcome some of these challenges but new approaches that facilitate the
commercial viability of lignocellulose valorization are needed. Biological systems are
complex with a plethora of regulatory systems that must be carefully modulated to
efficiently produce and excrete the desired metabolites. In this work, I explore metabolic
engineering strategies to address some of the biological constraints limiting
bioproduction such as redox, allosteric, and transport constraints to facilitate cost-effective
lignocellulose bioconversion.
ContributorsOnyeabor, Moses Ekenedilichukwu (Author) / Wang, Xuan (Thesis advisor) / Varman, Arul M (Committee member) / Nannenga, Brent (Committee member) / Nielsen, David R (Committee member) / Geiler-Samerotte, Kerry (Committee member) / Arizona State University (Publisher)
Created2023
Description
Clustered regularly interspace short palindromic repeats (CRISPR) and CRISPR associated (Cas) technologies have become integral to genome editing. Canonical CRISPR-Cas9 systems function as a ribonucleic acid (RNA)-guided nucleases. Single guide RNAs (sgRNA) can be easily designed to target Cas9’s nuclease activity towards protospacer deoxyribonucleic acid (DNA) sequences. The relatively ease and efficiency of CRISPR-Cas9 systems has enabled numerous technologies and DNA manipulations. Genome engineering in human cell lines is centered around the study of genetic contribution to disease phenotypes. However, canonical CRISPR-Cas9 systems are largely reliant on double stranded DNA breaks (DSBs). DSBs can induce unintended genomic changes including deletions and complex rearrangements. Likewise, DSBs can induce apoptosis and cell cycle arrest confounding applications of Cas9-based systems for disease modeling. Base editors are a novel class of nicking Cas9 engineered with a cytidine or adenosine deaminase. Base editors can install single letter DNA edits without DSBs. However, detecting single letter DNA edits is cumbersome, requiring onerous DNA isolation and sequencing, hampering experimental throughput. This document describes the creation of a fluorescent reporter system to detect Cytosine-to-Thymine (C-to-T) base editing. The fluorescent reporter utilizes an engineered blue fluorescent protein (BFP) that is converted to green fluorescent protein (GFP) upon targeted C-to-T conversion. The BFP-to-GFP conversion enables the creation of a strategy to isolate edited cell populations, termed Transient Reporter for Editing Enrichment (TREE). TREE increases the ease of optimizing base editor designs and assists in editing cell types recalcitrant to DNA editing. More recently, Prime editing has been demonstrated to introduce user defined DNA edits without the need for DSBs and donor DNA. Prime editing requires specialized prime editing guide RNAs (pegRNAs). pegRNAs are however difficult to manually design. This document describes the creation of a software tool: Prime Induced Nucleotide Engineering Creator of New Edits (PINE-CONE). PINE-CONE rapidly designs pegRNAs based off basic edit information and will assist with synthetic biology and biomedical research.
ContributorsStandage-Beier, Kylie S (Author) / Wang, Xiao (Thesis advisor) / Brafman, David A (Committee member) / Tian, Xiao-jun (Committee member) / Nielsen, David R (Committee member) / Arizona State University (Publisher)
Created2023
Description
Aromatic compounds have traditionally been generated via petroleum feedstocks and have wide ranging applications in a variety of fields such as cosmetics, food, plastics, and pharmaceuticals. Substantial improvements have been made to sustainably produce many aromatic chemicals from renewable sources utilizing microbes as bio-factories. By assembling and optimizing native and non-native pathways to produce natural and non-natural bioproducts, the diversity of biochemical aromatics which can be produced is constantly being improved upon. One such compound, 2-Phenylethanol (2PE), is a key molecule used in the fragrance and food industries, as well as a potential biofuel. Here, a novel, non-natural pathway was engineered in Escherichia coli and subsequently evaluated. Following strain and bioprocess optimization, accumulation of inhibitory acetate byproduct was reduced and 2PE titers approached 2 g/L – a ~2-fold increase over previously implemented pathways in E. coli. Furthermore, a recently developed mechanism to
allow E. coli to consume xylose and glucose, two ubiquitous and industrially relevant microbial feedstocks, simultaneously was implemented and systematically evaluated for its effects on L-phenylalanine (Phe; a precursor to many microbially-derived aromatics such as 2PE) production. Ultimately, by incorporating this mutation into a Phe overproducing strain of E. coli, improvements in overall Phe titers, yields and sugar consumption in glucose-xylose mixed feeds could be obtained. While upstream efforts to improve precursor availability are necessary to ultimately reach economically-viable production, the effect of end-product toxicity on production metrics for many aromatics is severe. By utilizing a transcriptional profiling technique (i.e., RNA sequencing), key insights into the mechanisms behind styrene-induced toxicity in E. coli and the cellular response systems that are activated to maintain cell viability were obtained. By investigating variances in the transcriptional response between styrene-producing cells and cells where styrene was added exogenously, better understanding on how mechanisms such as the phage shock, heat-shock and membrane-altering responses react in different scenarios. Ultimately, these efforts to diversify the collection of microbially-produced aromatics, improve intracellular precursor pools and further the understanding of cellular response to toxic aromatic compounds, give insight into methods for improved future metabolic engineering endeavors.
allow E. coli to consume xylose and glucose, two ubiquitous and industrially relevant microbial feedstocks, simultaneously was implemented and systematically evaluated for its effects on L-phenylalanine (Phe; a precursor to many microbially-derived aromatics such as 2PE) production. Ultimately, by incorporating this mutation into a Phe overproducing strain of E. coli, improvements in overall Phe titers, yields and sugar consumption in glucose-xylose mixed feeds could be obtained. While upstream efforts to improve precursor availability are necessary to ultimately reach economically-viable production, the effect of end-product toxicity on production metrics for many aromatics is severe. By utilizing a transcriptional profiling technique (i.e., RNA sequencing), key insights into the mechanisms behind styrene-induced toxicity in E. coli and the cellular response systems that are activated to maintain cell viability were obtained. By investigating variances in the transcriptional response between styrene-producing cells and cells where styrene was added exogenously, better understanding on how mechanisms such as the phage shock, heat-shock and membrane-altering responses react in different scenarios. Ultimately, these efforts to diversify the collection of microbially-produced aromatics, improve intracellular precursor pools and further the understanding of cellular response to toxic aromatic compounds, give insight into methods for improved future metabolic engineering endeavors.
ContributorsMachas, Michael (Author) / Nielsen, David R (Thesis advisor) / Haynes, Karmella (Committee member) / Wang, Xuan (Committee member) / Nannenga, Brent (Committee member) / Varman, Arul (Committee member) / Arizona State University (Publisher)
Created2019
Description
The fundamental building blocks for constructing complex synthetic gene networks are effective biological parts with wide dynamic range, low crosstalk, and modularity. RNA-based components are promising sources of such parts since they can provide regulation at the level of transcription and translation and their predictable base pairing properties enable large libraries to be generated through in silico design. This dissertation studies two different approaches for initiating interactions between RNA molecules to implement RNA-based components that achieve translational regulation. First, single-stranded domains known as toeholds were employed for detection of the highly prevalent foodborne pathogen norovirus. Toehold switch riboregulators activated by trigger RNAs from the norovirus RNA genome are designed, validated, and coupled with paper-based cell-free transcription-translation systems. Integration of paper-based reactions with synbody enrichment and isothermal RNA amplification enables as few as 160 copies/mL of norovirus from clinical samples to be detected in reactions that do not require sophisticated equipment and can be read directly by eye. Second, a new type of riboregulator that initiates RNA-RNA interactions through the loop portions of RNA stem-loop structures was developed. These loop-initiated RNA activators (LIRAs) provide multiple advantages compared to toehold-based riboregulators, exhibiting ultralow signal leakage in vivo, lacking any trigger RNA sequence constraints, and appending no additional residues to the output protein. Harnessing LIRAs as modular parts, logic gates that exploit loop-mediated control of mRNA folding state to implement AND and OR operations with up to three sequence-independent input RNAs were constructed. LIRA circuits can also be ported to paper-based cell-free reactions to implement portable systems with molecular computing and sensing capabilities. LIRAs can detect RNAs from a variety of different pathogens, such as HIV, Zika, dengue, yellow fever, and norovirus, and after coupling to isothermal amplification reactions, provide visible test results down to concentrations of 20 aM (12 RNA copies/µL). And the logic functionality of LIRA circuits can be used to specifically identify different HIV strains and influenza A subtypes. These findings demonstrate that toehold- and loop-mediated RNA-RNA interactions are both powerful strategies for implementing RNA-based computing systems for intracellular and diagnostic applications.
ContributorsMA, DUO (Author) / Green, Alexander (Thesis advisor) / Mangone, Marco (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2019
Description
Gene circuit engineering facilitates the discovery and understanding of fundamental biology and has been widely used in various biological applications. In synthetic biology, gene circuits are often constructed by two main strategies: either monocistronic or polycistronic constructions. The Latter architecture can be commonly found in prokaryotes, eukaryotes, and viruses and has been largely applied in gene circuit engineering. In this work, the effect of adjacent genes and noncoding regions are systematically investigated through the construction of batteries of gene circuits in diverse scenarios. Data-driven analysis yields a protein expression metric that strongly correlates with the features of adjacent transcriptional regions (ATRs). This novel mathematical tool helps the guide for circuit construction and has the implication for the design of synthetic ATRs to tune gene expression, illustrating its potential to facilitate engineering complex gene networks. The ability to tune RNA dynamics is greatly needed for biotech applications, including therapeutics and diagnostics. Diverse methods have been developed to tune gene expression through transcriptional or translational manipulation. Control of RNA stability/degradation is often overlooked and can be the lightweight alternative to regulate protein yields. To further extend the utility of engineered ATRs to regulate gene expression, a library of RNA modules named degradation-tuning RNAs (dtRNAs) are designed with the ability to form specific 5’ secondary structures prior to RBS. These modules can modulate transcript stability while having a minimal interference on translation initiation. Optimization of their functional structural features enables gene expression level to be tuned over a wide dynamic range. These engineered dtRNAs are capable of regulating gene circuit dynamics as well as noncoding RNA levels and can be further expanded into cell-free system for gene expression control in vitro. Finally, integrating dtRNA with synthetic toehold sensor enables improved paper-based viral diagnostics, illustrating the potential of using synthetic dtRNAs for biomedical applications.
ContributorsZhang, Qi (Author) / Wang, Xiao (Thesis advisor) / Green, Alexander (Committee member) / Brafman, David (Committee member) / Tian, Xiaojun (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2020