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One of the primary bottlenecks to chemical production in biological organisms is the toxicity of the chemical. Overexpression of efflux pumps has been shown to increase tolerance to aromatic compounds such as styrene and styrene oxide. Tight control of pump expression is necessary to maximize titers and prevent excessive strain

One of the primary bottlenecks to chemical production in biological organisms is the toxicity of the chemical. Overexpression of efflux pumps has been shown to increase tolerance to aromatic compounds such as styrene and styrene oxide. Tight control of pump expression is necessary to maximize titers and prevent excessive strain on the cells. This study aimed to identify aromatic-sensitive native promoters and heterologous biosensors for construction of closed-loop control of efflux pump expression in E. coli. Using a promoter library constructed by Zaslaver et al., activation was measured through GFP output. Promoters were evaluated for their sensitivity to the addition of one of four aromatic compounds, their "leaking" of signal, and their induction threshold. Out of 43 targeted promoters, 4 promoters (cmr, mdtG, yahN, yajR) for styrene oxide, 2 promoters (mdtG, yahN) for styrene, 0 promoters for 2-phenylethanol, and 1 promoter for phenol (pheP) were identified as ideal control elements in aromatic bioproduction. In addition, a series of three biosensors (NahR, XylS, DmpR) known to be inducible by other aromatics were screened against styrene oxide, 2-phenylethanol, and phenol. The targeted application of these biosensors is aromatic-induced activation of linked efflux pumps. All three biosensors responded strongly in the presence of styrene oxide and 2-phenylethanol, with minor activation in the presence of phenol. Bioproduction of aromatics continues to gain traction in the biotechnology industry, and the continued discovery of aromatic-inducible elements will be essential to effective pathway control.
ContributorsXu, Jimmy (Author) / Nielsen, David (Thesis director) / Wang, Xuan (Committee member) / School of Life Sciences (Contributor) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description
p-Coumaric acid is used in the food, pharmaceutical, and cosmetic industries due to its versatile properties. While prevalent in nature, harvesting the compound from natural sources is inefficient, requiring large quantities of producing crops and numerous extraction and purification steps. Thus, the large-scale production of the compound is both difficult

p-Coumaric acid is used in the food, pharmaceutical, and cosmetic industries due to its versatile properties. While prevalent in nature, harvesting the compound from natural sources is inefficient, requiring large quantities of producing crops and numerous extraction and purification steps. Thus, the large-scale production of the compound is both difficult and costly. This research aims to produce p-coumarate directly from renewable and sustainable glucose using a co-culture of Yeast and E. Coli. Methods used in this study include: designing optimal media for mixed-species microbial growth, genetically engineering both strains to build the production pathway with maximum yield, and analyzing the presence of p-Coumarate and its pathway intermediates using High Performance Liquid Chromatography (HPLC). To date, the results of this project include successful integration of C4H activity into the yeast strain BY4741 ∆FDC1, yielding a strain that completely consumed trans-cinnamate (initial concentration of 50 mg/L) and produced ~56 mg/L p-coumarate, a resting cell assay of the co-culture that produced 0.23 mM p-coumarate from an initial L-Phenylalanine concentration of 1.14 mM, and toxicity tests that confirmed the toxicity of trans-cinnamate to yeast for concentrations above ~50 mg/L. The hope for this project is to create a feasible method for producing p-Coumarate sustainably.
ContributorsJohnson, Kaleigh Lynnae (Author) / Nielsen, David (Thesis director) / Thompson, Brian (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
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Description
Escherichia coli is a bacterium that is used widely in metabolic engineering due to its ability to grow at a fast rate and to be cultured easily. E. coli can be engineered to produce many valuable chemicals, including biofuels and L-Phenylalanine—a precursor to many pharmaceuticals. Significant cell growth occurs in

Escherichia coli is a bacterium that is used widely in metabolic engineering due to its ability to grow at a fast rate and to be cultured easily. E. coli can be engineered to produce many valuable chemicals, including biofuels and L-Phenylalanine—a precursor to many pharmaceuticals. Significant cell growth occurs in parallel to the biosynthesis of the desired biofuel or biochemical product, and limits product concentrations and yields. Stopping cell growth can improve chemical production since more resources will go toward chemical production than toward biomass. The goal of the project is to test different methods of controlling microbial uptake of nutrients, specifically phosphate, to dynamically limit cell growth and improve biochemical production of E. coli, and the research has the potential to promote public health, sustainability, and environment. This can be achieved by targeting phosphate transporter genes using CRISPRi and CRISPR, and they will limit the uptake of phosphate by targeting the phosphate transporter genes in DNA, which will stop transcriptions of the genes. In the experiment, NST74∆crr∆pykAF, a L-Phe overproducer, was used as the base strain, and the pitA phosphate transporter gene was targeted in the CRISPRi and CRISPR systems with the strain with other phosphate transporters knocked out. The tested CRISPRi and CRISPR mechanisms did not stop cell growth or improved L-Phe production. Further research will be conducted to determine the problem of the system. In addition, the CRISPRi and CRISPR systems that target multiple phosphate transporter genes will be tested in the future as well as the other method of stopping transcriptions of the phosphate transporter genes, which is called a tunable toggle switch mechanism.
ContributorsPark, Min Su (Author) / Nielsen, David (Thesis director) / Machas, Michael (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description

Lignin is a naturally abundant source of aromatic carbon but is largely underutilized in industry because it is difficult to decompose. Under the current study we engineered Corynebacterium glutamicum for the depolymerization of lignin with the goal of using it as raw feed for the sustainable production of valuable chemicals.

Lignin is a naturally abundant source of aromatic carbon but is largely underutilized in industry because it is difficult to decompose. Under the current study we engineered Corynebacterium glutamicum for the depolymerization of lignin with the goal of using it as raw feed for the sustainable production of valuable chemicals. C. glutamicum is a standout candidate for the depolymerization and assimilation of lignin because of its performance as an industrial producer of amino acids, resistance to aromatic compounds in lignin, and low extracellular protease activity. Three different foreign and native ligninolytic enzymes were tested in combination with three signal peptides to assess lignin degradation efficacy. At this stage, six of the nine plasmid constructs have been constructed.

ContributorsEllis, Dylan Scott (Author) / Varman, Arul Mozhy (Thesis director) / Nannenga, Brent (Committee member) / Nowroozi, Farnaz (Committee member) / Chemical Engineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Ather Arop is bilingual. He is also fluent in Spanish and speaks some French. “Lost Boys Found” is an ongoing, interdisciplinary project that is collecting, recording and archiving the oral histories of the Lost Boys/Girls of Sudan. The collection is a work-in-progress, seeking to record the oral history of as

Ather Arop is bilingual. He is also fluent in Spanish and speaks some French. “Lost Boys Found” is an ongoing, interdisciplinary project that is collecting, recording and archiving the oral histories of the Lost Boys/Girls of Sudan. The collection is a work-in-progress, seeking to record the oral history of as many Lost Boys/Girls as are willing, and will be used in a future book.
ContributorsArop, Ather (Interviewee) / Amparano, Julie (Director) / Garcia, James (Interviewer) / MacNeill, MacNeill (Editor)
Created2017-10-14
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Description
Due to the wide range of health properties flavonoids possess, flavonoids are sold in health supplements to the general public. Flavonoids are also utilized in research but have a high cost due to current production techniques. This project focuses on engineering two DNA recombinants to develop new strains of Corynebacterium

Due to the wide range of health properties flavonoids possess, flavonoids are sold in health supplements to the general public. Flavonoids are also utilized in research but have a high cost due to current production techniques. This project focuses on engineering two DNA recombinants to develop new strains of Corynebacterium glutamicum that can produce flavonoids pinocembrin and naringenin. After culturing Escherichia coli colonies containing genes of interest, the genes were collected and purified by PCR reactions. The recombinant plasmid was assembled using CPEC and successfully transformed into Escherichia coli, with plans to transform Corynebacterium glutamicum to experiment and determine which recombinant can produce more pinocembrin and naringenin. Design work for other DNA recombinants, which were not the focus of this project, was also completed.
ContributorsWong, Adam (Co-author, Co-author) / Varman, Arul Mozhy (Thesis director) / Nielsen, David (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
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Description
Cyanobacteria have the potential to efficiently produce L-serine, an industrially important amino acid, directly from CO2 and sunlight, which is a more sustainable and inexpensive source of energy as compared to current methods. The research aims to engineer a strain of Cyanobacterium Synechococcus sp. PCC 7002 that increases L-serine production

Cyanobacteria have the potential to efficiently produce L-serine, an industrially important amino acid, directly from CO2 and sunlight, which is a more sustainable and inexpensive source of energy as compared to current methods. The research aims to engineer a strain of Cyanobacterium Synechococcus sp. PCC 7002 that increases L-serine production by mutating regulatory mechanisms that natively inhibit its production and encoding an exporter. While an excess of L-serine was not found in the supernatant of the cell cultures, with further fine tuning of the metabolic pathway and culture conditions, high titers of L-serine can be found. With the base strain engineered, the work can be extended and optimized by deleting degradation pathways, tuning gene expression levels, optimizing growth conditions, and investigating the effects of nitrogen supplementation for the strain.
ContributorsAbed, Omar (Author) / Nielsen, David (Thesis director) / Jones, Christopher (Committee member) / Chemical Engineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
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Description
The field of bioprocess engineering has become an increasingly popular route to produce chemicals and fuels in a sustainable fashion. Bioprocessing is an interdisciplinary field that joins chemical engineering, metabolic engineering, and synthetic biology to tackle problems that will arise from the ongoing use of products derived from non-renewable resources.

The field of bioprocess engineering has become an increasingly popular route to produce chemicals and fuels in a sustainable fashion. Bioprocessing is an interdisciplinary field that joins chemical engineering, metabolic engineering, and synthetic biology to tackle problems that will arise from the ongoing use of products derived from non-renewable resources. This study will overlook two effective tools that are widely used in the bioprocessing field. The first tool that was studied was strain optimization for biochemical production. This involves genetic manipulation of microbial hosts to create strains that are more efficient at producing the desired products. The second tool that was studied was adaptive laboratory evolution which is used to enhance overall cellular fitness. Enhancing the overall fitness and efficiency of these microbial production factories, allows for innovation and growth in the biochemical industry. Creating sustainable and renewable solutions for both specialty and commodity chemicals.
Strain optimization was specifically studied by enhancing inorganic carbon uptake in synechococcus sp. 7002. It is desired to have both high flux and high affinity transport for the rapid and efficient uptake of HCO3- for enhanced cell growth. The results found that the regulatory gene for carbon transporters in synechococcus genome was successfully deleted. Increasing the toxicity limits of 2-Phenylethanol was done by using adaptive laboratory evolution (ALE). ALE is a widely used practice in biotechnology studies to gain insights on mechanisms of molecular evolution and to better define the functionality of microbial cell factories. It was found that after growing E. coli BW25113 under selective conditions the genome evolved for a higher fitness medium with an increased concentration of 2-Phenylethanol. Overall, two key tools used in bioprocess engineering were successful studied to gain a better insight on the future of biochemical production industry.
ContributorsSteeber, Gavin Lyle (Author) / Nielsen, David (Thesis director) / Jones, Christopher (Committee member) / Chemical Engineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
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Description

Flavonoids are important biomolecules with a variety of pharmaceutical and agricultural applications. Currently, isolating these compounds is done by plant extraction, however this process is hindered by large land and energy requirements. Previous groups have aimed to overcome these challenges by engineering microbes to produce these important compounds, however this

Flavonoids are important biomolecules with a variety of pharmaceutical and agricultural applications. Currently, isolating these compounds is done by plant extraction, however this process is hindered by large land and energy requirements. Previous groups have aimed to overcome these challenges by engineering microbes to produce these important compounds, however this is largely bottlenecked by the lack of intercellular malonyl-CoA availability. To remedy this, the genes matB and matC have been identified as coding for malonyl-CoA synthase and a putative dicarboxylate carrier protein, respectively. Other works have successfully engineered two variants, Streptomyces coelicolor and Rhizobium trifolii, of these genes into Escherichia coli, however this has yet to be accomplished in Gram-positive Corynebacterium glutamicum. Additionally, other groups have neglected to attempt tuning these genes with respect to one another by inserting in front of different inducible promoters. This study has successfully assembled two plasmids containing the Streptomyces coelicolor and Rhizobium trifolii variants of both matB and matC. Preliminary fermentations and GCMS results confirmed that little to none naringenin was produced without the matB-matC module. Additionally, preliminary fermentations revealed that the DelAro1 and DelAro3 strains can be used to reduce metabolism of aromatics like naringenin.

ContributorsRonstadt, Jason (Author) / Varman, Arul (Thesis director) / Nielsen, David (Committee member) / Liu, Fang (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor)
Created2022-05
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Description

The outlying cities of Phoenix's West Metropolitan experienced rapid growth in the past ten years. This trend is only going to continue with an average expected growth of 449-891% between 2000 and 2035 (ADOT, 2012). Phoenix is not new to growth and has consistently seen swaths of people added to

The outlying cities of Phoenix's West Metropolitan experienced rapid growth in the past ten years. This trend is only going to continue with an average expected growth of 449-891% between 2000 and 2035 (ADOT, 2012). Phoenix is not new to growth and has consistently seen swaths of people added to its population. This raises the question of what happened to the people who lived in Phoenix's West Valley during this period of rapid change and growth in their communities? What are their stories and what do their stories reveal about the broader public history of change in Phoenix's West Valley? In consideration of these questions, the community oral histories of eight residents from the West Valley were collected to add historical nuance to the limited archival records available in the area. From this collection, the previous notion of "post-war boomtowns” describing Phoenix’s West Valley was revealed to be highly inaccurate and dismissive of the residents' experiences who lived and formed their lives there.

ContributorsGeiser, Samantha (Author) / Campanile, Isabella (Co-author) / Martinez Orozco, Rafael (Thesis director) / O'Flaherty, Katherine (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor) / School of Mathematical and Statistical Sciences (Contributor)
Created2022-05