Matching Items (14)
Description
Alzheimer’s disease is a major problem affecting over 5.7 million Americans. Although much is known about the effects of this neurogenerative disease, the exact pathogenesis is still unknown. One very important characteristic of Alzheimer’s is the accumulation of beta amyloid protein which often results in plaques. To understand these beta amyloid proteins better, antibody fragments may be used to bind to these oligomers and potentially reduce the effects of Alzheimer’s disease.
This thesis focused on the expression and crystallization the fragment antigen binding antibody fragment A4. A fragment antigen binding fragment was chosen to be worked with as it is more stable than many other antibody fragments. A4 is important in Alzheimer’s disease as it is able to identify toxic beta amyloid.
This thesis focused on the expression and crystallization the fragment antigen binding antibody fragment A4. A fragment antigen binding fragment was chosen to be worked with as it is more stable than many other antibody fragments. A4 is important in Alzheimer’s disease as it is able to identify toxic beta amyloid.
ContributorsColasurd, Paige (Author) / Nannenga, Brent (Thesis advisor) / Mills, Jeremy (Committee member) / Varman, Arul (Committee member) / Arizona State University (Publisher)
Created2018
Description
Increasing energy and environmental problems describe the need to develop renewable chemicals and fuels. Global research has been targeting using microbial systems on a commercial scale for synthesis of valuable compounds. The goal of this project was to refactor and overexpress b6-f complex proteins in cyanobacteria to improve photosynthesis under dynamic light conditions. Improvement in the photosynthetic system can directly relate to higher yields of valuable compounds such as carotenoids and higher yields of biomass which can be used as energy molecules. Four engineered strains of cyanobacteria were successfully constructed and overexpressed the corresponding four large subunits in the cytochrome b6-f complex. No significant changes were found in cell growth or pigment titer in the modified strains compared to the wild type. The growth assay will be performed at higher and/or dynamic light intensities including natural light conditions for further analysis.
ContributorsNauroth, Benjamin (Author) / Varman, Arul (Thesis director) / Singharoy, Abhishek (Committee member) / Li, Han (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Renewable bioproduction through fermentation of microbial species such as E. coli shows much promise in comparison to conventional fossil fuel based chemical production. Although Escherichia coli is a workhorse for bioproduction, there are inherent limitations associated with the use of this organism which negatively affect bioproduction. One example is E. coli fermentative growth being less robust compared to some microbes such as Lactobacilli under anaerobic and microaerobic fermentation conditions. Identification and characterization of its fermentative growth constraints will help in making E. coli a better fermentation host. In this thesis, I demonstrate that Lactobacillus plantarum WCFS1 has desirable fermentative capabilities that may be transferrable to E. coli through genetic engineering to alleviate growth restraints. This has led to the hypothesis that these L. plantarum DNA sequences are transferrable through a genomic library. A background of comparative genomics and complementary literature review has demonstrated that E. coli growth may be hindered by stress from many toxin-antitoxin systems. L. plantarum WCFS1 optimizes amino acid catabolism over glycolysis to generate high ATP levels from reducing agents and proton motive force, and Lactobacilli are resistant to acidic environments and encodes a wide variety of acid transporters that could help E. coli fermentative growth. Since a great variety of L. plantarum genes may contribute to its fermentative capabilities, a gDNA library containing L. plantarum WCFS1 genes has been successfully constructed for testing in E. coli bioproducers to search for specific genes that may enhance E. coli fermentative performance and elucidate the molecular basis of Lactobacillus fermentative success.
ContributorsDufault, Matthew Elijah (Co-author, Co-author) / Wang, Xuan (Thesis director) / Nielsen, David (Committee member) / Varman, Arul (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Fermentative bioproduction is an efficient production avenue for many small organic acids with less greenhouse gas emissions than petrochemical conversion. Export of these organic acids from the cell is proposed to be mediated by networks of transmembrane transport proteins. However characterization of full transporter networks or the substrate promiscuity of individual transporters is often incomplete. Here, we used a cheminformatic approach to predict previously unknown native activity of E. coli transporters based on substrate promiscuity. Experimental validation in characterized several major putative malate exporters, whereas others were characterized as weak putative lactate exporters. The lactate export network remains incompletely characterized and might be mediated by a large, evolved network of promiscuous transporters.
ContributorsSchneider, Aidan (Author) / Wang, Xuan (Thesis director) / Varman, Arul (Committee member) / Nielsen, David (Committee member) / Department of Finance (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
Description
The increased shift towards environmentalism has brought notable attention to a universal excessive plastic consumption and subsequent plastic overload in landfills. Among these plastics, polyethylene terephthalate, more commonly known as PET, constitutes a large percentage of the waste that ends up in landfills. Material and chemical/thermal methods for recycling are both costly, and inefficient, which necessitates a more sustainable and cheaper alternative. The current study aims at fulfilling that role through genetic engineering of Bacillus subtilis with integration of genes from LCC, Ideonella sakaiensis, and Bacillus subtilis. The plasmid construction was done through restriction cloning. A recombinant plasmid for the expression of LCC was constructed, and transformed into Escherichia coli. Future experiments for this study should include redesigning of primers, with possible combination of signal peptides with genes during construct design, and more advanced assays for effective outcomes.
ContributorsKalscheur, Bethany Ann (Author) / Varman, Arul (Thesis director) / Andino, Jean (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Polymers have played a pivotal role in building modern society. Polymers can be classified as synthetic and natural polymers. Accumulation of both synthetic and natural polymer waste leads to environmental pollution. This dissertation aims at developing one-pot bioprocesses for a breakdown of natural polymers like cellulose, and hemicellulose and synthetic polymers like polyethylene terephthalate (PET). First, a one-pot process was developed for hemicellulose breakdown. A signal peptide library of native SEC pathway signal peptides was developed for efficient secretion of endoxylanse enzyme. Furthermore, in situ, the process was successfully created for hemicellulose to xylose with the highest reported xylose titer of 7.1 g/L. In addition, E. coli: B. subtilis coculture bioprocess was developed to produce succinate, ethanol, and lactate from hemicellulose in one pot process.
Second, a one-pot process was developed for cellulose breakdown. In vitro enzyme assays were used to select SEC pathway signal peptides for endoglucanase and glucosidase secretion. Then, the breakdown of carboxymethyl cellulose (CMC), a cellulose derivative, was conducted in in situ conditions. U-13C fingerprinting study showed carbon enrichment from CMC when cultures were cofed with CMC and [U-13C] glucose. Further, Whatman filter paper sheets showed a change in shape in recombinant cocultures. SEM images showed continuous orientation in the case of two enzymes confirmed by fast Fourier transform (FFT), suggesting higher crystallinity of residues. Similarly, in microcrystalline cellulose breakdown in in situ conditions, a 72% reduction of avicel cellulose was achieved in a one pot bioprocess. SEM images revealed valleys and crevices on residues of coculture compared to smoother surfaces in monoculture residues pressing the importance of the synergistic activity of enzymes.
Finally, one pot deconstruction process was developed for synthetic polymer PET. First, the PET hydrolase secretion strain was developed by selecting a signal peptide library. The first bis(2-hydroxyethyl) terephthalate (BHET) consolidated bioprocess was developed, which produced a terephthalic acid titer of 7.4 g/L. PET breakdown was successfully demonstrated in in vitro conditions with a TPA titer of 4 g/L. Furthermore, PET breakdown was successfully demonstrated in in situ conditions. Consolidated bioprocesses can be an invaluable approach to waste utilization and making cost-effective processes.
ContributorsMhatre, Apurv (Author) / Varman, Arul (Thesis advisor) / Nielsen, David (Committee member) / Misra, Rajeev (Committee member) / Nannenga, Brent (Committee member) / Torres, Cesar (Committee member) / Arizona State University (Publisher)
Created2023
Description
Phenolic polymers such as polyphenols and polyphenylenes are generated industrially for several applications but are typically associated with harsh reaction conditions and environmentally hazardous chemicals, such as formaldehyde. Additionally, hydroxycinnamic acids, such as p-coumaric acid (CA), are found in high concentrations in underutilized lignin-derived hydrolysates and represent a renewable and sustainable feedstock for the production of various aromatics and phenolics. To that end, recently a strain of Corynebacterium glutamicum has been developed by the Joint Bioenergy Institute to express a Phenolic Acid Decarboxylase (PAD), which can convert CA into 4-vinylphenol (4VP). 4VP is cytotoxic but can be polymerized by ligninolytic enzymes such as laccases or peroxidases into less-toxic poly(4-vinylphenol) (PVP). This work investigates the potential of polymerizing 4VP in situ by adding ligninolytic enzymes into the fermentation media to polymerize 4VP into PVP as it is produced, while reducing cellular toxicity to aid in chemical conversion.
The engineered C. glutamicum strain was cultured in the presence of CA to produce 4VP, with a maximum yield of 80.75%. Simultaneously, two ligninolytic enzymes, laccase and horseradish peroxidase (HRP), were explored in an in vitro experiment for their ability to polymerize 4VP, with laccase achieving full polymerization within 45 minutes and HRP able to polymerize 54.06% of 4VP in 24 hours. The resulting polymers were further analyzed by using gas permeation chromatography - nuclear magnetic resonance, validating the synthesis of PVP from 4VP with the addition of laccase or HRP. Finally, the C. glutamicum strain was evaluated for its ability to grow in the presence of hydrogen peroxide, which is a necessary reagent for HRP functionality, and it was able to reach an optical density of 3.69 within 36 hours. These findings suggest that in situ polymerization may be possible. Further work is underway to explore the enzyme kinetics at different pH, validate the potential of polymerization in situ, and study the fermentative benefits associated with in situ polymerization. This will be followed by additional analytical studies to characterize the resulting PVP.
ContributorsEderer, William (Author) / Varman, Arul (Thesis director) / Long, Timothy (Committee member) / Rodriguez, Alberto (Committee member) / Barrett, The Honors College (Contributor) / School of Sustainability (Contributor) / Chemical Engineering Program (Contributor)
Created2022-05
Description
The current use of non-renewable fossil fuels for industry poses a threat for future generations. Thus, a pivot to renewable sources of energy must be made to secure a
sustainable future. One potential option is the utilization of metabolically engineered
bacteria to produce value-added chemicals during fermentation. Currently, numerous
strains of metabolically engineered Escherichia coli have shown great capacity to
specialize in the production of high titers of a desired chemical. These metabolic systems,
however, are constrained by the biological limits of E. coli itself. During fermentation, E.
coli grows to less than one twentieth of the density that aerobically growing cultures can
reach. I hypothesized that this decrease in growth during fermentation is due to cellular
stress associated with fermentative growth, likely caused by stress related genes. These
genes, including toxin-antitoxin (TA) systems and the rpoS mediated general stress
response, may have an impact on fermentative growth constraints. Through
transcriptional analysis, I identified that the genes pspC and relE are highly expressed in
fermenting strains of both wild type and metabolically engineered E. coli. Fermentation
of toxin gene knockouts of E. coli BW25113 revealed their potential impacts on E. coli
fermentation. The inactivation of ydcB, lar, relE, hipA, yjfE, chpA, ygiU, ygjN, ygfX,
yeeV, yjdO, yjgK and ydcX did not lead to significant changes in cell growth when tested
using sealed tubes under microaerobic conditions. In contrast, inactivation of pspC, yafQ,
yhaV, yfjG and yoeB increased cell growth after 12 hours while inactivation yncN
significantly arrested cell growth in both tube and fermentation tests, thus proving these
toxins’ roles in fermentative growth. Moreover, inactivation of rpoS also significantly hindered the ability of E. coli to ferment, suggesting its important role in E. coli
fermentation
ContributorsHernandez, Michaella (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Varman, Arul (Committee member) / Arizona State University (Publisher)
Created2022
Description
Single and double deletion strains of Escherichia coli were grown in paired co-cultures with an intent to identify examples of metabolite exchange and cooperative interactions between strains. The essential genes pheA, argA, tyrA, and trpC, as well as the non- essential genes pykF, pykA, mdh, ppc, and nuoN were deleted from Escherichia coli strains Bw25113 and ATCC 9637. Cultures were paired at three different initial ratios and grown at plate and flask scale. Optical density measurements were used to observe the performance of tested co-cultures, with changes in maximum optical density and growth rate used as indicators of interaction or lack thereof between tested pairs. Auxotrophic strains unable to produce essential amino acids were observed to grow in co-culture but not in monoculture, indicative of metabolite exchange facilitating growth. An increase in optical density for non-essential pairs when compared to the prototrophic parent and precursor monocultures was indicative of metabolite exchange. The initial frequency of paired mutants with non-essential deletions appeared to have an impact on growth performance, but whether this was indicative of any beneficial exchange was not able to be determined from data.
ContributorsFenner, Alexander James (Author) / Nielsen, David (Thesis advisor) / Wang, Xuan (Committee member) / Varman, Arul (Committee member) / Arizona State University (Publisher)
Created2022
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