Matching Items (64)
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 cyanobacterium Synechocystis sp. PCC 6803 performs oxygenic photosynthesis. Light energy conversion in photosynthesis takes place in photosystem I (PSI) and photosystem II (PSII) that contain chlorophyll, which absorbs light energy that is utilized as a driving force for photosynthesis. However, excess light energy may lead to formation of reactive oxygen species that cause damage to photosynthetic complexes, which subsequently need repair or replacement. To gain insight in the degradation/biogenesis dynamics of the photosystems, the lifetimes of photosynthetic proteins and chlorophyll were determined by a combined stable-isotope (15N) and mass spectrometry method. The lifetimes of PSII and PSI proteins ranged from 1-33 and 30-75 hours, respectively. Interestingly, chlorophyll had longer lifetimes than the chlorophyll-binding proteins in these photosystems. Therefore, photosynthetic proteins turn over and are replaced independently from each other, and chlorophyll is recycled from the damaged chlorophyll-binding proteins. In Synechocystis, there are five small Cab-like proteins (SCPs: ScpA-E) that share chlorophyll a/b-binding motifs with LHC proteins in plants. SCPs appear to transiently bind chlorophyll and to regulate chlorophyll biosynthesis. In this study, the association of ScpB, ScpC, and ScpD with damaged and repaired PSII was demonstrated. Moreover, in a mutant lacking SCPs, most PSII protein lifetimes were unaffected but the lifetime of chlorophyll was decreased, and one of the nascent PSII complexes was missing. SCPs appear to bind PSII chlorophyll while PSII is repaired, and SCPs stabilize nascent PSII complexes. Furthermore, aminolevulinic acid biosynthesis, an early step of chlorophyll biosynthesis, was impaired in the absence of SCPs, so that the amount of chlorophyll in the cells was reduced. Finally, a deletion mutation was introduced into the sll1906 gene, encoding a member of the putative bacteriochlorophyll delivery (BCD) protein family. The Sll1906 sequence contains possible chlorophyll-binding sites, and its homolog in purple bacteria functions in proper assembly of light-harvesting complexes. However, the sll1906 deletion did not affect chlorophyll degradation/biosynthesis and photosystem assembly. Other (parallel) pathways may exist that may fully compensate for the lack of Sll1906. This study has highlighted the dynamics of photosynthetic complexes in their biogenesis and turnover and the coordination between synthesis of chlorophyll and photosynthetic proteins.
ContributorsYao, Cheng I Daniel (Author) / Vermaas, Wim (Thesis advisor) / Fromme, Petra (Committee member) / Roberson, Robert (Committee member) / Webber, Andrew (Committee member) / Arizona State University (Publisher)
Created2011
Description
Photosynthesis is a critical process that fixes the carbon utilized in cellular respiration. In higher plants, the immutans gene codes for a protein that is both involved in carotenoid biosynthesis and plastoquinol oxidation (Carol et al 1999, Josse et al 2003). This plastoquinol terminal oxidase (PTOX) is of great interest in understanding electron flow in the plastoquinol pool. In order to characterize this PTOX, polyclonal antibodies were developed. Expression of Synechococcus WH8102 PTOX in E. coli provided a useful means to harvest the protein required for antibody production. Once developed, the antibody was tested for limit of concentration, effectiveness in whole cell lysate, and overall specificity. The antibody raised against PTOX was able to detect as low as 10 pg of PTOX in SDS-PAGE, and could detect PTOX extracted from lysed Synechococcus WH8102. The production of this antibody could determine the localization of the PTOX in Synechococcus.
ContributorsKhan, Mohammad Iqbal (Author) / Moore, Thomas (Thesis director) / Redding, Kevin (Committee member) / Roberson, Robert (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
Methane (CH4) is very important in the environment as it is a greenhouse gas and important for the degradation of organic matter. During the last 200 years the atmospheric concentration of CH4 has tripled. Methanogens are methane-producing microbes from the Archaea domain that complete the final step in breaking down organic matter to generate methane through a process called methanogenesis. They contribute to about 74% of the CH4 present on the Earth's atmosphere, producing 1 billion tons of methane annually. The purpose of this work is to generate a preliminary metabolic reconstruction model of two methanogens: Methanoregula boonei 6A8 and Methanosphaerula palustris E1-9c. M. boonei and M. palustris are part of the Methanomicrobiales order and perform hydrogenotrophic methanogenesis, which means that they reduce CO2 to CH4 by using H2 as their major electron donor. Metabolic models are frameworks for understanding a cell as a system and they provide the means to assess the changes in gene regulation in response in various environmental and physiological constraints. The Pathway-Tools software v16 was used to generate these draft models. The models were manually curated using literature searches, the KEGG database and homology methods with the Methanosarcina acetivorans strain, the closest methanogen strain with a nearly complete metabolic reconstruction. These preliminary models attempt to complete the pathways required for amino acid biosynthesis, methanogenesis, and major cofactors related to methanogenesis. The M. boonei reconstruction currently includes 99 pathways and has 82% of its reactions completed, while the M. palustris reconstruction includes 102 pathways and has 89% of its reactions completed.
ContributorsMahendra, Divya (Author) / Cadillo-Quiroz, Hinsby (Thesis director) / Wang, Xuan (Committee member) / Stout, Valerie (Committee member) / Barrett, The Honors College (Contributor) / Computing and Informatics Program (Contributor) / School of Life Sciences (Contributor) / Biomedical Informatics Program (Contributor)
Created2014-05
Description
Transmission electron microscopy has been used to identify poly-3-hydroxybutyrate (PHB) granules in cyanobacteria for over 40 years. Electron-transparent (sometimes containing a slightly electron-dense area in the inclusions) or slightly electron-dense spherical inclusions found in transmission electron micrographs of cyanobacteria are often assumed to be PHB granules. The aim of this study was to test this assumption in Synechocystis sp. PCC 6803, and to determine whether all inclusions of this kind are indeed PHB granules. Based on the results gathered, it is concluded that not all of the slightly electron-dense spherical inclusions are PHB granules in Synechocystis sp. PCC 6803. This result is potentially applicable to other cyanobacteria.
ContributorsTsang, Tin Ki (Author) / Vermaas, Willem F. J. (Thesis director) / Nielsen, David (Committee member) / Roberson, Robert (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
There is a wide intersection where animal and human lives interact or mimic each other behaviorally or biologically. A lot of the products that are part of our day-to-day were first validated by animals, and eventually found their way to us. From food to beauty products to scientific developments, animals deal with a lot behind the scenes. Some humans are cognizant of what is happening backstage, while others only see the final presentation. Either way, all of us have our opinions in support or against animal treatment. The project is heavily inspired from my experience in a neurorehabilitation lab, so the foundation is similar to the structure and function of neurons. Through this project, I am focusing on one aspect of this debate, which is animal testing in the scietific setting. The goal of the project is not to force the viewer to choose one side, but to understand the big picture and the reasoning of the opposing side.
ContributorsSharma, Bhavya (Author) / Beiner, Susan (Thesis director) / Roberson, Robert (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Amphibians around the world are suffering the effects of the chytrid fungus, Batrachochytrium dendrobatidis (Bd). Whenever amphibians are housed in captivity, they must go through a decontamination protocol to ensure they are not infected with diseases such as Bd. Itraconazole is the most commonly used fungicide used in these protocols. This study set out to determine if Bd could develop resistance or tolerance to itraconazole. Two 24 well plates were prepared with different concentrations of itraconazole with Bd zoospores added. Plate 1 had concentrations similar to what animals are currently being treated with in decontamination protocols. Plate 2 had concentrations at and below the published minimum inhibitory concentration values (MIC). Plate 1 displayed the ability of itraconazole to kill Bd sporangia with higher concentrations and Plate 2 showed that even under published MIC values, Bd still struggled to complete its reproductive cycle. I find the evolution of a resistant/tolerant strain of Bd unlikely given the efficacy of this drug, the sensitivity of Bd to itraconazole, and the lack of evidence of the completion of Bd’s reproductive cycle under the conditions used in this study.
ContributorsSommer, Bradley Dean (Author) / Collins, James (Thesis director) / Roberson, Robert (Committee member) / Brus, Evan (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Traditional methods of genetic engineering are often limited to relatively few rounds of gene additions, deletions, or alterations due to a lack of additional available antibiotic resistance markers. Counter-selection marker methods can be used to remove and reuse marker genes as desired, resulting in markerless engineered strains and allowing for theoretically unlimited rounds of genetic modifications. The development of suitable counter-selection markers is vital for the development of model organisms such as cyanobacteria as biotechnological platforms.
In the hopes of providing other researchers with a new tool for markerless genetic engineering of cyanobacteria, the toxin MazF from E. coli was developed as a counter-selection marker in the most widely used cyanobacterium, Synechocystis sp. PCC 6803. The mazF gene from E. coli was cloned and inserted into a plasmid vector for downstream transformation of Synechocystis. The plasmid construct also contained two homologous flanking regions for integration of the insert into the Synechocystis genome, a nickel-inducible response regulator and promoter to control MazF expression, and a kanamycin resistance gene to serve as the antibiotic marker. In order to ensure the mazF plasmids could be cloned in a MazF-sensitive E. coli host even with slight promoter leakage, MazF expression was toned down by decreasing the efficiency of translation initiation by inserting base pairs between the ribosome binding site and the start codon of the mazF gene. Following successful cloning by E. coli, the mazF plasmids were then used to transform Synechocystis to create mazF mutant strains. Genomic analysis confirmed the successful transformation and segregation of mazF mutant strains containing the desired marker cassette. Phenotypic analysis revealed both growth arrest and production of mazF transcripts in mazF mutant strains following the addition of nickel to the cell cultures, indicating successful nickel-induced MazF expression as desired.
In the hopes of providing other researchers with a new tool for markerless genetic engineering of cyanobacteria, the toxin MazF from E. coli was developed as a counter-selection marker in the most widely used cyanobacterium, Synechocystis sp. PCC 6803. The mazF gene from E. coli was cloned and inserted into a plasmid vector for downstream transformation of Synechocystis. The plasmid construct also contained two homologous flanking regions for integration of the insert into the Synechocystis genome, a nickel-inducible response regulator and promoter to control MazF expression, and a kanamycin resistance gene to serve as the antibiotic marker. In order to ensure the mazF plasmids could be cloned in a MazF-sensitive E. coli host even with slight promoter leakage, MazF expression was toned down by decreasing the efficiency of translation initiation by inserting base pairs between the ribosome binding site and the start codon of the mazF gene. Following successful cloning by E. coli, the mazF plasmids were then used to transform Synechocystis to create mazF mutant strains. Genomic analysis confirmed the successful transformation and segregation of mazF mutant strains containing the desired marker cassette. Phenotypic analysis revealed both growth arrest and production of mazF transcripts in mazF mutant strains following the addition of nickel to the cell cultures, indicating successful nickel-induced MazF expression as desired.
ContributorsNewell, Phoebe Quynh (Co-author) / Newell, Phoebe (Co-author) / Vermaas, Willem (Thesis director) / Wang, Xuan (Committee member) / Li, Shuqin (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The primary objective of this project is to further the knowledge about SCL26 family of anion transporters. The goals of the experiment were to find the lowest sulfate concentration where the yeast without Sulp1 and Sulp2 is able to grow, but it grows very slowly, and to find a higher sulfate concentration where the yeast grows quickly, with or without the sulfate transporters. The lowest sulfate concentration where the yeast without the sulfate transporters is able to grow was determined to be 2-4 mM, however, this range can likely be refined by more quantitative analytical methods. At a sulfate concentration of 20 mM sulfate or higher, the yeast is able to grow quickly without high-affinity sulfate transporters. The next step in the project is to re-introduce the Sulp1 and Sulp2 genes into the yeast, so that growth in low and high sulfate conditions can be compared with and without the Sulp1 and Sulp2 proteins. The long-term goals of the project are to bring experience with yeast to Dr. Nannenga’s structural discovery lab, to determine if yeast sulfate transporters respond in the same way to drug candidates as human sulfate transporters, and to determine the structure of the proteins using cryo-electron microscopy.
ContributorsCall, Nicolas I (Author) / Nannenga, Brent (Thesis director) / Wang, Xuan (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Abiotic stresses, such as heat, can drive protein misfolding and aggregation, leading to inhibition of cellular function and ultimately cell death. Unexpectedly, a thermotolerant Escherichia coli was identified from a pool of antibiotic resistant RNA polymerase β subunit (rpoB) mutants. This stress tolerant phenotype was characterized through exposure to high temperature and ethanol. After 30-minute exposure of cells to 55°C or 25% ethanol, the mutant displayed 100 times greater viability than the wild-type, indicating that the rpoB mutation may have broadly affected the cellular environment to reduce protein misfolding and/or prevent protein aggregation. To further test this hypothesis, we examined thermotolerance of cells lacking heat shock chaperone DnaJ (Hsp40), which is a cochaperone of one of the most abundant and conserved chaperones, DnaK (Hsp70). The deletion of dnaJ led to severe growth defects in the wild-type, namely a slower growth rate and extreme filamentation at 42°C. The severity of the growth defects increased after additionally deleting DnaJ analog, CbpA. However, these defects were significantly ameliorated by the rpoB mutation. Finally, the rpoB mutant was found to be minimally affected by the simultaneous depletion of DnaK and DnaJ compared to the wild-type, which failed to form single colonies at 37°C and 42°C. Based on these observations, it is proposed that the rpoB mutant’s robust thermotolerant phenotype results from a cellular environment protective against protein aggregation or improper folding. The folding environment of the rpoB mutants should be further examined to elucidate the mechanism by which both antibiotic resistance and thermotolerance can be conferred.
ContributorsYeh, Melody (Author) / Misra, Rajeev (Thesis director) / Wang, Xuan (Committee member) / Kelly, Keilen (Committee member) / School of Life Sciences (Contributor) / School of International Letters and Cultures (Contributor) / School of Human Evolution & Social Change (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05