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- Creators: School of Life Sciences
- Status: Published
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
Sexually transmitted diseases like gonorrhea and chlamydia, standardly treated with antibiotics, produce over 1.2 million cases annually in the emergency department (Jenkins et al., 2013). To determine a need for antibiotics, hospital labs utilize bacterial cultures to isolate and identify possible pathogens. Unfortunately, this technique can take up to 72 hours, leading to several physicians presumptively treating patients based solely on history and physical presentation. With vague standards for diagnosis and a high percentage of asymptomatic carriers, several patients undergo two scenarios; over- or under-treatment. These two scenarios can lead to consequences like unnecessary exposure to antibiotics and development of secondary conditions (for example: pelvic inflammatory disease, infertility, etc.). This presents a need for a laboratory technique that can provide reliable results in an efficient matter. The viability of DNA-based chip targeted for C. trachomatis, N. gonorrhoeae, and other pathogens of interest were evaluated. The DNA-based chip presented several advantages as it can be easily integrated as a routine test given the process is already well-known, is customizable and able to target multiple pathogens within a single test and has the potential to return results within a few hours as opposed to days. As such, implementation of a DNA-based chip as a diagnostic tool is a timely and potentially impactful investigation.
ContributorsCharoenmins, Patherica (Author) / Penton, Christopher (Thesis director) / Moore, Marianne (Committee member) / College of Integrative Sciences and Arts (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
“Extremophile” is used to describe life that has adapted to extreme conditions and the conditions they live in are often used to understand the limits of life. In locations with low precipitation and high solar radiation, photosynthetic cyanobacteria can colonize the underside of quartz fragments, forming ‘hypoliths.’ The quartz provides protection against wind, reduces solar radiation, and slows the rate of evaporation following infrequent rain or fog events. In most desert systems, vascular plants are the main primary producers. However, hypoliths might play a key role in carbon fixation in hyperarid deserts that are mostly devoid of vegetation. I investigated hypolith distribution and carbon fixation at six sites along a rainfall and fog gradient in the central Namib Desert in Namibia. I used line point intersect transects to assess ground cover (bare soil, colonized quartz fragment, non-colonized quartz fragment, non-quartz rock, grass, or lichen) at each site. Additionally, at each site I selected 12 hypoliths and measured cyanobacteria colonization on quartz and measured CO2 flux of hypoliths at five of the six sites.
Ground cover was fairly similar among sites, with bare ground > non-colonized quartz fragments > colonized quartz fragments > non-quartz rocks. Grass was present only at the site with the highest mean annual precipitation (MAP) where it accounted for 1% of ground cover. Lichens were present only at the lowest MAP site, where they accounted for 30% of ground cover. The proportion of quartz fragments colonized generally increased with MAP, from 5.9% of soil covered by colonized hypoliths at the most costal (lowest MAP) site, to 18.7% at the most inland (highest MAP) site. There was CO2 uptake from most hypoliths measured, with net carbon uptake rates ranging from 0.3 to 6.4 μmol m-2 s-1 on well hydrated hypoliths. These carbon flux values are similar to previous work in the Mojave Desert. Our results suggest that hypoliths might play a key role in the fixation of organic carbon in hyperarid ecosystems where quartz fragments are abundant, with MAP constraining hypolith abundance. A better understanding of these extremophiles and the niche they fill could give an understanding of how microbial life might exist in extraterrestrial environments similar to hyperarid deserts.
Ground cover was fairly similar among sites, with bare ground > non-colonized quartz fragments > colonized quartz fragments > non-quartz rocks. Grass was present only at the site with the highest mean annual precipitation (MAP) where it accounted for 1% of ground cover. Lichens were present only at the lowest MAP site, where they accounted for 30% of ground cover. The proportion of quartz fragments colonized generally increased with MAP, from 5.9% of soil covered by colonized hypoliths at the most costal (lowest MAP) site, to 18.7% at the most inland (highest MAP) site. There was CO2 uptake from most hypoliths measured, with net carbon uptake rates ranging from 0.3 to 6.4 μmol m-2 s-1 on well hydrated hypoliths. These carbon flux values are similar to previous work in the Mojave Desert. Our results suggest that hypoliths might play a key role in the fixation of organic carbon in hyperarid ecosystems where quartz fragments are abundant, with MAP constraining hypolith abundance. A better understanding of these extremophiles and the niche they fill could give an understanding of how microbial life might exist in extraterrestrial environments similar to hyperarid deserts.
ContributorsMonus, Brittney Daniel (Author) / Throop, Heather (Thesis director) / Hall, Sharon (Committee member) / Cadillo-Quiroz, Hinsby (Committee member) / School of Life Sciences (Contributor) / School of Earth and Space Exploration (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Acyl Carrier Protein (ACP) is a small, acidic protein that plays an essential role in fatty acid synthesis by elongating fatty acid chains. ACP was isolated from an extract of a modified strain of Synechocystis sp. PCC 6803 that contains a thioesterase and from which the acyl-ACP synthetase has been deleted. Using ammonium sulfate precipitation to isolate a crude protein fraction containing ACP, immunoblot analysis was performed to determine relative amounts of free and acylated-ACP in the cell. The nature of fatty acids attached to ACP was determined by creating butylamide derivatives that were analyzed using GC/MS. Immunoblot analysis showed a roughly 1:1 ratio of acylated ACP to free ACP in the cell depending on the nutritional state of the cell. From GC/MS data it was determined that palmitic acid was the predominate component of acyl groups attached to ACP. The results indicate that there is a significant amount of acyl-ACP, a feedback inhibitor of early steps in the fatty acid biosynthesis pathway, in the cell. Moreover, the availability of free ACP may also limit fatty acid biosynthesis. Most likely it is necessary for ACP to be overexpressed or to have the palmitic acid cleaved off in order to synthesize optimal amounts of lauric acid to be used for cyanobacterial biofuel production.
ContributorsWu, Sharon Gao (Author) / Vermaas, Willem (Thesis director) / Redding, Kevin (Committee member) / School of Sustainability (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Cyanobacteria and microalgae help reduce the environmental impact of human energy consumption by playing a vital role in carbon and nitrogen cycling. They are also used in various applications like biofuel production, food, medicine, and bioremediation. Understanding how these organisms respond to stress is important for efficient recovery strategies and sustainable outcomes. This study investigated the effects of low-level bleaching and thermal stress on cyanobacteria and microalgae, specifically Synechocystis, Chlorella, and Scenedesmus. The role of ferroptosis, an iron-dependent form of cell death, in the degradation of cellular components under these stressors was examined. Flow cytometry and spectrophotometry were used to measure changes in cellular health and viability. The results showed that temperature influences the type of cell death mechanism and can impact photosynthetic organisms. When treated with Liproxstatin-1, an inhibitor of ferroptosis, both Synechocystis and Chlorella experienced a decrease in oxidative damage, suggesting a potential protective role for the compound. Further investigation into ferroptosis and other forms of cell death, as well as identifying additional inhibitory molecules, could lead to strategies for mitigating oxidative stress and enhancing the resilience of cyanobacteria and microalgae.
ContributorsRayes, Rammy (Author) / Rittmann, Bruce (Thesis director) / Eustance, Everett (Committee member) / Lewis, Christine (Committee member) / Khdour, Omar (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2023-05
Description
Desert organisms lead harsh lives owing to the extreme, often unpredictable environmental conditions they endure. Climate change will likely make their existence even harsher. Predicting the ecological consequences of future climate scenarios thus requires understanding how the biota will be affected by climatic shifts. Biological soil crusts (biocrusts) are an important ecosystem component in arid lands, one that covers large portions of the landscape, improving soil stability and fertility. Because cyanobacteria are biocrust’s preeminent primary producers, eking out an existence during short pulses of precipitation, they represent a relevant global change object of study. I assessed how climate scenarios predicted for the Southwestern United States (US) will affect biocrusts using long-term, rainfall-modifying experimental set-ups that imposed either more intense drought, a seasonally delayed monsoon season, or a shift to smaller but more frequent precipitation events. I expected drought to be detrimental, but not a delay in the monsoon season. Surprisingly, both treatments showed similar effects on cyanobacterial community composition and population size after four years. While successionally incipient biocrusts were unaffected, mature biocrusts lost biomass and diversity with treatment, especially among nitrogen-fixing cyanobacteria. In separate experiments, I assessed the effect of rainfall with modified event size and frequency after a decade of treatment. Small, frequent rainfall events surprisingly enhanced the diversity and biomass of bacteria and cyanobacteria, with clear winners and losers: nitrogen-fixing Scytonema sp. benefited, while Microcoleus vaginatus lost its dominance. As an additional finding, I could also show that water addition is not always beneficial to biocrusts, calling into question the notion that these are strictly water-limited systems.
Finally, results interpretation was severely hampered by a lack of appropriate systematic treatment for an important group of biocrust cyanobacteria, the “Microcoleus steenstrupii complex”. I characterized the complex using a polyphasic approach, leading to the formal description of a new family (Porphyrosiphonaceae) of desiccation resistant cyanobacteria that includes 11 genera, of which 5 had to be newly described. Under the new framework, the distribution and abundance of biocrust cyanobacteria with respect to environmental conditions can now be understood. This body of work contributes significantly to explain current distributional patterns of biocrust cyanobacteria and to predict their fate in the face of climate change.
Finally, results interpretation was severely hampered by a lack of appropriate systematic treatment for an important group of biocrust cyanobacteria, the “Microcoleus steenstrupii complex”. I characterized the complex using a polyphasic approach, leading to the formal description of a new family (Porphyrosiphonaceae) of desiccation resistant cyanobacteria that includes 11 genera, of which 5 had to be newly described. Under the new framework, the distribution and abundance of biocrust cyanobacteria with respect to environmental conditions can now be understood. This body of work contributes significantly to explain current distributional patterns of biocrust cyanobacteria and to predict their fate in the face of climate change.
ContributorsMoreira Camara Fernandes, Vanessa (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Rudgers, Jennifer (Committee member) / Sala, Osvaldo (Committee member) / Penton, Christopher (Committee member) / Arizona State University (Publisher)
Created2020
Description
Prochlorococcus marinus (MED4), a genus of marine picocyanobacteria that proliferates in open oligotrophic ocean, is one of the most abundant photosynthetic microbes in the world, estimated to contribute up to 10% of the ocean’s primary production. The productivity of these microorganisms is controlled by macronutrient availability in the surface waters. The ratio of macronutrients in the ocean was defined, by Alfred Redfield, as an elemental ratio of 106C:16N:1P. However, the C:N:P ratio varies based on region, season, temperature and irradiance, as well as the composition of the primary producers. In oligotrophic gyres, these nutrient ratios are elevated from the Redfield stoichiometry, but whether this ratio exerts influence on the growth rate of the organism has not been investigated. Elemental stoichiometry of available nutrients can affect the aggregation of organic carbon and exportation of the particles to the ocean depths. The purpose of this study was to investigate the effects of nutrient limitation on aggregation and transparent exopolymeric particle (TEP) production which aids in aggregation. My findings suggested that nutrient limitation reduces TEP production and does not increase aggregate volume concentration. With continued warming, certain regions of the ocean will become more oligotrophic, which further decreases the nutrient supply available for Prochlorococcus. My research shows that this could lead to decreased exportation of organic carbon matter to the depths of the sea.
ContributorsRoy, Kevin Thomas (Author) / Neuer, Susanne (Thesis director) / Cadillo-Quiroz, Hinsby (Committee member) / Cruz, Bianca (Committee member) / Department of Psychology (Contributor) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The changes in marine ecological conditions brought on by warming and stratification of the oceans have radically shifted many marine environments around the globe. This project aimed to better characterize the aggregation behavior of the abundant picocyanobacterium Prochlorococcus marinus, which is hypothesized to dominate over other phytoplankton as the primary autotroph in increasingly warmer and nutrient poor oceans. This aggregation, believed to be mediated through the secretion of sticky Transparent Exopolymeric Substances (TEP), might be key for Prochlorococcus to sink throughout the ocean and serve as a source of carbon to other communities within its environment. Considering the relatively low concentration of TEP secreted by Prochlorococcus when on its own, this study explored the synergistic effect that heterotrophic bacteria and inorganic minerals in the surrounding seawater may have on the aggregation of P. marinus. This was done by inoculating P. marinus and the model heterotroph Marinobacter adhaerens HP15 individually and mixed in cylindrical roller tanks with the addition of ballasting clay minerals into roller tanks to simulate constant sinking for 7 days. The aggregates which formed after rolling were quantified and their sinking velocities and excess densities were measured. Our results indicate that the most numerous and densest aggregates formed when Prochlorococcus was in the presence of both M. adhaerens and kaolinite clay particles. I will discuss how methodology, particularly cell number, may play a role in the enhanced aggregation that I found when Prochlorococcus was cultured together with the Marinobacter.
ContributorsAouad, Samer Ghassan (Author) / Neuer, Susanne (Thesis director) / Cadillo-Quiroz, Hinsby (Committee member) / Cruz, Bianca (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05