Matching Items (10)
Filtering by
- Creators: Arizona State University
Description
With the aid of metabolic pathways engineering, microbes are finding increased use as biocatalysts to convert renewable biomass resources into fine chemicals, pharmaceuticals and other valuable compounds. These alternative, bio-based production routes offer distinct advantages over traditional synthesis methods, including lower energy requirements, rendering them as more "green" and "eco-friendly". Escherichia coli has recently been engineered to produce the aromatic chemicals (S)-styrene oxide and phenol directly from renewable glucose. Several factors, however, limit the viability of this approach, including low titers caused by product inhibition and/or low metabolic flux through the engineered pathways. This thesis focuses on addressing these concerns using magnetic mesoporous carbon powders as adsorbents for continuous, in-situ product removal as a means to alleviate such limitations. Using process engineering as a means to troubleshoot metabolic pathways by continuously removing products, increased yields are achieved from both pathways. By performing case studies in product toxicity and reaction equilibrium it was concluded that each step of a metabolic pathway can be optimized by the strategic use of in-situ adsorption as a process engineering tool.
ContributorsVasudevan, Anirudh (Author) / Nielsen, David R (Thesis advisor) / Torres, César I (Committee member) / Wang, Xuan (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
The need for rapid, specific and sensitive assays that provide a detection of bacterial indicators are important for monitoring water quality. Rapid detection using biosensor is a novel approach for microbiological testing applications. Besides, validation of rapid methods is an obstacle in adoption of such new bio-sensing technologies. In this study, the strategy developed is based on using the compound 4-methylumbelliferyl glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β-D-glucuronidase (GUD) enzyme to yield a fluorogenic product that can be quantified and directly related to the number of E. coli cells present in water samples. The detection time required for the biosensor response ranged from 30 to 120 minutes, depending on the number of bacteria. The specificity of the MUG based biosensor platform assay for the detection of E. coli was examined by pure cultures of non-target bacterial genera and also non-target substrates. GUD activity was found to be specific for E. coli and no such enzymatic activity was detected in other species. Moreover, the sensitivity of rapid enzymatic assays was investigated and repeatedly determined to be less than 10 E. coli cells per reaction vial concentrated from 100 mL of water samples. The applicability of the method was tested by performing fluorescence assays under pure and mixed bacterial flora in environmental samples. In addition, the procedural QA/QC for routine monitoring of drinking water samples have been validated by comparing the performance of the biosensor platform for the detection of E. coli and culture-based standard techniques such as Membrane Filtration (MF). The results of this study indicated that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. The procedural QA/QC of the biosensor will provide both industry and regulatory authorities a useful tool for near real-time monitoring of E. coli in drinking water samples. Furthermore, this system can be applied independently or in conjunction with other methods as a part of an array of biochemical assays in order to reliably detect E. coli in water.
ContributorsHesari, Nikou (Author) / Abbaszadegan, Morteza (Thesis advisor) / Alum, Absar (Committee member) / Fox, Peter (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2015
Description
Advanced oxidation processes (AOP’s) are water/wastewater treatment processes simultaneously providing disinfection and potential oxidation of contaminants that may cause long-term adverse health effects in humans. One AOP involves injecting peracetic acid (PAA) upstream of an ultraviolet (UV) irradiation reactor. Two studies were conducted, one in pilot-scale field conditions and another under laboratory conditions. A pilot-scale NeoTech UV reactor (rated for 375 GPM) was used in the pilot study, where a smaller version of this unit was used in the laboratory study (20 to 35 GPM). The pilot study analyzed coliform disinfection and also monitored water quality parameters including UV transmittance (UVT), pH and chlorine residual. Pilot study UV experiments indicate the unit is effectively treating flow streams (>6 logs total coliforms) twice the 95% UVT unit capacity (750 GPM or 17 mJ/cm2 UV Dose). The results were inconclusive on PAA/UV inactivation due to high data variability and field operation conditions creating low inlet concentrations.Escherichia coli (E. coli) bacteria and the enterobacteria phage P22—a surrogate for enteric viruses—were analyzed. UV inactivated >7.9 and 4 logs of E. coli and P22 respectively at a 16.8 mJ/cm2 UV dose in test water containing a significant organics concentration. When PAA doses of 0.25 and 0.5 mg/L were injected upstream of UV at approximately the same UV Dose, the average E.coli log inactivation increased to >8.9 and >9 logs respectively, but P22 inactivation decreased to 2.9 and 3.0 logs, respectively. A bench-scale study with PAA was also conducted for 5, 10 and 30 minutes of contact time, where 0.25 and 0.5 mg/L had <1 log inactivation of E. coli and P22 after 30 minutes of contact time. In addition, degradation of the chemical N-Nitrosodimethylamine (NDMA) in tap water was analyzed, where UV degraded NDMA by 48 to 97% for 4 and 0.5 GPM flowrates, respectively. Adding 0.5 mg/L PAA upstream of UV did not significantly improve NDMA degradation.
The results under laboratory conditions indicate that PAA/UV have synergy in the inactivation of bacteria, but decrease virus inactivation. In addition, the pilot study demonstrates the applicability of the technology for full scale operation.
The results under laboratory conditions indicate that PAA/UV have synergy in the inactivation of bacteria, but decrease virus inactivation. In addition, the pilot study demonstrates the applicability of the technology for full scale operation.
ContributorsCooper, Samantha (Author) / Abbaszadegan, Morteza (Thesis advisor) / Alum, Absar (Committee member) / Fox, Peter (Committee member) / Arizona State University (Publisher)
Created2017
Description
This study was designed to provide insight into microbial transport kinetics which might be applied to bioremediation technology development and prevention of groundwater susceptibility to pathogen contamination. Several pilot-scale experiments were conducted in a saturated, 2 dimensional, packed porous media tank to investigate the transport of Escherichia coli bacteria, P22 bacteriophage, and a visual tracer and draw comparisons and/or conclusions. A constructed tank was packed with an approximate 3,700 cubic inches (in3) of a fine grained, homogeneous, chemically inert sand which allowed for a controlled system. Sampling ports were located at 5, 15, 25, and 25 vertical inches from the base of the 39 inch saturated zone and were used to assess the transport of the selected microorganisms. Approximately 105 cells of E. coli or P22 were injected into the tank and allowed to move through the media at approximately 10.02 inches per day. Samples were collected intermittently after injection based off of an estimated sampling schedule established from the visual tracer.
The results suggest that bacteriophages pass through soil faster and with greater recovery than bacteria. P22 in the tank reservoir experienced approximately 1 log reduction after 36 hours. After 85 hours, P22 was still detected in the reservoir after experiencing a 2 log reduction from the start of the experiment. E. coli either did not reach the outlet or died before sampling, while P22 was able to be recovered. Bacterial breakthrough curves were produced for the microbial indicators and illustrate the peak concentrations found for each sampling port. For E. coli, concentrations at the 5 inch port peaked at a maximum of 5170 CFU/mL, and eventually at the 25 inch port at a maximum of 90 CFU/mL. It is presumed that E. coli might have experienced significant filtration, straining and attachment, while P22 might have experienced little adsorption and instead was transported rapidly in long distances and was able to survive for the duration of the experiment.
The results suggest that bacteriophages pass through soil faster and with greater recovery than bacteria. P22 in the tank reservoir experienced approximately 1 log reduction after 36 hours. After 85 hours, P22 was still detected in the reservoir after experiencing a 2 log reduction from the start of the experiment. E. coli either did not reach the outlet or died before sampling, while P22 was able to be recovered. Bacterial breakthrough curves were produced for the microbial indicators and illustrate the peak concentrations found for each sampling port. For E. coli, concentrations at the 5 inch port peaked at a maximum of 5170 CFU/mL, and eventually at the 25 inch port at a maximum of 90 CFU/mL. It is presumed that E. coli might have experienced significant filtration, straining and attachment, while P22 might have experienced little adsorption and instead was transported rapidly in long distances and was able to survive for the duration of the experiment.
ContributorsAcosta, Jazlyn Cauren (Author) / Abbaszadegan, Morteza (Thesis advisor) / Dahlen, Paul (Committee member) / Fox, Peter (Committee member) / Arizona State University (Publisher)
Created2017
Description
Biomass synthesis is a competing factor in biological systems geared towards generation of commodity and specialty chemicals, ultimately limiting maximum titer and yield; in this thesis, a widely generalizable, modular approach focused on decoupling biomass synthesis from the production of the phenylalanine in a genetically modified strain of E. coli BW25113 was explored with the use of synthetic trans-encoded small RNA (sRNA) to achieve greater efficiency. The naturally occurring sRNA MicC was used as a scaffold, and combined on a plasmid with a promoter for anhydrous tetracycline (aTc) and a T1/TE terminator. The coding sequence corresponding to the target binding site for fourteen potentially growth-essential gene targets as well as non-essential lacZ was placed in the seed region of the of the sRNA scaffold and transformed into BW25113, effectively generating a unique strain for each gene target. The BW25113 strain corresponding to each gene target was screened in M9 minimal media; decreased optical density and elongated cell morphology changes were observed and quantified in all induced sRNA cases where growth-essential genes were targeted. Six of the strains targeting different aspects of cell division that effectively suppressed growth and resulted in increased cell size were then screened for viability and metabolic activity in a scaled-up shaker flask experiment; all six strains were shown to be viable during stationary phase, and a metabolite analysis showed increased specific glucose consumption rates in induced strains, with unaffected specific glucose consumption rates in uninduced strains. The growth suppression, morphology and metabolic activity of the induced strains in BW25113 was compared to the bacteriostatic additives chloramphenicol, tetracycline, and streptomycin. At this same scale, the sRNA plasmid targeting the gene murA was transformed into BW25113 pINT-GA, a phenylalanine overproducer with the feedback resistant genes aroG and pheA overexpressed. Two induction times were explored during exponential phase, and while the optimal induction time was found to increase titer and yield amongst the BW25113 pINT-GA murA sRNA variant, overall this did not have as great a titer or yield as the BW25113 pINT-GA strain without the sRNA plasmid; this may be a result of the cell filamentation.
ContributorsHerschel, Daniel Jordan (Author) / Nielsen, David R (Thesis advisor) / Torres, César I (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2016
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
Metabolic engineering of bacteria has become a viable technique as a sustainable and efficient method for the production of biochemicals. Two main goals were explored: investigating styrene tolerance genes in E. coli and engineering cyanobacteria for the high yield production of L-serine. In the first study, genes that were shown to be highly differentially expressed in E. coli upon styrene exposure were further investigated by testing the effects of their deletion and overexpression on styrene tolerance and growth. It was found that plsX, a gene responsible for the phospholipid formation in membranes, had the most promising results when overexpressed at 10 µM IPTG, with a relative OD600 of 706 ± 117% at 175 mg/L styrene when compared to the control plasmid at the same concentration. This gene is likely to be effective target when engineering styrene- and other aromatic-producing strains, increasing titers by reducing their cytotoxicity.In the second study, the goal is to engineer the cyanobacterium Synechococcus sp. PCC 7002 for the overproduction of L-serine. As a robust, photosynthetic bacteria, it has potential for being used in such-rich states to capture CO2 and produce industrially relevant products. In order to increase L-serine titers, a key degradation gene, ilvA, must be removed. While ilvA is responsible for degrading L-serine into pyruvate, it is also responsible for initiating the only known pathway for the production of isoleucine. Herein, we constructed a plasmid containing the native A0730 gene in order to investigate its potential to restore isoleucine production. If functional, a Synechococcus sp. PCC 7002 ΔilvA strain can then be engineered with minimal effects on growth and an expected increase in L-serine accumulation.
ContributorsAbed, Omar (Author) / Nielsen, David R (Thesis advisor) / Varman, Arul M (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2021
Description
When exposed to abiotic stresses, Escherichia coli responds by activating various stress-mitigating pathways. Initiation of stress responses partially relies on the RNA polymerase (RNAP) to transcribe genes necessary to tolerate various stresses, including nutritional deprivation and heat exposure. Consequently, RNAP mutations impacting transcription can have pleiotropic effects on the cell physiology and the ability to tolerate stress. Previously, while investigating antibiotic-resistant mutations arising in the absence of major antibiotic efflux pumps, four mutants containing alterations in the RNA polymerase beta subunit gene (rpoB) were isolated (Cho & Misra, 2021). Of the four mutants, one (RpoB58) was found to be thermotolerant, permitting homogenous, stable growth at temperatures up to 47°C, whereas the parental rpoB wildtype (RpoB-WT) was only able to do so up to 45°C. Additionally, RNA-Seq analysis indicated that the RpoB58 mutant had a ‘stringent’ profile that is normally seen under nutritionally deprived conditions. To better understand the regulatory pathways used to confer stress tolerance, this thesis sought to further characterize and investigate the intracellular mechanisms contributing to the thermotolerance conferred by the rpoB58 mutation. The RpoB58 mutant was found to be significantly more tolerant to both continuous heat stress (up to 47°C) and short-term heat (55°C) and ethanol (25%) exposure. Additionally, the RpoB58 mutant tolerated the absence or depletion of major heat shock chaperones DnaJ and DnaK that normally play key roles during temperature stresses by reducing protein misfolding. RNA-Seq data and reporter gene assays showed reduced expression of genes involved in protein synthesis. A similar reduction in the expression of protein synthesis genes was observed when cells were grown in growth-limiting minimal media. Interestingly, growth in minimal medium rescued the ΔdnaJ defect like the rpoB58 mutation. Based on these data, it was proposed that a decrease in protein synthesis, whether caused by rpoB58 or the growth medium, would result in less growth-inhibiting protein misfolding and aggregation, especially at higher growth temperatures where proteins are susceptible to denaturation and aggregation. As a result of these investigations, a possible mechanistic insight was provided as to how the rpoB58 mutation confers thermotolerance.
ContributorsYeh, Melody (Author) / Misra, Rajeev RM (Thesis advisor) / Wang, Xuan XW (Committee member) / Muralinath, Maneesha MM (Committee member) / Arizona State University (Publisher)
Created2021
Description
Pathogenic contamination is a significant factor contributing to the degradation of surface water both globally and within the United States. This leads to negative economic impacts, sickness, and, in severe cases, fatalities. As the world's population grows, pollution increases, placing more stress on water resources, particularly in arid regions. The situation is made worse by climate change. The forecasted expansion of arid and semi-arid land areas and alterations in precipitation patterns could have a significant impact on those living in poverty and dry regions. This dissertation aims to investigate previously undocumented threats to water quality through understanding pathogen drivers in arid and semi-arid environments and documenting wastewater infrastructure on Tribal lands. Specifically, I first investigated how ephemeral streams (common in arid and semiarid areas) impact the presence of pathogens in surface waters by identifying the main drivers of E. coli concentration from a series of proposed predictors. Second, I identified unknown potential sources of water quality impairments on Tribal lands, which are mainly rural and in arid or semiarid areas, focusing on wastewater infrastructure in these systems. I specifically quantified populations served by wastewater treatment plants and then used a remote sensing approach to identify possible unpermitted wastewater lagoons that often serve as the only wastewater infrastructure in some areas. The findings revealed unique insights that could help aid water management in arid and semiarid regions as well as in rural areas.
ContributorsMenchu Maldonado, Maria Elena (Author) / Muenich, Rebecca L. (Thesis advisor) / Vivoni, Enrique R. (Committee member) / Conroy-Ben, Otakuye (Committee member) / Hamilton, Kerry (Committee member) / Arizona State University (Publisher)
Created2023