Matching Items (14)
Filtering by
- Creators: Barrett, The Honors College
Description
Increasing energy and environmental problems describe the need to develop renewable chemicals and fuels. Global research has been targeting using microbial systems on a commercial scale for synthesis of valuable compounds. The goal of this project was to refactor and overexpress b6-f complex proteins in cyanobacteria to improve photosynthesis under dynamic light conditions. Improvement in the photosynthetic system can directly relate to higher yields of valuable compounds such as carotenoids and higher yields of biomass which can be used as energy molecules. Four engineered strains of cyanobacteria were successfully constructed and overexpressed the corresponding four large subunits in the cytochrome b6-f complex. No significant changes were found in cell growth or pigment titer in the modified strains compared to the wild type. The growth assay will be performed at higher and/or dynamic light intensities including natural light conditions for further analysis.
ContributorsNauroth, Benjamin (Author) / Varman, Arul (Thesis director) / Singharoy, Abhishek (Committee member) / Li, Han (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
This thesis focused on the development of a system that can sense light intensity and then control a smart film to provide the optimal light intensity for cyanobacteria. The overarching goal of this project is to further the study of biofuels as an alternative energy source by increasing growth rates. If more algae or cyanobacteria can be grown per day, then the cost to produce the biofuel will decrease. To achieve this goal, PDLC (polymer dispersed liquid crystal) film was selected to be controlled due to its unique properties. It can be controlled with electricity and has variable states, in other words, not restricted to simply on or off. It also blocks 80% ultraviolet light and reduces thermal heat gain by 40% which is an important consideration for outdoor growing situations. To control the film, a simple control system was created using an Arduino Uno, SainSmart 8 channel relay board, an inverter, and a power supply. A relay board was utilized to manage the 40 volts required by the PDLC film and protected the electronics on the Arduino Uno. To sense the light intensity, the Arduino Uno was connected to a photoresistor, which changes resistance with light intensity. A 15 day test of two flasks of Cyanobacteria Synechocycstis sp. 6803, one shaded by the PDLC film, and the other unshaded, yielded 65% difference in optical densities. Overall, the experiment showed promise for controlling light intensity for photobioreactors. Ideally, this research will help to optimize light intensities when growing cyanobacteria or algae outdoors or it will help to discover what an ideal light intensity is by allowing a researcher unprecedented control.
ContributorsRoney, Kitt Alicia (Author) / Nielsen, David (Thesis director) / Middleton, James (Committee member) / Barrett, The Honors College (Contributor) / Mechanical and Aerospace Engineering Program (Contributor)
Created2015-05
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
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
Measuring changes in concentration within a dynamic system can be accomplished with a simple Arduino powered system. Currently, the system is utilized in cyanobacteria CO2 fixation experiments, where the fixation rates of multiple cultures can be measured simultaneously. The system employs solenoids in parallel and can be applied for n number of outlet streams, all are connected to one large manifold which feeds to a CO2 concentration probe. In the future, the system can be modified to fit other simple dynamic gas systems.
ContributorsInnes, Sean (Author) / Nielsen, David (Thesis director) / Jones, Christopher (Committee member) / Barrett, The Honors College (Contributor)
Created2021-12
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
The production of sustainable biochemicals has been a major topic of discussion in recent years. Using microbial cells for their production through genetic engineering has been a major topic of research. Cyanobacteria have been considered as a viable candidate for such production. However, the slow growth rate of the cells presents a challenge for the possibility of scaling for use in industrial settings. This project focuses on two different solutions for this problem. The first is using four different engineered strains of Synechocystis sp. PCC 6803 that overexpress the proteins in the b6f complex to improve photosynthetic efficiency. It was found that the strains PetB and PetD showed an increase in growth rate compared to wild type cells. This was especially true under mixotrophic conditions and with a light intensity of 100 µmol photons*m-2s-1 for 3 days. The second solution is by using a newly discovered marine strain of cyanobacteria, Synechococcus sp. PCC 11901, which has a higher reported growth rate. Higher growth rates were achieved for this strain when it was grown mixotrophically with glycerol, and when grown in bubble cultures with aeration.
ContributorsWinsor, Kira Varga (Author) / Varman, Arul Mohzy (Thesis director) / Vermaas, Wim (Committee member) / School of International Letters and Cultures (Contributor) / Chemical Engineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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