The use of enzyme-catalyst interfaces is underexplored in the field of biocatalysis, particularly in studies on enabling novel reactivity of enzymes. For this thesis, the HaloTag® protein tagging platform was proposed as a bioconjugation method for a pinacol coupling reaction using lipases, as a model for novel reactivities proceeding via ketyl radical intermediates and hydrogen-bonding-facilitated redox attenuation. After an initial lipase screening of 9 lipases, one lipase (Candida rugosa) was found to perform the pinacol coupling of p-anisaldehyde under standard conditions (fluorescein and 530nm light, 3% yield). Based on a retrosynthetic analysis for the photocatalyst-incorporated HaloTag® linker, the intermediates haloamine 1 and aldehyde 6 were synthesized. Further experiments are underway or planned to complete linker synthesis and conduct pinacol coupling experiments with a bioconjugated system. This project underscores the promising biocatalytic promiscuity of lipases for performing reactions proceeding through ketyl radical intermediates, as well as the underdeveloped potential of incorporating bioengineering principles like bioconjugation into biocatalysis to overcome kinetic barriers to electron transfer and optimize biocatalytic reactions.
Interactions driving the collapse of islet amyloid polypeptide: implications for amyloid aggregation
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.
coronavirus 2 (SARS-CoV-2), has been responsible for significant social and economic
disruption, prompting an urgent search for therapeutic solutions. The spike protein of the virus
has been examined as an immunogenic target because of its role in viral binding and fusion
necessary for infection of host cells. Previous studies have identified a recombinant protein
(denoted as S1) that has been shown to potentially induce a neutralizing antibody response by
mimicking the structure of the SARS-CoV-2 spike protein. We have produced the S1 in plants
using agroinfiltration, a plant transformation technique whereby plasmid-containing
Agrobacterium tumefaciens is injected into Nicotiana benthamiana plants, resulting in transfer of
the desired gene from bacteria to plant cells. S1 was expressed to high levels within 5 days of
infiltration, and Western blot analysis showed recognition of the S1 by an anti-S1 antibody.
ELISA results exhibited increased binding activity to anti-S1 with increasing concentrations of
S1, indicating their specific interaction. This ongoing study will demonstrate the potential of a
plant-produced S1 as a vaccine, therapeutic, and diagnostic tool against COVID-19 that is not
only effective, but also cost-efficient and scalable in comparison to conventional mammalian cell
culture production methods.
While taking a fashion technology course under the instruction of Galina Mihaleva, I developed a tracksuit incorporating concealed LED displays that are capable of scrolling customizable text on the sides of the garment. I expanded on this futuristic execution of politically charged clothes by utilizing a more realistic application of the LED technology in the Bouis Vuitton project. This project is a collection of six white vinyl bags with semi-flexible LED displays projecting revolutionary slogans through the vinyl textile.
The bags act as an appropriate housing for technology that is intended for significantly longer use, as bags have a longer lifespan in wardrobes than clothes and return to trend more frequently. The production investment in the technology is more equitable to the investment in the production of a bag and facilitates the wearer’s broadcasting of concise messages. The result is a collection of functional, utilitarian pieces with a clean, futuristic look and a mixed modern and vintage silhouette scrolling pro-revolutionary messages.
Broadcasting the knock-off name ‘BOUIS VUITTON’, I’ve inserted only my first initial into the reputable luxury company and paired it with slogans: ‘EAT THE RICH’ and ‘HEADS WILL ROLL’. The collection articulates a sense of nihilism felt by the youngest generations growing up on the outside of a very exclusive economic and political sphere. Three upcycled vintage luggage pieces evoke associations with the white American upper-class society of the 1960s. The luggage pieces were retrofitted in white vinyl and white-enameled metal fixtures. Three additional soft bags made of the same material reflect a utilitarian style of functional bags on trend with Spring/Summer 2019 streetwear. For the runway presentation of the bags, the models are dressed in navy-colored Dickies boiler suits, white retro-style Fila sneakers, and white ascots reminiscent of the historical male ruffled cravat. The contradictions of iconic silhouettes from both upper and lower-class American fashion history further the juxtaposition of anti-capitalist slogans posted on luxury goods.
Bouis Vuitton: Bags for the Revolution is intended to embody an unapologetic disregard for established wealth and political power in the most public of venues: the sidewalk, the mall, the high and the low-income neighborhoods – wherever people are wearing clothes. Fashion is the modern protest that requires no permit, and the new poster is a luxury bag.
Therefore, it can be concluded that downstream processes are limiting the electron flow to the hydrogenase. It was also shown that the use of a PSII inhibitor, 3-(3,4-dichlorophenyl)-1,1- dimethylurea (DCMU), at sub-saturating concentrations under light exposure during growth temporarily improves the duration of the H2 evolution phase. The maximal hydrogen production rate was found to be approximately 32 nmol h-1 (µg Chl)-1. Although downregulation of PSII activity with DCMU improves the long-term hydrogen production, future experiments must be focused on improving oxygen tolerance of the hydrogenase as a means for higher hydrogen yields.