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Biochemistry And Molecular Biology

  • A three-dimensional model of methionine is superimposed on a phase contrast micrograph of Saccharomyces cerevisiae from a log phase culture.

    Follow the Sulfur: Using Yeast Mutants to Study a Metabolic Pathway

    Learning Objectives
    At the end of this lesson, students will be able to:
    • use spot plating techniques to compare the growth of yeast strains on solid culture media.
    • predict the ability of specific met deletion strains to grow on media containing various sulfur sources.
    • predict how mutations in specific genes will affect the concentrations of metabolites in the pathways involved in methionine biosynthesis.
  • Binding pocket diagram The image suggests that by providing appropriate non-covalent interactions at sites A, B and C, students can create a binding pocket selective for the neurotransmitter molecule serotonin.

    Serotonin in the Pocket: Non-covalent interactions and neurotransmitter binding

    Learning Objectives
    • Students will design a binding site for the neurotransmitter serotonin.
    • Students will be able to determine the effect of a change in molecular orientation on the affinity of the molecule for the binding site.
    • Students will be able to determine the effect of a change in molecular charge on the affinity of the molecule for the binding site.
    • Students will be able to better differentiate between hydrogen bond donors and acceptors.
    • Students can use this knowledge to design binding sites for other metabolites.
  • Abelson kinase signaling network. The image shows many connections between genes and illustrates that signaling molecules and pathways function within networks. It emphasizes the indispensability of computational tools in understanding the molecular functioning of cells. The image was generated with Cytoscape from publicly accessible protein-protein interactions databases.

    Investigating Cell Signaling with Gene Expression Datasets

    Learning Objectives
    Students will be able to:
    • Explain the hierarchical organization of signal transduction pathways.
    • Explain the role of enzymes in signal propagation and amplification.
    • Recognize the centrality of signaling pathways in cellular processes, such as metabolism, cell division, or cell motility.
    • Rationalize the etiologic basis of disease in terms of deranged signaling pathways.
    • Use software to analyze and interpret gene expression data.
    • Use an appropriate statistical method for hypotheses testing.
    • Produce reports that are written in scientific style.
  • Using QIIME to Interpret Environmental Microbial Communities in an Upper Level Metagenomics Course

    Learning Objectives
    Students will be able to:
    • list and perform the steps of sequence processing and taxonomic inference.
    • interpret microbial community diversity from metagenomic sequence datasets.
    • compare microbial diversity within and between samples or treatments.
  • Students using the Understanding Eukaryotic Genes curriculum to construct a gene model. Students are working as a pair to complete each Module using classroom computers.

    An undergraduate bioinformatics curriculum that teaches eukaryotic gene structure

    Learning Objectives
    Module 1
    • Demonstrate basic skills in using the UCSC Genome Browser to navigate to a genomic region and to control the display settings for different evidence tracks.
    • Explain the relationships among DNA, pre-mRNA, mRNA, and protein.
    Module 2
    • Describe how a primary transcript (pre-mRNA) can be synthesized using a DNA molecule as the template.
    • Explain the importance of the 5' and 3' regions of the gene for initiation and termination of transcription by RNA polymerase II.
    • Identify the beginning and the end of a transcript using the capabilities of the genome browser.
    Module 3
    • Explain how the primary transcript generated by RNA polymerase II is processed to become a mature mRNA, using the sequence signals identified in Module 2.
    • Use the genome browser to analyze the relationships among:
    • pre-mRNA
    • 5' capping
    • 3' polyadenylation
    • splicing
    • mRNA
    Module 4
    • Identify splice donor and acceptor sites that are best supported by RNA-Seq data and TopHat splice junction predictions.
    • Utilize the canonical splice donor and splice acceptor sequences to identify intron-exon boundaries.
    Module 5
    • Determine the codons for specific amino acids and identify reading frames by examining the Base Position track in the genome browser.
    • Assemble exons to maintain the open reading frame (ORF) for a given gene.
    • Define the phases of the splice donor and acceptor sites and describe how they impact the maintenance of the ORF.
    • Identify the start and stop codons of an assembled ORF.
    Module 6
    • Demonstrate how alternative splicing of a gene can lead to different mRNAs.
    • Show how alternative splicing can lead to the production of different polypeptides and result in drastic changes in phenotype.
  • CRISPR/Cas9 in yeast experimental overview

    CRISPR/Cas9 in yeast: a multi-week laboratory exercise for undergraduate students

    Learning Objectives
    Week 1: CRISPR design
    • Locate the coding sequence, flanking sequence, protein product, and characteristics of a given gene from the Saccharomyces Genome Database (https://www.yeastgenome.org/).
    • Design and defend the design of guide RNA and single stranded template for DNA repair in CRISPR/Cas9 gene editing studies to generate Saccharomyces cerevisiae auxotrophic mutants.
    Week 3-4: Cloning
    • Describe the qualities of the vector, pML104, that allow replication and selection in bacteria and yeast as well as allow expression of necessary factors in CRISPR/Cas9 genome editing, including Cas9 and sgRNA.
    • Describe the rationale of and perform procedures necessary for cloning a small cassette (i.e., sgRNA gene) into a vector (i.e., pML104) including; restriction digest, annealing of DNA strands, removal of 5’ phosphates, ligation, and transformation.
    • Recognize and design appropriate controls for cloning procedures such as ligation and transformation.
    Week 5: Screening clones
    • Describe the method of polymerase chain reaction (PCR), including the rationale for essential components of a reaction mixture and thermal-cycling conditions.
    • Locate the binding sites of and design primers for PCR, then report the expected size of the amplification product.
    • Describe and perform isolation of plasmid DNA from E. coli.  
    Week 6: Selection of clones and transformation of yeast
    • Describe the rationale for and perform procedures to transform yeast, including the essential components of a transformation mixture and conditions necessary for transformation.
    • Describe the basic conditions required for cultivating yeast.
    • Describe the rationale for and perform agarose gel electrophoresis of a given size of DNA.
    • Analyze DNA separated by agarose gel electrophoresis, including size estimation.
    • Recognize and describe the qualities of a template for DNA repair that allows efficient DNA repair. 
    Week 7: Phenotyping
    • Design an experiment to determine auxotrophic phenotypes.
    • Predict the outcome of multi-step experiments.
    Multiweek
    • Recognize and describe conditions necessary for growth of E. coli and S. cerevisiae.
    • Qualitatively and quantitatively analyze scientific data from scientific experiments, including bacterial and yeast transformation, agarose gel electrophoresis, extraction of plasmid DNA from bacteria, PCR, and auxotroph phenotypic analysis.
    • Communicate science to peers through maintenance of a laboratory notebook, verbal communication with group members, and writing of a formal laboratory report written in a format acceptable for journal publication.
    • Troubleshoot scientific protocols by identifying procedures that are prone to error, comparing recommended protocols to actual procedure, and using positive and negative controls to narrow the location of a potential error.
    • Communicate specific potential or actual uses of CRISPR/Cas9 in science and/or medicine.
    Alignment with Society-Generated Learning Objectives - From Biochemistry and Molecular Biology, and Genetics Learning Frameworks
    • Use various bioinformatics approaches to analyze macromolecular primary sequence and structure.
    • Illustrate how DNA is replicated and genes are transmitted from one generation to the next in multiple types of organisms including bacteria, eukaryotes, viruses, and retroviruses.
    • Define what a genome consists of and how the information in various genes and other sequence classes within each genome are used to store and express genetic information.
    • Explain the meaning of ploidy (haploid, diploid, aneuploid etc.) and how it relates to the number of homologues of each chromosome. 
    • Predict the effects of mutations on the activity, structure, or stability of a protein and design appropriate experiments to assess the effects of mutations.
    • Predict the growth behavior of microbes based on their growth conditions, e.g., temperature, available nutrient, aeration level, etc.
    • Discuss the benefits of specific tools of modern biotechnology that are derived from naturally occurring microbes (e.g. cloning vectors, restriction enzymes, Taq polymerase, etc.)
    • Accurately prepare and use reagents and perform experiments.
    • When presented with an observation, develop a testable and falsifiable hypothesis.
    • When provided with a hypothesis, identify the appropriate experimental observations and controllable variables.
  • Enzymatic avocado browning is driven by polyphenol oxidase. Mashed avocado pulp is bright green but turns dark brown over the course of two hours at room temperature in the presence of air and salt. This reaction can be accelerated or inhibited by more than 20 different testable reagents, allowing students to explore experimental design.

    The Avocado Lab: An Inquiry-Driven Exploration of an Enzymatic Browning Reaction

    Learning Objectives
    Students will be able to:
    • develop a testable research question and supportive hypothesis regarding the browning of damaged avocado flesh caused by the activity of avocado polyphenol oxidase (aPPO).
    • design and execute a well-controlled experiment to test aPPO hypotheses.
    • evaluate qualitative enzyme activity data.
    • create a figure and legend to present qualitative data that tests multiple hypotheses and variables.
    • search for and correctly cite primary literature to support or refute hypotheses.
    • know the role of reducing reagents, pH, chelators, and temperature in reactions catalyzed by aPPO.
    • explain why the effects of salt and detergent differ for aPPO experiments conducted in situ
    • (in mashed avocado flesh) as compared to in vitro (on purified protein).
    • discuss how substrate and cofactor availability affect aPPO reactions.
    • describe how endogenous subcellular organization restricts aPPO reactions in a healthy avocado.
    • evaluate food handling practices for fruits expressing PPO.