Biochemistry, Biophysics, and Structural Biology Commons^™

Developing Regulated Crispr Systems To Control Bacterial Microbiomes, Gregory M. Pellegrino

Electronic Thesis and Dissertation Repository

Changes to the human microbiome’s composition and metabolome are associated with numerous diseases and alterations to xenobiotic metabolism. As such, targeting the human microbiome is an increasingly popular option for therapeutic interventions. However, traditional therapies that target the microbiome such as antibiotics lack specificity, which can affect the beneficial species of the microbiome and cause adverse health outcomes such as the rise of antimicrobial-resistant bacteria. Therefore, the research and development of specific, targeted antimicrobial therapies is crucial to effectively treating microbiome dysbioses.
CRISPR and CRISPRi provide easily modifiable, RNA-guided mechanisms mediated by the Cas9 or dCas9 enzymes to induce sequence-specific …

Flavonol Glucosylation: A Structural Investigation Of The Flavonol Specific 3-O Glucosyltransferase Cp3gt, Aaron S. Birchfield

Electronic Theses and Dissertations

Flavonoid glycosyltransferases (GTs), enzymes integral to plant ecological responses and human pharmacology, necessitate rigorous structural elucidation to decipher their mechanistic function and substrate specificity, particularly given their role in the biotransformation of diverse pharmacological agents and natural products. This investigation delved into a comprehensive exploration of the flavonol 3-O GT from Citrus paradisi (Cp3GT), scrutinizing the impact of a c-terminal c-myc/6x histidine tag on its enzymatic activity and substrate specificity, and successfully achieving its purification to apparent homogeneity. This established a strong foundation for potential future crystallographic and other structure/function analyses. Through the strategic implementation of site-directed mutagenesis, a thrombin …

Characterization Of Dna Regulatory Elements Of The Highly Abundant Secreted Protein 1 (Hasp1) Promoter And Analysis Of Transcriptomic Responses Under Phosphate Depletion In The Diatom Phaeodactylum Tricornutum, Mahsa Farmanbar

Electronic Thesis and Dissertation Repository

Phosphorus (P) is essential for all life. The bioavailability of phosphorus in oceans impacts diatoms like Phaeodactylum tricornutum (P. tricornutum). P. tricornutum is extensively used in transcriptomic studies to understand pathways involved in P-acquisition. However, activation mechanisms and roles of regulatory elements in P-acquisition responses remains unclear. Here, I deleted predicted phosphate regulatory sites in P.tricornutum’s HASP1 promoter to create different HASP1-eGFP constructs. Under P-depletion, two constructs showed increased eGFP secretion. Additionally, a HASP1 knockout strain was grown under different phosphorus sources to determine if HASP1 is a phytase. Cells grown in full phosphorus utilized organic …

Advances In Phaeodactylum Tricornutum Nuclear Engineering, Mark Pampuch

Electronic Thesis and Dissertation Repository

The marine diatom Phaeodactylum tricornutum has the potential to become an excellent platform for the sustainable production of valuable compounds and pharmaceuticals, but currently large-scale engineering of this organism remains a challenge due factors like inefficient genetic transformation protocols and a lack of accurate genomic data. This thesis addresses these two bottlenecks by (i) optimizing an electroporation protocol to P. tricornutum and (ii) remapping genomic data from a scaffolded genome assembly to a telomere-to-telomere genome assembly. An optimized transformation protocol was developed that could consistently transform blunt-ended and DNA with overhangs and yielded up to 1000+ colony forming units per …

Functionalizing Conjugative Systems To Deliver Crispr Nucleases For Targeted Bacterial Killing, Thomas A. Hamilton

Electronic Thesis and Dissertation Repository

The interactions between humans and microbes are intimately important to human health, with both commensal and pathogenic bacteria affecting homeostasis and disease. Increasing concern over antibiotic resistance in bacterial pathogens represents a significant threat to human health, and use of traditional antibiotics to treat infections can be detrimental to commensal bacteria as well as pathogens, demonstrating a need for more specific antibacterial reagents. RNA-guided CRISPR nucleases, which can target and cleave genomes of interest, are a potential tool for specific bacterial targeting. A key limitation to the use of CRISPR antimicrobials is effective and robust delivery to the target bacteria. …

Improving The Ribozyme Toolbox: From Structure-Function Insights To Synthetic Biology Applications, Jessica Michelle Roberts

Boise State University Theses and Dissertations

Self-cleaving ribozymes are a naturally occurring class of catalytically active RNA molecules which cleave their own phosphate backbone. In nature, self-cleaving ribozymes are best known for their role in processing concatamers of viral genomes into monomers during viral replication in some RNA viruses, but to a lesser degree have also been implicated in mRNA regulation and processing in bacteria and eukaryotes. In addition to their biological relevance, these RNA enzymes have been harnessed as important biomolecular tools with a variety of applications in fields such as bioengineering. Self-cleaving ribozymes are relatively small and easy to generate in the lab using …

Methanogen Metabolic Flexibility, Sean Carr

School of Biological Sciences: Dissertations, Theses, and Student Research

Methanogens are obligately anaerobic archaea which produce methane as a byproduct of their respiration. They are found across a wide diversity of environments and play an important role in cycling carbon in anaerobic spaces and the removal of harmful fermentation byproducts which would otherwise inhibit other organisms. Methanogens subsist on low-energy substrates which requires them to utilize a highly efficient central metabolism which greatly favors respiratory byproducts over biomass. This metabolic strategy creates high substrate:product conversion ratios which is industrially relevant for the production of biomethane, but may also allow for the production of value-added commodities. Particularly of interest are …

Crowd Control: Regulating The Spatial Organization Of Biopolymers And Gene Expression By Macromolecular Crowding, Gaurav Chauhan

Doctoral Dissertations

The intracellular environment is crowded with macromolecules that can occupy a significant fraction of the cellular volume. This can give rise to attractive depletion interactions that impact the conformations and interactions of biopolymers, as well as their interactions with confining surfaces. We used computer simulations to study the effects of crowding on biologically-inspired models of polymers. We showed that crowding can lead to attractive interactions between two flexible ring polymers, and we further characterized the adsorption of both flexible and semiflexible polymers onto confining surfaces. These results indicate that crowding-induced depletion interactions could play a role in the spatial organization …

Leveraging Queueing Theory To Develop Advanced Synthetic Biological Circuits, Prajakta K. Jadhav

Electronic Theses and Dissertations

The complexity and redundancy in the network topology of the cell hinder our understanding of the natural system, making it challenging to engineer living organisms with the desired functionality. The long-term goal of synthetic biology and bioengineering is to engineer cells to perform specific functions with increased robustness. The robust engineered systems can be easily applied to different species with the same outcome. An effective approach is a bottom-up approach where we tease apart the biological pathways and study them independently. Understanding the underlying mechanisms of biological pathways can be further applied to construct complex biological circuits that function similarly …

Assessing Selective Plasmids For Bradyrhizobium Sp. Doa9 And Mesorhizobium Loti, Aiden J. Wilcox, Rebecca Meaney, Bogumil Karas

Undergraduate Student Research Internships Conference

No abstract provided.

Engineering Modularity Of Ester Biosynthesis Across Biological Scales, Hyeongmin Seo

Doctoral Dissertations

Metabolic engineering and synthetic biology enable controlled manipulation of whole-cell biocatalysts to produce valuable chemicals from renewable feedstocks in a rapid and efficient manner, helping reduce our reliance on the conventional petroleum-based chemical synthesis. However, strain engineering process is costly and time-consuming that developing economically competitive bioprocess at industrial scale is still challenging. To accelerate the strain engineering process, modular cell engineering has been proposed as an innovative approach that harnesses modularity of metabolism for designing microbial cell factories. It is important to understand biological modularity and to develop design principles for effective implementation of modular cell engineering. In this …

Isoprene Production From Municipal Wastewater Biosolids By Engineered Archaeon Methanosarcina Acetivorans, Sean Carr, Jared Aldridge, Nicole R. Buan

Department of Biochemistry: Faculty Publications

Wastewater biosolids are a promising feedstock for production of value-added renewable chemicals. Methane-producing archaea (methanogens) are already used to produce renewable biogas via the anaerobic treatment of wastewater. The ability of methanogens to efficiently convert dissolved organic carbon into methane makes them an appealing potential platform for biorefining using metabolic engineering. We have engineered a strain of the methanogen Methanosarcina acetivorans to produce the volatile hemiterpene isoprene in addition to methane. The engineered strain was adapted to grow in municipal wastewater through cultivation in a synthetic wastewater medium. When introduced to municipal wastewater the engineered methanogens were able to compete …

Implications And Applications Of Transfer Rna Variants That Mistranslate The Genetic Code, Matthew D. Berg

Electronic Thesis and Dissertation Repository

Genetic information is passed from DNA to RNA to protein through the processes of transcription and translation. Transfer RNAs (tRNA) are the adaptors that bring amino acids to the growing polypeptide chain during translation and decode the three base codons that define protein sequence. Mistranslation occurs when an amino acid different from what is specified by the genetic code is inserted into a protein. tRNA variants cause mistranslation by decreasing the accuracy of amino acid charging or by altering decoding at the ribosome. My goal was to characterize mistranslating tRNA variants, identify their effects on cells and determine mechanisms used …

Practical Applications And Future Directions Of Genetic Code Expansion: Validation Of Novel Akt1 Substrates And The Design Of A Synthetic Auxotroph Strain Of B. Subtilis, Mcshane M. Mckenna

Electronic Thesis and Dissertation Repository

In Chapter 1, site-specifically phosphorylated variants of the oncogene Akt1 were made in Escherichia coli using the orthogonal translation system that enable genetic code expansion with phosphoserine. The differentially phosphorylated variants of Akt1 were used to validate newly predicted Akt1 substrates. The predicted target sites of the peptide substrates were synthesized and subjected to in vitro kinase assays to quantify the activity of each Akt1 phosphorylated variant towards the predicted peptide. A previously uncharacterized kinase-substrate interaction between Akt1 and a peptide derived from RAB11 Family Interacting Protein 2 (RAB11FIP2) was validated in vitro. Chapter 2 describes the preliminary development of …

Towards The Synthetic Design Of Camelina Oil Enriched In Tailored Acetyl-Triacylglycerols With Medium-Chain Fatty Acids, Sunil Bansal, Hae Jin Kim, Gunnam Na, Megan E. Hamilton, Edgar B. Cahoon, Chaofu Lu, Timothy P. Durrett

Department of Biochemistry: Faculty Publications

The ability to manipulate expression of key biosynthetic enzymes has allowed the development of genetically modified plants that synthesise unusual lipids that are useful for biofuel and industrial applications. By taking advantage of the unique activities of enzymes from different species, tailored lipids with a targeted structure can be conceived. In this study we demonstrate the successful implementation of such an approach by metabolically engineering the oilseed crop Camelina sativa to produce 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) with medium-chain fatty acids (MCFAs). Different transgenic camelina lines that had been genetically modified to produce MCFAs through the expression of MCFA-specific thioesterases and …

Development Of In Vivo Systems For Detecting And Studying Ribosome Inhibition By Small Molecules, Shijie Huang

Chemistry and Chemical Biology ETDs

The ribosome is the quintessential antibacterial drug target, with many structurally and mechanistically distinct classes of antibacterial agents acting by inhibiting ribosome function. Detecting and quantifying ribosome inhibition by small molecules and investigating their binding modes and mechanisms of action are critical to antibacterial drug discovery and development efforts. To develop a ribosome inhibition assay that is operationally simple, yet provides direct information on the drug target and the mechanism of action, we have developed engineered E. coli strains harboring an orthogonal ribosome controlled green fluorescent protein reporter that produce fluorescent signal when the O-ribosome is inhibited. As a proof …

A Novel Synthetic Yeast For Enzymatic Biodigester Pretreatment, Tianyu Tan, Mark S. Aronson, Arren Liu, Jill H. Osterhus, Melissa Robins, Suraj Mohan, Erich Leazer, Bowman Clark, Alexa Petrucciani, Katherine Lowery, James Welch, Casey Martin, Helena Lysandrou, Michael E. Scharf, Jenna Rickus

The Summer Undergraduate Research Fellowship (SURF) Symposium

Lignin, a complex organic polymer, is a major roadblock to the efficiency of biofuel conversion as it both physically blocks carbohydrate substrates and poisons biomass degrading enzymes, even if broken down to monomer units. A pretreatment process is often applied to separate the lignin from biomass prior to biofuel conversion. However, contemporary methods of pretreatment require large amounts of energy, which may be economically uncompelling or unfeasible. Taking inspiration from several genes that have been isolated from termites and fungi which translate to enzymes that degrade lignin, we want to establish a novel “enzymatic pretreatment” system where microbes secrete these …

Ex Vivo Dna Cloning, Adam B. Fisher

Theses and Dissertations

Genetic engineering of microbes has developed rapidly along with our ability to synthesize DNA de novo. Yet, even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. While technological advances have resulted in powerful techniques for in vitro and in vivo assembly of DNA, each suffers inherent disadvantages. Here, an ex vivo DNA cloning suite using crude cellular lysates derived from E. coli is demonstrated to amplify and assemble DNA containing small sequence homologies. Further, the advantages of an ex vivo approach are …

Determining The Genotype-Phenotype Connection In Synthetic Inducible Gene Expression Systems, Rhys M. Adams

Dissertations & Theses (Open Access)

Introduction Gene expression is an important process whereby the genotype controls an individual cell’s phenotype. However, even genetically identical cells display a variety of phenotypes, which may be attributed to differences in their environment. Yet, even after controlling for these two factors, individual phenotypes still diverge due to noisy gene expression. Synthetic gene expression systems allow investigators to isolate, control, and measure the effects of noise on cell phenotypes. I used mathematical and computational methods to design, study, and predict the behavior of synthetic gene expression systems in S. cerevisiae, which were affected by noise.

Methods I created probabilistic …