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- All Subjects: Molecular Biology
- All Subjects: Alzheimer's Disease
Description
Synechocystis sp PCC 6803 is a photosynthetic cyanobacterium that can be easily transformed to produce molecules of interest; this has increased Synechocystis’ popularity as a clean energy platform. Synechocystis has been shown to produce and excrete molecules such as fatty acids, isoprene, etc. after appropriate genetic modification. Challenges faced for large–scale growth of modified Synechocystis include abiotic stress, microbial contamination and high processing costs of product and cell material. Research reported in this dissertation contributes to solutions to these challenges. First, abiotic stress was addressed by overexpression of the heat shock protein ClpB1. In contrast to the wild type, the ClpB1 overexpression mutant (Slr1641+) tolerated rapid temperature changes, but no difference was found between the strains when temperature shifts were slower. Combination of ClpB1 overexpression with DnaK2 overexpression (Slr1641+/Sll0170+) further increased thermotolerance. Next, we used a Synechocystis strain that carries an introduced isoprene synthase gene (IspS+) and that therefore produces isoprene. We attempted to increase isoprene yields by overexpression of key enzymes in the methyl erythritol phosphate (MEP) pathway that leads to synthesis of the isoprene precursor. Isoprene production was not increased greatly by MEP pathway induction, likely because of limitations in the affinity of the isoprene synthase for the substrate. Finally, two extraction principles, two–phase liquid extraction (e.g., with an organic and aqueous phase) and solid–liquid extraction (e.g., with a resin) were tested. Two–phase liquid extraction is suitable for separating isoprene but not fatty acids from the culture medium. Fatty acid removal required acidification or surfactant addition, which affected biocompatibility. Therefore, improvements of both the organism and product–harvesting methods can contribute to enhancing the potential of cyanobacteria as solar–powered biocatalysts for the production of petroleum substitutes.
ContributorsGonzalez Esquer, Cesar Raul (Author) / Vermaas, Willem (Thesis advisor) / Chandler, Douglas (Committee member) / Bingham, Scott (Committee member) / Nielsen, David (Committee member) / Arizona State University (Publisher)
Created2013
Description
Alzheimer's disease (AD) is the most common type of dementia, affecting one in nine people age 65 and older. One of the most important neuropathological characteristics of Alzheimer's disease is the aggregation and deposition of the protein beta-amyloid. Beta-amyloid is produced by proteolytic processing of the Amyloid Precursor Protein (APP). Production of beta-amyloid from APP is increased when cells are subject to stress since both APP and beta-secretase are upregulated by stress. An increased beta-amyloid level promotes aggregation of beta-amyloid into toxic species which cause an increase in reactive oxygen species (ROS) and a decrease in cell viability. Therefore reducing beta-amyloid generation is a promising method to control cell damage following stress. The goal of this thesis was to test the effect of inhibiting beta-amyloid production inside stressed AD cell model. Hydrogen peroxide was used as stressing agent. Two treatments were used to inhibit beta-amyloid production, including iBSec1, an scFv designed to block beta-secretase site of APP, and DIA10D, a bispecific tandem scFv engineered to cleave alpha-secretase site of APP and block beta-secretase site of APP. iBSec1 treatment was added extracellularly while DIA10D was stably expressed inside cell using PSECTAG vector. Increase in reactive oxygen species and decrease in cell viability were observed after addition of hydrogen peroxide to AD cell model. The increase in stress induced toxicity caused by addition of hydrogen peroxide was dramatically decreased by simultaneously treating the cells with iBSec1 or DIA10D to block the increase in beta-amyloid levels resulting from the upregulation of APP and beta-secretase.
ContributorsSuryadi, Vicky (Author) / Sierks, Michael (Thesis advisor) / Nielsen, David (Committee member) / Dai, Lenore (Committee member) / Arizona State University (Publisher)
Created2014
Description
In our modern world the source of for many chemicals is to acquire and refine oil. This process is becoming an expensive to the environment and to human health. Alternative processes for acquiring the final product have been developed but still need work. One product that is valuable is butanol. The normal process for butanol production is very intensive but there is a method to produce butanol from bacteria. This process is better because it is more environmentally safe than using oil. One problem however is that when the bacteria produce too much butanol it reaches the toxicity limit and stops the production of butanol. In order to keep butanol from reaching the toxicity limit an adsorbent is used to remove the butanol without harming the bacteria. The adsorbent is a mesoporous carbon powder that allows the butanol to be adsorbed on it. This thesis explores different designs for a magnetic separation process to extract the carbon powder from the culture.
ContributorsChabra, Rohin (Author) / Nielsen, David (Thesis director) / Torres, Cesar (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor)
Created2015-05
Description
Alzheimer's disease (AD), which currently affects approximately 5.4 million Americans, is a type of dementia, which causes memory, cognitive, and behavioral problems. AD is among the top 10 leading causes of death in the United States, typically affecting people ages 65 and older. Beta-Amyloid (Aβ) is an Alzheimer's target protein, which starts as a single protein, but can misfold and bind to itself, forming larger chains and eventually fibrils and plaques of Aβ in the brain. Antibodies that bind to different regions and sizes of Aβ may prevent progression into a more toxic stage. The antibody worked with in this thesis, A4 scFv, binds to oligomeric Aβ. The objective of this antibody research is to optimize the production of functional antibodies, specifically A4, through modifications in the scFv growth process, in order to enhance the discovery of possible diagnostics and therapeutics for Alzheimer's disease. In order to produce functional A4 antibody, four complex sugars were tested in the E. Coli bacterial culture growth media that expresses the desired antibody. The sugars: sucrose, glucose, mannitol, and sorbitol were used in the growth process to improve the yield of functional antibody. Through the steps of growth, purification, and dialysis, the sugar sorbitol was found to provide the optimal results of ending functional antibody concentration. Once an ample amount of functional A4 scFv is produced, it can be used in assays as a biomarker for Alzheimer's disease.
ContributorsDolberg, Taylor Brianne (Author) / Sierks, Michael (Thesis director) / Nielsen, David (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor) / School of Sustainability (Contributor)
Created2014-05
Description
Enhancing the expression levels of Fabs (antigen-binding antibody fragments) in Escherichia coli is a difficult field that has a variety of potential exciting implications. The field has grown substantially in the past twenty years. The main area of difficulty is facilitating the entry of the antibody fragments into the periplasm of E. Coli, where the antibody fragments can be successfully expressed. Entry into the periplasm is difficult for antibody fragments due to their inability to fold in any other section besides the periplasm. Therefore it is necessary for the antibody to enter the periplasm in an unfolded state. Background research was done into inspecting the three primary methods of periplasmic entry: the Sec-dependent pathway, the SRP-dependent pathway (signal recognition particle) and the TAT-dependent pathway (twin arginine translocase). The Sec-dependent and SRP-dependent pathways were deemed more viable for expressing antibodies due to their ability to transfer an unfolded protein into the periplasm, which the TAT-dependent pathway cannot do. Academic research showed that the Sec-dependent and SRP-dependent pathways were equally viable methods, with more research being done into the Sec-dependent pathway, particularly of the OmpA signal sequence. Physical experiments were done using typical cloning procedures with slight modifications to the ligation step (Gibson Assembly was performed instead of normal ligation). These physical experiments showed that the Sec-dependent and SRP-dependent pathways were equally viable methods of periplasmic entry. The A4 and C6 antibodies were successfully expressed using these pathways. These antibodies were expressed on an SDS gel using 10% SDS. It was hypothesized that with further experimental modifications, using different signal sequences, Fabs can be expressed at higher and more consistent level.
ContributorsParker, Matthew David (Author) / Nannenga, Brent (Thesis director) / Nielsen, David (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
This dissertation focuses on the biosynthetic production of aromatic fine chemicals in engineered Escherichia coli from renewable resources. The discussed metabolic pathways take advantage of key metabolites in the shikimic acid pathway, which is responsible for the production of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. For the first time, the renewable production of benzaldehyde and benzyl alcohol has been achieved in recombinant E. coli with a maximum titer of 114 mg/L of benzyl alcohol. Further strain development to knockout endogenous alcohol dehydrogenase has reduced the in vivo degradation of benzaldehyde by 9-fold, representing an improved host for the future production of benzaldehyde as a sole product. In addition, a novel alternative pathway for the production of protocatechuate (PCA) and catechol from the endogenous metabolite chorismate is demonstrated. Titers for PCA and catechol were achieved at 454 mg/L and 630 mg/L, respectively. To explore potential routes for improved aromatic product yields, an in silico model using elementary mode analysis was developed. From the model, stoichiometric optimums maximizing both product-to-substrate and biomass-to-substrate yields were discovered in a co-fed model using glycerol and D-xylose as the carbon substrates for the biosynthetic production of catechol. Overall, the work presented in this dissertation highlights contributions to the field of metabolic engineering through novel pathway design for the biosynthesis of industrially relevant aromatic fine chemicals and the use of in silico modelling to identify novel approaches to increasing aromatic product yields.
ContributorsPugh, Shawn (Author) / Nielsen, David (Thesis advisor) / Dai, Lenore (Committee member) / Torres, Cesar (Committee member) / Lind, Mary Laura (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2016