Matching Items (55)
Description
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.
ContributorsHart, Hayden (Author) / Chiu, Po-Lin (Thesis director) / Redding, Kevin (Committee member) / Van Horn, Wade (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor) / Department of Military Science (Contributor)
Created2022-05
Description
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.
ContributorsLieberman, Emma (Author) / Bartelle, Benjamin (Thesis director) / Redding, Kevin (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2022-05
Description
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.
ContributorsNikkel, Zachary (Author) / Redding, Kevin (Thesis director) / Ghirlanda, Giovanna (Committee member) / Borges, Chad (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2022-05
Description
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.
ContributorsHeeres, Nicholas (Author) / Mazor, Yuval (Thesis director) / Chiu, Po Lin (Committee member) / Redding, Kevin (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2022-05
Description
The FOF1 ATP synthase is responsible for generating the majority of adenosine triphosphate (ATP) in almost all organisms on Earth. A major unresolved question is the mechanism of the FO motor that converts the transmembrane flow of protons into rotation that drives ATP synthesis. Using single-molecule gold nanorod experiments, rotation of individual FOF1 were observed to measure transient dwells (TDs). TDs occur when the FO momentarily halts the ATP hydrolysis rotation by the F1-ATPase. The work presented here showed increasing TDs with decreasing pH, with calculated pKa values of 5.6 and 7.5 for wild-type (WT) Escherichia coli (E. coli) subunit-a proton input and output half-channels, respectively. This is consistent with the conclusion that the periplasmic proton half-channel is more easily protonated than the cytoplasmic half-channel. Mutation in one proton half-channel affected the pKa values of both half-channels, suggesting that protons flow through the FO motor via the Grotthuss mechanism. The data revealed that 36° stepping of the E. coli FO subunit-c ring during ATP synthesis consists of an 11° step caused by proton translocations between subunit-a and the c-ring, and a 25° step caused by the electrostatic interaction between the unprotonated c-subunit and the aR210 residue in subunit-a. The occurrence of TDs fit to the sum of three Gaussian curves, which suggested that the asymmetry between the FO and F1 motors play a role in the mechanism behind the FOF1 rotation. Replacing the inner (N-terminal) helix of E. coli c10-ring with sequences derived from c8 to c17-ring sequences showed expression and full assembly of FOF1. Decrease in anticipated c-ring size resulted in increased ATP synthesis activity, while increase in c-ring size resulted in decreased ATP synthesis activity, loss of Δψ-dependence to synthesize ATP, decreased ATP hydrolysis activity, and decreased ACMA quenching activity. Low levels of ATP synthesis by the c12 and c15-ring chimeras are consistent with the role of the asymmetry between the FO and F1 motors that affects ATP synthesis rotation. Lack of a major trend in succinate-dependent growth rates of the chimeric E. coli suggest cellular mechanisms that compensates for the c-ring modification.
ContributorsYanagisawa, Seiga (Author) / Frasch, Wayne D (Thesis advisor) / Misra, Rajeev (Committee member) / Redding, Kevin (Committee member) / Singharoy, Abhishek (Committee member) / Wideman, Jeremy (Committee member) / Arizona State University (Publisher)
Created2023