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Heterologous expression and purification of Nicotiana benthamiana Cellulose synthase-like B (NbCslB)Ståhl, Olivia January 2020 (has links)
Hemicelluloses are synthesized by proteins encoded by genes from the cellulose synthasegene superfamily. One subgroup of this gene family is the cellulose synthase-like B, which islargely uncharacterized and unexplored. The common model organism Nicotianabenthamiana has one such gene in its genome, NbCslB, encoding a membrane protein. Theexpression of this gene has previously been studied in vivo, but in order to study the protein invitro a viable solubilization and purification protocol is required. This study evaluated the useof the detergent n-Dodecyl β-D-maltoside (DDM) for solubilization, followed by purificationusing immobilized metal ion affinity chromatography (IMAC), and thereafter reconstitutionof the protein into proteoliposomes. SDS-PAGE as well as Western blot analyses showed thatthe purification was successful and provided a pure sample of protein. Throughout theanalyses performed, an anti-FLAG antibody was discovered to bind well to the protein, andthereby be especially useful for analysis. An activity assay was performed on the purifiedprotein, to characterize its function and evaluate whether the protein had maintained itsactivity and conformation after the steps of purification and reconstitution. No activity couldbe detected in the enzymatic assay, which indicated that the purification protocol may havebeen too rough on the protein, that the reconstitution was not successful, or that the assayconditions were not optimal. These results can be used as a base for future research, where theprotocols for solubilization, purification, and reconstitution should be further refined in orderto obtain an end result where the purified protein is active. When an active and pure proteinsample is achieved, it will be possible to perform further attempts at characterizing thefunction of the protein using enzymatic activity assays. Additionally, the results showed thatthe choice of antibody can be crucial for proper analysis of this protein. / Hemicellulosa syntetiseras av proteiner vars gener återfinns i genfamiljen cellulosasyntas. Enundergrupp till denna genfamilj är cellulosasyntasliknande B, en grupp som till stor del ärokarakteriserad och outforskad. Den vanliga modellorganismen Nicotiana benthamiana haren sådan gen i sitt genom, NbCslB, som kodar för ett membranprotein. Hur denna genuttrycks har tidigare studerats in vivo, men for att kunna studera proteinet in vitro krävs etthållbart protokoll för solubilisering och rening. Denna studie utvärderade användningen avlösningsmedlet n-Dodecyl β-D-maltoside (DDM) för solubilisering, följt av rening medimmobiliserad metalljon-affinitetskromatografi (IMAC), och efter det rekonstitution avproteinet till proteoliposomer. SDS-PAGE och Western blot analyser visade att reningen varlyckad, och att ett rent proteinprov erhållits. När analyserna genomfördes upptäcktes att enanti-FLAG antikropp band särskilt väl till proteinet, och därmed var mycket användbar vidanalys. En aktivitetsanalys genomfördes med det renade proteinet för att karakterisera dessfunktion och utvärdera huruvida proteinet hade bevarat sin aktivitet och konformation efterrening och rekonstitution. Ingen aktivitet kunde detekteras i den enzymatiskaaktivitetsanalysen, vilket indikerade att reningen eventuellt var för hård mot proteinet,alternativt att rekonstitutionen inte var lyckad, eller att förhållandena för analysen inte varoptimala. Dessa resultat kan användas som en bas för framtida forskning om proteinet, därprotokollen för solubilisering, rening och rekonstitution bör vidareutvecklas för att uppnå ettslutresultat där det renade proteinet är aktivt. När ett aktivt och rent proteinprov uppnåtts ärdet möjligt att genomföra ytterligare försök att karakterisera proteinets funktion medenzymatiska aktivitetsanalyser. Resultaten visade också att valet av antikropp kan varaavgörande för att ordentligt kunna analysera detta protein.
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Undergraduate Students' Understanding and Interpretation of Carbohydrates and Glycosidic BondsJennifer Garcia (16510035) 10 July 2023 (has links)
<p>For the projects titled Undergraduate Students’ Interpretation of Fischer and Haworth Carbohydrate Projections and Undergraduate Students' Interpretation of Glycosidic Bonds – there is a prevalent issue in biochemistry education in which students display fragmented knowledge of the biochemical concepts learned when asked to illustrate their understandings (via drawings, descriptions, analysis, etc.). In science education, educators have traditionally used illustrations to support students’ development of conceptual understanding. However, interpreting a representation is dependent on prior knowledge, ability to decode visual information, and the nature of the representation itself. With a prevalence of studies conducted on visualizations, there is little research with a focus on the students’ interpretation and understanding of carbohydrates and/or glycosidic bonds. The aim of these projects focuses on how students interpret representations of carbohydrates and glycosidic bonds. This study offers a description of undergraduate students’ understanding and interpretation using semi-structured interviews through Phenomenography, Grounded Theory and the Resources Frameworks. The data suggests that students have different combinations of (low or high) accuracy and productivity for interpreting and illustrating carbohydrates and glycosidic bonds, among other findings to be highlighted in their respective chapters. More effective teaching strategies can be designed to assist students in developing expertise in proper illustrations and guide their thought process in composing proper explanations in relation to and/or presence of illustrations.</p>
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<p>For the project titled Impact of the Pandemic on Student Readiness: Laboratories, Preparedness, and Support – it was based upon research by Meaders et. al (2021) published in the International Journal of STEM Education. Messaging during the first day of class is highly important in establishing positive student learning environments. Further, this research suggests that students are detecting the messages that are communicated. Thus, attention should be given to prioritizing what information and messages are most important for faculty to voice. There is little doubt that the pandemic has had a significant impact on students across the K-16 spectrum. In particular, for undergraduate chemistry instructors’, data on the number of laboratories students completed in high school and in what mode would be important information in considering what modifications could be implemented in the laboratory curriculum and in messaging about the laboratory activities – additionally on how prepared students feel to succeed at college work, how the pandemic has impacted their preparedness for learning, and what we can do to support student learning in chemistry can shape messaging on the first day and for subsequent activities in the course. An initial course survey that sought to highlight these student experiences and perspectives will be discussed along with the impact on course messaging and structure. </p>
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