Biochemistry, Biophysics, and Structural Biology Commons^™

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 …

Development Of A Thermosensitive Endonuclease To Act As A Plasmid Kill-Switch, Christopher D. Leichthammer

Electronic Thesis and Dissertation Repository

Biocontainment is an integral part of biomedical research that aims to protect the environment and human health by containing hazardous or invasive organisms in the laboratory. Containment systems often rely on elaborate genetic circuits; however, cells may escape containment by developing mutations that render the genetic circuits inviable or resistant to killing mechanisms. The aim of this thesis was to create a site-specific nuclease for biocontainment of plasmids in the mammalian gastrointestinal tract. LAGLIDADG homing endonucleases would be good candidate nucleases for a biocontainment system as they are resistant to mutations in their coding sequence and their target sequence in …

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 …

Production Of A Candidate Recombinant Protein Vaccine For Mannheimia Haemolytica In Lettuce And Tobacco Chloroplasts, Coby K. Martin

Electronic Thesis and Dissertation Repository

The cattle industry worldwide is ravaged by bovine respiratory disease (BRD), a bacterial disease caused by Mannheimia haemolytica. Recent efforts to design vaccines against M. haemolytica focus on a virulence factor, leukotoxin, in addition to surface lipoproteins. Plant-based protein production is a safe and inexpensive alternative to traditional methods. Edible vaccines deliver antigens to pharyngeal tissues, which can provide local immunization against M. haemolytica prior to its progression into the lungs. In this project, a chimeric protein containing M. haemolytica antigens was produced in tobacco chloroplasts as a candidate edible vaccine for BRD. Attempts were made to transform lettuce …

Deciphering Sulfur Amino Acid Metabolism In Developing Seeds Of Common Bean, Jaya Joshi

Electronic Thesis and Dissertation Repository

With increasing food insecurity in the populated world, the number of people affected by chronic undernourishment is also increasing. Alone, protein energy malnutrition is linked to 6 million deaths annually. Despite being a good source of protein and dietary fibre, the quality of bean protein is limited because of sub optimal levels of essential sulfur amino acids: methionine and cysteine. Levels of cysteine and methionine in developing seeds have an inverse relationship with the non-protein sulfur amino acid S-methyl-cysteine (S-methylCys) and dipeptide g-glutamyl-S-methyl-cysteine (g-Glu-S-methylCys).

One of the strategies to improve protein quality in …

Genetic Engineering Studies Of Escherichia Coli And Microalgae For Expression Of Hydrolytic Enzymes And Development Of High Throughput Screening Technique, Shreyas S. Yedahalli

Electronic Thesis and Dissertation Repository

The field of biochemical engineering has made substantial progress through major advances in genetic and metabolic engineering with applications in various sectors such as energy, food science, pharmaceuticals, etc. The hosts used for this work are constantly broadening. A host particularly important for energy applications are microalgae. The potential to enhance microalgae genetically for energy applications is not well explored and was therefore investigated in this thesis. Non-photosynthetic micro-organisms and photosynthetic microalgae offer a potential approach to enhance sustainable biochemical production. In this study expression vectors for Escherichia coli (E. coli) and Chlorella vulgaris (C. vulgaris) …

Examining The Nucleotide Preference Of The Linker Domain In Engineered Tev-Mtalens, Brendon C. Mcdowell

Electronic Thesis and Dissertation Repository

Tev-mTALENs are genome-editing nucleases which combine the nuclease and linker domains of I-TevI with the DNA-binding domain of a TAL effector. The linker domain interacts with a portion of the Tev-mTALEN target site called the DNA Spacer, facilitating DNA cleavage. Linker-DNA Spacer interactions are poorly understood but necessary for Tev-mTALEN activity. I examined the DNA Spacer sequence requirements of the linker by assaying Tev-mTALEN activity on targets with mutated DNA Spacer sequences. I also performed activity assays using Tev-mTALENs with mutations to the I-TevI linker domain. My results indicate that the linker DNA Spacer sequence requirements are highly cryptic. No …

Translesion Synthesis And Mutations: On The Mutagenic Properties Of The Two Dna Lesions, 8-Oxo-G And Pt-Gg, And The Functions Of Y-Family Dna Polymerases And Rev3l On The Bypass Of Each Of The Dna Lesions In Mammalian Cells, Lizhen Guo

Electronic Thesis and Dissertation Repository

I studied the capabilities of the two DNA lesions 8-oxo-guanine and cisplatin intrastrand crosslinked 1,2-d(GpG) or Pt-GG to cause mutations in mammalian cells. Using isogenic cell lines generated from mice with selective gene knockouts of distinct DNA polymerases as models, I deduced the biological functions of the translesion DNA polymerases Pol eta, Pol kappa, Pol iota, Rev1 and Rev3L on bypassing each of the lesions 8-oxo-G and Pt-GG. My study takes advantage of the Next Generation Sequencing (NGS) technology to determine mutagenic effects of the DNA lesions in vivo and effects of translesion DNA polymerases on bypassing the lesions. Through …

Uncovering The Molecular Link Between Mir156.Spl15 And Carotenoid Accumulation In Arabidopsis, Davood Emami Meybodi

Electronic Thesis and Dissertation Repository

Carotenoid Cleavage Dioxygenases (CCDs) are an enzyme family that cleaves specific double bonds in carotenoids. MicroR156 in Arabidopsis regulates a network of genes by repressing 10 SPL genes, among which, SPL15 was found to regulate shoot branching and carotenoid accumulation. The expression of CCD1, CCD4, CCD7, CCD8, NCED2, NCED3, NCED5, NCED6, NCED9 and SPL15 was evaluated in siliques at 10 days post anthesis and in 10-day-old roots in Arabidopsis wild type, sk156 (miR156 overexpression mutant), RS105 (miR156 overexpression line), spl15 (SPL15 knockout mutant) and two 35S:SPL15 lines. Results showed that most of CCD/NCED genes were …

Identification Of Cyclophilin Gene Family In Soybean And Characterization Of Gmcyp1, Hemanta Raj Mainali

Electronic Thesis and Dissertation Repository

I identified members of the Cyclophilin (CYP) gene family in soybean (Glycine max) and characterized the GmCYP1, one of the members of soybean CYP. CYPs belong to the immunophilin superfamily with peptidyl-prolyl cis-trans isomerase (PPIase) activity. PPIase catalyzes the interconversion of the cis- and trans-rotamers of the peptidyl-prolyl amide bond of peptides. After extensive data mining, I identified 62 different CYP genes in soybean (GmCYP1 to GmCYP62), of which 8 are multi-domain proteins and 54 are single domain proteins. At least 25% of the GmCYP genes are expressed in soybean. GmCYP1 …

Dna Hydrolysis And Genome Editing Applications Of Giy-Yig Family Homing Endonucleases, Benjamin P. Kleinstiver

Electronic Thesis and Dissertation Repository

The ability to manipulate complex genomes in a precise manner is essential for studying biological processes in model systems, engineering plant strains for agriculture, or advancing human cellular therapies to treat diseases. Genomic alterations are most efficient when a double-strand DNA break is introduced at the loci where the modification is desired. Different classes of naturally occurring DNA endonucleases, including homing endonucleases, have therefore been explored as candidates for genome modification studies as they target long stretches of DNA. Homing endonucleases are mobile genetic elements whose biological role is to introduce site-specific double-strand breaks into naïve genomes, ultimately resulting in …

Protein Body Formation In Stable Transgenic Plants Of Nicotiana Tabacum Expressing Elastin-Like Polypeptide And Hydrophobin Fusion Proteins, Sonia P. Gutierrez

Electronic Thesis and Dissertation Repository

Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. However, the use of plants still faces two main limitations: the low accumulation levels of some recombinant proteins and the lack of efficient protein purification methods. Two fusion partners, elastin-like polypeptides (ELP) and hydrophobin I (HFBI) were found to increase the accumulation of recombinant proteins and induce the formation of protein bodies (PBs) when targeted to the ER in transient expression assays. In this study I examined the effect of these tags in stable transgenic plants of two Nicotiana tabacum cultivars when fused to green fluorescent …