Synthesis of the Intermediate of a Catalytic Reaction: An NHC-Stabilized, First-Row Transition Metal Complex part of CUREnet:Institutes:Other Institutes (2019-2020):Examples
The advanced synthesis laboratory course object allows students to study the synthesis, purification, and characterizations of a new diamagnetic organometallic complex of a first-row transition metal. The air-stable complex is stabilized by an N-heterocyclic carbene spectator ligand. It also bears an actor ligand and therefore, is potentially a reactive intermediate of a catalytic reaction. The synthesis of a reactive intermediate is the key to elucidate the mechanism of catalysis. The instructor chooses the first-row transition metal and the actor ligand based on his or her interests. The CURE starts from an NHC-ligated complex that does not bear this actor ligand but is otherwise similar. In our CURE, an anion ligand-replacement reaction was used to install the actor ligand, but an instructor may choose other approaches. The students will evaluate their results by standard spectroscopic analyses using UV-vis, FT-IR, and proton NMR (60 MHz or above) analysis.

Analysis of the effects of protein-protein interactions on signaling through a team-based undergraduate biochemistry laboratory course part of CUREnet:CURE Collection
We developed a research-based laboratory course centered on a biological problem involving the B-Raf kinase, specifically the mutant that is commonly found in melanomas. One of the major goals of the project for the students is to generate mutants to determine whether a particular region of the B-Raf protein is critical for the interaction with MEK kinase, a downstream target in the pathway. Students analyze the published B-Raf-MEK crystal structure and choose a mutation to generate in B-Raf or MEK that might alter the dissociation constant (KD) of the complex. They design primers, perform PCR to generate their desired mutant, transform and purify the resulting DNA, express the DNA in E. coli, and purify the protein, all before characterizing it. Characterizing the mutant proteins consist of performing basic pull-downs, western blots, spectroscopic absorbance assays, and biolayer interferometry for binding kinetics. Students also engage in group meeting presentations and journal clubs in which they discuss their work and related primary literature, respectively. Group meeting and journal club discussions provide a forum for students to come up with new ideas to analyze their results, or for future work. Students summarize key results in a final presentation and paper, and develop a research proposal based on their work. Data that students obtain from their mutants provide evidence of the importance of a binding region for B-Raf-MEK complex formation, as well as downstream phosphorylation events. Such data will inform future drug discovery programs, as well as form the foundation for students' work in the course the following year. Because working with mutants can result in unpredictable data and results, students sometimes have to adjust their protocols and repeat experiments. Thus, the CURE format of this course also gives students an opportunity to learn to troubleshoot when things do not work as expected, which helps them learn resiliency in science.

BASIL (Biochemistry Authentic Scientific Inquiry Laboratory) part of CUREnet:CURE Collection
This curriculum from the BASIL (Biochemistry Authentic Scientific Inquiry Laboratory) biochemistry consortium aims to get students to transition from thinking like students to thinking like scientists. Students will analyze proteins with known structure but unknown function using computational analyses and wet-lab techniques. BASIL is designed for undergraduate biochemistry lab courses, but can be adapted to first year (or even high school) settings, as well as upper-level undergraduate or graduate coursework. It is targeted to students in biology, biochemistry, chemistry, or related majors. Further details about the BASIL biochemistry consortium can be found on the BASIL blog, http://basiliuse.blogspot.com/ The curriculum is flexible and can be adapted to match the available facilities, the strengths of the instructor and the learning goals of a course and institution. These lessons are often used as part of upper-level laboratory coursework with at least one semester of biochemistry as a pre-requisite or co-requisite. The lab has been designed for classes ranging from 10-24 students (working in teams of two or three) per lab section. This lesson can be adapted to laboratory courses for introductory biology, cell and molecular biology, or advanced biology labs.

MCC: Malate Dehydrogenase CUREs Community part of CUREnet:CURE Collection
The Malate Dehydrogenase CUREs Community (MCC) project is designed to facilitate the adoption of effective, protein‐centric, Course Based Undergraduate Research Experiences (CUREs) into teaching labs at a wide variety of undergraduate serving institutions. (Primarily Undergraduate Institutions, Research Intensive Universities and Community Colleges) MCC coordinates and conducts pedagogical research into two major features of CUREs:1) their duration (whole semester versus 5‐6 week modules incorporated into a lab class), and 2) the impact of scientific collaboration between institutions (a key aspect of much modern research). Using validated assessment tools we seek to establish their effects on student confidence, persistence in STEM, and ability to design research experiments and interprete data. To facilitate faculty adoption of CURE approaches the project provides a number of resources. These focus on a variety of research areas related to Malate Dehydrogenase including mechanisms of catalysis and regulation, adaptation and evolution, cofactor specificity, folding and stability and interactions in metabolons. Resources include biologics, experimental protocols and assessment tools. The project also coordinates interactions between courses at different institutions to allow incorporation of scientific collaboration into CUREs. These collaborations also facilitate the use of more sophisticated experimental approaches and broaden the experimental scope of the CUREs.

Biochemistry II lab: Crithidia parasite metabolism part of CUREnet:CURE Collection
Course Undergraduate Research Experiences (CUREs) have been shown to increase student engagement, skills, and retention in STEM. We developed a CURE using non-pathogenic Crithidia fasciculata parasites, which are insect trypanosomes related to the causative agents of Leishmaniasis, African Trypanosomiasis, and Chagas' diseases. This parasite is ideal for undergraduate CUREs because it grows to high density in serum-free inexpensive media, and has not been well studied in the literature, providing opportunities for novel discoveries. Metabolically labelled 1-13C-glucose was added to the parasites, and changes in peak position was monitored over time (either in real time, or in the supernatant). The main fermentation products observed were ethanol and succinate. Student groups then designed a novel project investigating metabolism in Crithidia. Students produced novel data on metabolism in a little-studied parasite.

Biomass conversion into highly useful chemicals part of CUREnet:Institutes:Alabama State University:Examples
This is CURE based course that aims at bridging the gap between theoretical knowledge in chemistry and its practical applications at solving real-world problems. It gives students an opportunity to construct and synthesize their knowledge and skills by learning to apply theoretical knowledge to practice by the laboratory research. The purpose of this course is to acquaint students with the fundamental concepts of chemistry, synthetic methods and techniques. The emphasis will be on novel catalysts synthesis and evaluating their activity towards biomass conversion to liquid fuel and useful chemicals. Students will design synthesize, deduce identities of the biomass conversion products from chemical and spectral clues, and predict reaction products.

The HICA project part of CUREnet:CURE Collection
In this CURE, inspired by the work of Hoffmann, et al., students prepare mutant Haemophilus influenzae carbonic anhydrase (HICA) proteins. Using PyMOL to visualize the three-dimensional structure of the HICA protein, students choose one or more surface amino acid residues to mutate to histidine residues in order to create a surface histidine cluster that will allow the mutant protein to bind to a nickel affinity column. Using site-directed mutagenesis, recombinant plasmids are constructed and are then used to transform an E. coli expression vector. The mutant HICA protein is overexpressed, cells are lysed, and students load the cell lysate onto Ni-NTA columns and determine the imidazole concentration required to elute the mutant protein. The construction of a library of mutant proteins will allow the development of a general method in which specific surface histidine residues of any protein can be mutated in order to facilitate affinity purification. The Haemophilus influenzae bacterium described herein is a respiratory pathogen that causes meningitis (in its encapsulated form) and mucosal infections such as otitis media, sinusitis and conjunctivitis (in its unencapsulated form). A recent study showed that the carbonic anhydrase enzyme is absolutely required for pathogenesis. Furthermore, expression of the HICA enzyme allows the pathogen to survive in host immune cells (Langereis, et al.). These observations make the study of HICA itself particularly attractive, in addition to the overall goal of contributing to a body of work that will allow the minimal histidine character required for nickel affinity to be ascertained.

Exploring eukaryotic protein structure and post-translational modifications. part of CUREnet:Institutes:Bowie State University:Examples
This CURE will provide opportunity for students to think and act as researchers by using computational, biochemical, and bioanalytical techniques to examine tick antigen proteins. The CURE is designed as a lab for upper-level students who are taking or have taken a one-semester introductory biochemistry course, but two semesters would be even better. It could also be adapted for cell/molecular biology or (bio) analytical chemistry instrumentational analysis labs. It has been taught for classes ranging from 12-24 students. Ticks are notorious vectors of viral, protozoan, and bacterial diseases, including Lyme disease. While an anti-vector vaccine capable of protecting people from diseases transmitted by a particular tick species is an alluring goal, only one such anti-tick vaccine is currently available. This vaccine targets Bm86, a protein from the midgut of Rhipicephalus microplus, a cattle tick. Not only does the vaccine limit parasitism of the cattle by ticks, data suggests that it can also prevent transmission of tick-borne diseases including bovine anaplasmosis and babesiosis. However, similar vaccination approaches have not succeeded thus far against ticks that transmit diseases to humans, and little is known about the antibody response to the antigen, or about the protein itself. Since the protein's structure and function are unknown, the research goal of this CURE is to purify Bm86 using an insect cell/baculovirus expression system and characterize it, including domain structure and post-translational modifications (glycosylation sites). There are homologs to Bm86 in every sequenced tick species examined, and future CUREs will characterize some of the homologs including those in Ixodes scapularis, the tick that is mainly responsible for transmitting Lyme in the eastern US, and Haemaphysalis longicornis, the Asian longhorned tick, a newly-discovered invasive species in the area that also has significant disease-transmitting potential. By understanding the structure and post-translational modifications of this protein, we hope to gain a better understanding of how to make effective anti-tick vaccines, including those for humans, that may prevent transmission of Lyme disease. Importantly, the basic parameters of this CURE can be used to examine other proteins besides tick antigens. For example, during the pandemic, the CURE pivoted from the tick antigen to the SARS-CoV-2 nucleocapsid protein, which was also expressed in an insect cell system. Instead of characterizing glycosylation sites, we characterized phosphorylation sites. It's therefore possible to use this same framework for many different eukaryotic proteins that may be of research interest.

Design2Data part of CUREnet:Institutes:Other Institutes (2019-2020):Examples
The D2D program is centered around an undergraduate-friendly protocol workflow that follows the design-build-test-learn engineering framework. This protocol has served as the scaffold for a successful undergraduate training program and has been further developed into courses that range from a 10-week freshman seminar to a year-long, upper-division molecular biology course. The overarching research goal of this CURE probes the current predictive limitations of protein-modeling software by functionally characterizing single amino acid mutants in a robust model system. The most interesting outcomes of this project are dependent on large datasets, and, as such, the project is optimal for multi-institutional collaborations.

A Bioinformatic Look at Iron Uptake in Insects part of CUREnet:Institutes:CU Denver:Examples
Students will perform BLAST searches, make phylogenetic trees, identify putative orthologs, and investigate secondary structure elements of 5' untranslated regions (UTRs). The sequences used will be related to iron uptake in insects.