Separation of proteins using metal ligand complexes is a well-established practice in the field of bioengineering and biochemistry.  electrospray ionization mass spectrometry (ESI-MS) can be used to identify bio and organic molecules. Previous studies have detected chelated metal ions using ESI-MS, this project focuses on the identification of a metal ligand complex comprised of a tridentate chelating agent Iminodiacetic acid (IDA) and a bidentate ligand, Histidine. By coordinating copper with IDA a binary complex is formed, this allows for the detection of copper by proxy of the IDA. By selecting a unique fragment related to IDA we can target in tandem mass spectrometry (MS/MS) for greater sensitivity; the complex can be selected for analysis out of solution. We will use this novel approach to build a parent-ion scanning technique to monitor metal-ligand complexes extracted from environmental matrices. first the ideal solution parameters are determined to maximize the complex formation and detection of the Cu-IDA complex. So far, a high ratio of copper to IDA coupled with a basic buffer have yielded the best data. Creating a ternary complex comprised of copper IDA and an imidazole ring containing compound, histidine. Selectively tuning to the peaks associated with the copper IDA complex, the ternary complexes can be selected for in depth analysis of its structure and bonding properties. Future work could focus on identification of metal ligand complexes from soil samples with other compounds containing imidazole rings such as the neonicotinoid imidacloprid which has been indicated in bee colony collapse. By chelating solid with IDA complex formed from pentacoordinate copper ions could be detected despite the low relative concentration.

Mitosis results in two genetically identical daughter cells, each containing their own nucleus and set of replicated chromosomes from the original parent cell. Inaccurate chromosome segregation can result in severe consequences like cancer and developmental defects. During mitosis, the replicated chromosomes line up at the center of the cell in preparation to be pulled apart by microtubules. Microtubules are dynamic cytoskeletal components that provide the forces necessary to pull the chromosomes towards their respective daughter cells. The ends of the microtubules attach to the kinetochore, which is an assembly of protein complexes located on the chromosome. Accurate segregation of these chromosomes relies on the ability of the kinetochore to strongly bind chromosomes to microtubule ends. Ndc80 complex is an outer-kinetochore component that binds microtubule ends and is required for proper segregation. Emerging cellular data suggests that multiple Ndc80 complexes interact with one microtubule end. We seek to assemble a particle of multiple Ndc80 complexes in vitro, which may model the native kinetochore-microtubule interface more closely. We utilized a designed protein, HBRP, that forms a hexamer in solution and was modified to couple with and artificially oligomerize any protein of interest. We optimized the coupling rate and completion degree of three different variants of the HBRP protein with Ndc80 complex to ensure a complete hexamer particle assembly. Successful formation of this particle assembly will allow us to better understand the binding mechanism of the kinetochore to microtubule ends.

Computational protein design is an emerging field that takes advantage of first principles derived from biological protein-protein interactions and explores the protein space that nature has yet to evolve. The Baker Lab developed a software called Rosetta that enables researchers to explore this space and create brand new proteins more stable than those produced in biological systems via evolution. This software has been adapted to take advantage of a concept that exists everywhere in nature- symmetry. Using this concept, I am building higher-order protein assemblies including protein nanocages and three-dimensional protein crystals. Researchers at the Baker Lab have developed a hierarchical approach to engineer these highly symmetric, complex structures. This hierarchical approach involves combining protein building blocks with different symmetric topologies multiple times to facilitate higher-order symmetric assembly of a three-dimensional protein crystal. By breaking up crystal symmetries into their constituent building blocks, we can design these higher-order symmetries with greater accuracy and troubleshoot experimental difficulties by pin-pointing structural deviations along the way. Here, I will describe my experience using this approach to create a protein crystal from symmetric building blocks.

The effectiveness of different catalysts during biofuel production was investigated to determine if a heterogeneous catalyst, eggshells, could perform as well as a traditional homogenous catalyst, Sodium Hydroxide. Eggshells add up to a significant amount of waste each year. If they were repurposed, that would not only get rid of that waste factor but also serve as an alternative to fossil fuels. The biodiesel was synthesized from sunflower oil. The amount of each catalyst was fixed at 2.25 grams and the yield of each fuel was documented. Fifty milliliters of the fuels produced were individually tested to measure their efficiency. The fuels were tested in a pop-can calorimeter. The pre-calculated combustion of ethanol in said calorimeter was used as a baseline for calculating relative efficiency. From there, we were able to determine the richness of the fuels produced by the two catalysts. The biodiesel catalyzed by Sodium Hydroxide produced 38.17 kilojoules per gram and the one catalyzed by Waste Egg shells produced 34.81 kilojoules per gram.

Wastewater treatment has been a major issue in undeveloped and developing countries due to the lack of access to water filtration systems to treat wastewater. Without clean water resources, it has affected agriculture, water storage, and daily activities. According to Global Affairs Canada, eighty percent of illnesses in developing countries are linked to a lack of proper wastewater treatment. In this project, an eco-friendly and lower cost of chemical, ferrate, has been generated and analyzed to combat global wastewater treatment.To generate more stable and user friendly ferrate. The precursors of ferrate are synthesized in solutions. These solutions are freeze dried to form stable precursors in solid form. These precursors can be mixed to give an easy route to make ferrate on demand. More importantly, they can be added directly to wastewater to generate ferrate for disinfection. In the second part of this project we have come up with an analytical technique to quantify the concentration of ferrate in solutions using UV instrument.The ferrate solution can be easily transferred to developing countries to use in wastewater treatment without generating harmful chemicals to humans or the environment. In future research, the research will focus on the mass production of ferrate as well as creating a more stable form.

In recent decades, there has been massive growth in consumer demand for products containing live bacterial cultures, or "probiotics", driving a $75B market. Yet, even as market share has expanded, the relative effectiveness of different probiotic products is still not fully understood. Such products require further scientific substantiation before manufacturers can claim health benefits. Few studies have been conducted on how wide-ranging and adverse conditions in the gastro-intestinal tract can influence ingested "pro-biotic" culture function and viability. This research attempts to close this knowledge gap, providing a formal method of characterizing bacterial function under various gut conditions through the identification of biomarkers that are indicative of healthy “probiotic” cultures. L. Bulgaricus, L. Acidophilus and S. thermophilus cultures were evaluated after exposure to conditions simulating major components of the gastro-intestinal tract, their protein expression analyzed and correlated with growth. Simulated colonic conditions maximized bacterial growth, while simulated gastric conditions minimized it. The validity of the experimental model was thus reinforced, as it accurately reflected previous in vivo analysis of bacterial growth in different components of the GI tract. By linking growth and protein expression, the gene, oppa1, was identified as a possible biomarker of cell growth. This gene, activated in conditions that conferred sub-standard growth relative to a positive control, seems to present a key to understanding bacterial population health. This research presents a step forward in the evaluation of the quality of various “probiotic” products by understanding the influence of the human digestive system on live cultures.

 Chemically induced dimerization (CID) systems, in which a pair of proteins only dimerizes in the presence of a specific ligand, have wide use for the biosensing of small molecules, such as drugs, metabolites, and signaling molecules. However, few CID systems are currently available, and thus, until recently, little work has been done to design effective and highly specific CID-based biosensors, limiting the diversity of our small molecule detection toolkit. Previously, we established a highly efficient and generalizable method for de novo engineering of new CID systems, and demonstrated its effectiveness by designing a CID system specific to cannabidiol (CBD). Here, we engineer circularly permutated green fluorescent proteins (cpGFP’s) to have the cannabidiol CID system flanking it, allowing for small molecule detection via protein dimerization to be translated into measurable changes in fluorescent signals. As an experimental platform to screen high-performance designs, we employ the yeast display technique, where yeast cells are engineered to display on their surfaces any cpGFP-CID biosensor constructs we wish to screen. The lengths and compositions of the linker regions connecting the cpGFP and the dimerization proteins play a crucial role in the efficiency of the biosensor. Currently, we seek to understand what linker lengths and contributions help optimize biosensor performance. Initially, we introduced linkers with lengths of about 20 residues that are composed of alternating glycine and serine residues. Biosensor constructs with these linkers exhibited minimal, baseline performance. We generate biosensor construct libraries by mutating the linker regions, displaying this library on yeast cells, and performing high-throughput screening to identify optimal linker lengths and compositions for our biosensor constructs. Afterwards, we will validate the biosensor’s performance in vivo and in vitro. Our research will not only contribute to fluorescent and CID-based biosensor design and study, but will also aid in the expansion of clinical small-molecule detection toolkits.

Cannabidiol (CBD), an active compound in marijuana that provides diverse health benefits such as treating epilepsy, anxiety, inflammation, and chronic pain, is increasingly used in the United States. However, little is known about the pharmacological effects of CBD on neurological diseases. Although the chemical-induced dimerization (CID) system, in which dimerization binder and anchor binder dimerize only in the presence of small molecules, has been well established, very few studies have applied it as a biosensor, especially to detect CBD. This hinders our exploration of the medical values of CBD. Here we utilize the nanobody-based CID system to develop a novel CBD biosensor, consisting of split nano luciferase - a reporter protein, CBD induced CID - a sensor protein, and glycine-serine linker - a protein linker, which ensures the biosensor efficiency. We validated the biosensor between Dimerization binder 1(DB1) and CBD anchor 14 (CA-14) both in vitro and in vivo. From our previous in vitro data, we predict that the biosensor containing Dimerization binder 4 (DB4), an analog to DB1, can detect CBD at a higher concentration in vivo than DB1, which detects better at lower CBD concentrations. We use split-luciferase assays to test the binding affinities of DB4 for a better biosensor with a broader range of drug concentration-response evaluation. This study demonstrates an effective method to maximize the CBD biosensor system, extending the applications onto detecting drugs in several brain regions, different cell populations, and even subcellular components, thus, furthering the understanding of physiological mechanisms and therapeutic potentials of CBD.