Matching Items (5)
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- All Subjects: Cyanobacteria
- Creators: Wang, Xuan
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
The basic scheme for photosynthesis suggests the two photosystems existing in parity with one another. However, cyanobacteria typically maintain significantly more photosystem I (PSI) than photosystem II (PSII) complexes. I set out to evaluate this disparity through development and analysis of multiple mutants of the genetically tractable cyanobacterium Synechocystis sp. PCC 6803 that exhibit a range of expression levels of the main proteins present in PSI (Chapter 2). One hypothesis was that the higher abundance of PSI in this organism is used to enable more cyclic electron flow (CEF) around PSI to contribute to greater ATP synthesis. Results of this study show that indeed CEF is enhanced by the high amount of PSI present in WT. On the other hand, mutants with less PSI and less cyclic electron flow appeared able to maintain healthy levels of ATP synthesis through other compensatory mechanisms. Reduction in PSI abundance is naturally associated with reduced chlorophyll content, and mutants with less PSI showed greater primary productivity as light intensity increased due to increased light penetration in the cultures. Another question addressed in this research project involved the effect of deletion of flavoprotein 3 (an electron sink for PSI-generated electrons) from mutant strains that produce and secrete a fatty acid (Chapter 3). Removing Flv3 increased fatty acid production, most likely due to increased abundance of reducing equivalents that are key to fatty acid biosynthesis. Additional components of my dissertation research included examination of alkane biosynthesis in Synechocystis (Chapter 4), and effects of attempting to overexpress fibrillin genes for enhancement of stored compounds (Chapter 5). Synechocystis is an excellent platform for metabolic engineering studies with its photosynthetic capability and ease of genetic alteration, and the presented research sheds light on multiple aspects of its fundamental biology.
ContributorsMoore, Vickie (Author) / Vermaas, Willem (Thesis advisor) / Wang, Xuan (Committee member) / Roberson, Robert (Committee member) / Gaxiola, Roberto (Committee member) / Bingham, Scott (Committee member) / Arizona State University (Publisher)
Created2017
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
To efficiently produce biofuels and meet the planet’s rising energy demands, different biofuel production methods need to be developed and improved. One of the ways is to produce fatty acid methyl esters (FAMEs) in Synechocystis sp. PCC 6803, a versatile strain of cyanobacteria. In this thesis, Synechocystis was engineered to produce and excrete methyl laurate. In this pathway, first, lauroyl-ACP from fatty acid biosynthesis is converted to laurate by a thioesterase (TE) from Umbellularia californica. Then, the laurate is methylated to methyl laurate by a juvenile hormone acid O-methyltransferase (DmJHAMT) from Drosophila melanogaster. The TE/∆slr1609 strain of Synechocystis sp. PCC 6803 contains the TE gene and lacks the slr1609 gene encoding an acyl–acyl carrier protein synthetase, which functions in free fatty acid reuptake. The DmJHAMT gene was introduced into this strain for FAME production.
The DmJHAMT gene was cloned into a vector that contains neutral sites from the Synechocystis genome, making it suitable for homologous recombination, and a kanamycin resistance gene, for selection. The obtained plasmid was verified using restriction digests and Sanger sequencing. The sequence analysis and comparison of the cDNA in the obtained plasmid and the mRNA transcript of the same gene revealed three amino acid differences. Subsequent comparison with homologous genes in other Drosophila species revealed the differences in the cDNA match those of the other species, and thus, the gene most likely is functional.
The plasmid was transformed into Synechocystis, and PCRs were used to confirm proper integration and segregation. The TE/∆slr1609/DmJHAMT strain produced 62 mg/L methyl laurate in 12 days under a light intensity of 150 µmol photons m-2 s-1, bubbled with 0.5% CO2 at a rate of 30 mL/min, and supplemented with 0.5 mM methionine. The laurate levels did not decrease over time, but instead, remained stagnant after day 3. When the strain was grown in the same conditions without methionine, the laurate concentrations continued to increase above 400 µM, suggesting minimal methyl laurate production and thus a strong need for methionine supplementation. This work provides further evidence of the viability and success of the introduced FAME production pathway, and improved efficiency may be gained in the future.
The DmJHAMT gene was cloned into a vector that contains neutral sites from the Synechocystis genome, making it suitable for homologous recombination, and a kanamycin resistance gene, for selection. The obtained plasmid was verified using restriction digests and Sanger sequencing. The sequence analysis and comparison of the cDNA in the obtained plasmid and the mRNA transcript of the same gene revealed three amino acid differences. Subsequent comparison with homologous genes in other Drosophila species revealed the differences in the cDNA match those of the other species, and thus, the gene most likely is functional.
The plasmid was transformed into Synechocystis, and PCRs were used to confirm proper integration and segregation. The TE/∆slr1609/DmJHAMT strain produced 62 mg/L methyl laurate in 12 days under a light intensity of 150 µmol photons m-2 s-1, bubbled with 0.5% CO2 at a rate of 30 mL/min, and supplemented with 0.5 mM methionine. The laurate levels did not decrease over time, but instead, remained stagnant after day 3. When the strain was grown in the same conditions without methionine, the laurate concentrations continued to increase above 400 µM, suggesting minimal methyl laurate production and thus a strong need for methionine supplementation. This work provides further evidence of the viability and success of the introduced FAME production pathway, and improved efficiency may be gained in the future.
ContributorsSharma, Shuchi (Author) / Vermaas, Willem (Thesis director) / Wang, Xuan (Committee member) / Li, Shuqin (Committee member) / Computer Science and Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05