Ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme (Rubisco) is responsible for the majority of carbon fixation and is also the least efficient enzyme on Earth. Rubisco assists 1,5-ribulose bisphosphate (RuBP) in binding CO2, however CO2 and oxygen have similar binding affinities to Rubisco, resulting in a low enzymatic efficiency. Rubisco activase (Rca) is an enzyme that removes inhibiting molecules from Rubisco’s active sites, promoting the Rubisco activity. The binding of Rubisco and Rca stimulates a high-rate of carbon fixation and lowers the overall CO2 concentration in the atmosphere. To study the interaction between the two complexes, Rubisco was extracted from baby spinach (Spinacia oleracea) and purified using anion-exchange chromatography and size-exclusion chromatography. Rca was designed to use a recombinant gene and overexpressed in Escherichia coli (E. coli). The purified proteins were verified using SDS-PAGE. The two proteins were assembled in vitro and the interaction of the protein complex was stabilized using glutaraldehyde cross-linking. The samples were then deposited on a carbon-coated electron microscopy (EM) grid, stained with uranyl formate, and observed under a transmission electron microscope (TEM). The ultimate goal is to image the specimen and reconstruct the structure of the protein complex at high resolution.

Arsenic contamination in groundwater is a serious problem both in local Arizonan communities and abroad: prolonged exposure to arsenic contamination can cause cancer, vascular damage, and liver failure. This project aims to engineer the microalgae Chlamydomonas reinhardtii to sequester arsenic out of water. Metallothionein, arsenate reductase, and ferritin were integrated into the microalgae via the pASapI plasmid. The plasmid rescues function of the photosystem II gene, leveraging the ability to photosynthesize as a selective trait. Metallothionein and ferritin bind the two most common forms of arsenic: arsenite and arsenate, respectively. Arsenate reductase catalyzes the reduction of arsenate to arsenite, allowing for the ultimate sequestration of the toxic metal to occur in the chloroplast. The algae was transformed using a biolistic device, to create three mutant strains, expressing Metallothionein (MT), Arsenate Reductase (ArsC)-HA, and MT-6xHIS plasmids respectively. When testing the fluorescence output of these three strains, they showed a maximum quantum yield of photosystem II comparable to that of the wildtype algae, indicating that the rescue gene had been incorporated into the chloroplast genome properly. Strains were exposed to arsenic-containing media at 50ppb and 500 ppb for 48 and 72 hours to determine the arsenic sequestration rate. Arsenic concentration in the supernatant was measured using ICP-MS analysis and sequestration rate was calculated in terms of arsenic concentration per fold growth of algae. The normalized arsenic sequestration rates of tagged protein expressing strains at 50 ppb were significantly higher than wildtype.

With needs for carbon sequestration and sustainable chemical feedstocks increasing formate stands out as a real possibility in addressing these growing problems. One of the principal issues with positioning formate as the central compound of a bioeconomy is establishing a sustainable and reliable method for producing it. The goal of this project was to take the first steps towards engineering a formate production cell factory in Chlamydomonas reinhardtii by introducing the biosynthetic pathway necessary for the creation of molybdenum cofactor which would later be used as an integral part of the function of a formate dehydrogenase enzyme capable of reducing carbon dioxide to make formate. I was able to get some seemingly successful transformants but unable to confidently confirm whether or not these transformants hardboard the molybdenum cofactor synthesis genes.

In algae, the Mutant Affecting Retrograde Signaling (MARS1) Kinase plays a critical role in the chloroplast unfolded protein response (cpUPR) when the chloroplast faces proteotoxic stress4. The MARS1 protein is relatively unknown in terms of structure and function. However, there has been ample research performed on the main pathway associated with the MARS1 protein, the cpUPR. The exact mechanism of why MARS1 is necessary for the cpUPR is still unknown. Our structural and biochemical studies will help develop a better understanding of the MARS1 structure, and the role it plays in the cpUPR. The MARS1 expression construct will be assembled following the yeast golden gate (yGG) assembly protocol. Here, we will attempt to recombinantly express MARS1 kinase in Saccharomyces cerevisiae to provide insights into the protein.

Exerting bias on a diverse pool of random short single stranded oligonucleotides (ODNs) by favoring binding to a specific target has led to the identification of countless high affinity aptamers with specificity to a single target. By exerting this same bias without prior knowledge of targets generates libraries to capture the complex network of molecular interactions presented in various biological states such as disease or cancer. Aptamers and enriched libraries have vast applications in bio-sensing, therapeutics, targeted drug delivery, biomarker discovery, and assay development. Here I describe a novel method of computational biophysical characterization of molecular interactions between a single aptamer and its cognate target as well as an alternative to next generation sequencing (NGS) as a readout for a SELEX-based assay. I demonstrate the capability of an artificial neural network (ANN) trained on the results of screening an aptamer against a random sampling of a combinatorial library of short synthetic 11mer peptides to accurately predict the binding intensities of that aptamer to the remainder of the combinatorial space originally sampled. This machine learned comprehensive non-linear relationship between amino acid sequence and aptamer binding to synthetic peptides can also make biologically relevant predictions for probable molecular interactions between the aptamer and its cognate target. Results of SELEX-based assays are determined by quantifying the presence and frequency of informative species after probing patient specimen. Here I show the potential of DNA microarrays to simultaneously monitor a pool of informative sequences within a diverse library with similar variability and reproducibility as NGS.

Fluoroquinolone antibiotics have been known to cause severe, multisystem adverse side effects, termed fluoroquinolone toxicity (FQT). This toxicity syndrome can present with adverse effects that vary from individual to individual, including effects on the musculoskeletal and nervous systems, among others. The mechanism behind FQT in mammals is not known, although various possibilities have been investigated. Among the hypothesized FQT mechanisms, those that could potentially explain multisystem toxicity include off-target mammalian topoisomerase interactions, increased production of reactive oxygen species, oxidative stress, and oxidative damage, as well as metal chelating properties of FQs. This review presents relevant information on fluoroquinolone antibiotics and FQT and explores the mechanisms that have been proposed. A fluoroquinolone-induced increase in reactive oxygen species and subsequent oxidative stress and damage presents the strongest evidence to explain this multisystem toxicity syndrome. Understanding the mechanism of FQT in mammals is important to aid in the prevention and treatment of this condition.

Heliobacteria are an anaerobic phototroph that require carbon sources such as pyruvate, <br/>lactate, or acetate for growth (Sattley, et. al. 2008). They are known for having one of the <br/>simplest phototrophic systems, the central component of which is a Type I reaction center (RC) <br/>that pumps protons to generate the electrochemical gradient for making ATP. Heliobacteria <br/>preform cyclic electron flow (CEF) with the RC in the light but can also grow chemotropically in <br/>the dark. Many anaerobes like heliobacteria, such as other members of the class Clostridia, <br/>possess the capability to produce hydrogen via a hydrogenase enzyme in the cell, as protons can <br/>serve as an electron acceptor in anaerobic metabolism. However, the species of heliobacteria <br/>studied here, H. modesticaldum have been seen to produce hydrogen via their nitrogenase <br/>enzyme but not when this enzyme is inactive. This study aimed to investigate if the reason for <br/>their lack of hydrogen production was due to a lack of an active hydrogenase enzyme, possibly <br/>indicating that the genes required for activity were lost by an H. modesticaldum ancestor. This <br/>was done by introducing genes encoding a clostridial [FeFe] hydrogenase from C. thermocellum<br/>via conjugation and measuring hydrogen production in the transformant cells. Transformant cells <br/>produced hydrogen and cells without the genes did not, meaning that the heliobacteria ferredoxin <br/>was capable of donating electrons to the foreign hydrogenase to make hydrogen. Because the <br/>[FeFe] hydrogenase must receive electrons from the cytosolic ferredoxin, it was hypothesized <br/>that hydrogen production in heliobacteria could be used to probe the redox state of the ferredoxin <br/>pool in conditions of varying electron availability. Results of this study showed that hydrogen <br/>production was affected by electron availability variations due to varying pyruvate <br/>concentrations in the media, light vs dark environment, use acetate as a carbon source, and being <br/>provided external electron donors. Hydrogen production, therefore, was predicted to be an <br/>effective indicator of electron availability in the reduced ferredoxin pool.

The heliobacteria, a family of anoxygenic phototrophs, are significant to photosynthesis evolution research, as they possess the simplest known photosynthetic apparatus. Although they are photoheterotrophs in the light, the heliobacteria may also grow chemotrophically via pyruvate metabolism in the absence of light. In Heliobacterium modesticaldum, the cytochrome bc complex is responsible for oxidizing menaquinol and reducing cytochrome c553 in the electron flow cycle used for phototrophy. However, there is no known electron acceptor for cytochrome c553 other than the photosynthetic reaction center. Therefore, it was hypothesized that the cytochrome bc complex is necessary for phototrophy, but unnecessary for chemotrophic growth in the dark. Under this hypothesis, a mutant of H. modesticaldum lacking the cytochrome bc complex was predicted to be viable, but non-phototrophic. In this project, a two-step method for CRISPR-based genome editing was used in H. modesticaldum to delete the genes encoding the cytochrome bc complex. Genotypic analysis verified the deletion of the petC, B, D, and A genes encoding the catalytic components of complex. Spectroscopic studies revealed that re-reduction of cytochrome c553 after flash-induced photo-oxidation was ~130 to 190 times slower in the ∆petCBDA mutant compared to wildtype, phenotypically confirming the removal of the cytochrome bc complex. The resulting ∆petCBDA mutant was unable to grow phototrophically, instead relying on pyruvate metabolism to grow chemotrophically as does wildtype in the dark.

Due to analytical limitations, thermodynamic modeling is a lucrative alternative for obtaining metal speciation in chemically complex systems like life. However, such modeling is limited by the lack of equilibrium constant data for metal-complexation reactions, particularly for metal-organic species. These problems were ameliorated estimating these properties from 0-125°C for ~18,000 metal complexes of small molecules, proteins and peptides.

The estimates of metal-ligand equilibrium constants at 25°C and 1 bar were made using multiple linear free energy relationships in accordance with the metal-coordinating properties of ligands such as denticity, identity of electron donor group, inductive effects and steric hindrance. Analogous relationships were made to estimated metal-ligand complexation entropy that facilitated calculation of equilibrium constants up to 125°C using the van’t Hoff equation. These estimates were made for over 250 ligands that include carboxylic acids, phenols, inorganic acids, amino acids, peptides and proteins.

The stability constants mentioned above were used to obtain metal speciation in several microbial growth media including past bioavailability studies and compositions listed on the DSMZ website. Speciation calculations were also carried out for several metals in blood plasma and cerebrospinal fluid that include metals present at over micromolar abundance (sodium, potassium, calcium, magnesium, iron, copper and zinc) and metals of therapeutic or toxic potential (like gallium, rhodium and bismuth). Metal speciation was found to be considerably dependent on pH and chelator concentration that can help in the selection of appropriate ligands for gallium & rhodium based anticancer drugs and zinc-based antidiabetics. It was found that methanobactin can considerably alter copper speciation and is therefore a suitable agent for the treatment of Wilson Disease. Additionally, bismuth neurotoxicity was attributed to the low transferrin concentration in cerebrospinal fluid and the predominance of aqueous bismuth trihydroxide. These results demonstrate that metal speciation calculations using thermodynamic modeling can be extremely useful for understanding metal bioavailability in microbes and human bodily fluids.

Exoelectrogenic microorganisms can grow by transferring electrons from their internal metabolism to extracellular substrates in a process known as extracellular electron transfer (EET). This dissertation explores the mechanisms of EET by both chemotrophic and phototrophic organisms and constructs a novel supramolecular structure that can be used as a model for microbial, long-range electron transfer. Geobacter sulfurreducens has been hypothesized to secrete and use riboflavin as a soluble, extracellular redox shuttle in conjunction with multi-heme, outer membrane, c-type cytochromes, but the required proteins and their properties have not been defined. To address the mechanism of extracellular electron transfer by G. sulfurreducens, the first part of this work explores the interaction between an outer membrane, octaheme, c-type cytochrome OmcZs from G. sulfurreducens and riboflavin. Interrogation via multiple physical techniques shows that OmcZs transfers electrons to riboflavin. By analogy to other characterized systems, riboflavin then likely interacts with extracellular acceptors directly. The second part of this work addresses the mechanisms of EET by the model cyanobacterium Synechocystis sp. PCC 6803. It has been hypothesized that Synechocystis employs conductive pili for production of extracellular current. However, the results herein show that a strain that does not have pili produces extracellular photocurrent in a direct electrochemical cell at a level similar to that by wild type cells. Furthermore, conductive atomic force microscopy (AFM) imaging is used to show that pili produced by the wild type organism are not conductive. Thus, an alternative EET mechanism must be operable. In the third part of this work, a supramolecular structure comprised of peptide and cytochromes designed to serve as a model for long-range electron transfer through cytochrome rich environments is described. The c-type cytochromes in this synthetic nanowire retain their redox activity after assembly and have suitable characteristics for long-range electron transfer. Taken together, the results of this dissertation not only inform on natural microbial mechanisms for EET but also provide a starting point to develop novel, synthetic systems.