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Biochemistry And Molecular Biology

  • DNA Detective: Genotype to Phenotype. A Bioinformatics Workshop for Middle School to College. In this image, students are selecting the mutant Arabidopsis plant defective for the “mystery” gene that they identified and annotated through the DNA Subway Red Line.
  • DNA

    Using CRISPR-Cas9 to teach the fundamentals of molecular biology and experimental design

    Learning Objectives
    Module 1
    • Generate a testable hypothesis that requires a creative design of reagents based on critical reading of and review of prior research.
    • Demonstrate proficiency in using molecular cloning software to analyze, manipulate and verify DNA sequences.
    • Predict the downstream effect on the mRNA and protein after successfully inserting a DNA repair template into the genome of a cell/organism.
    • Compare and contrast the processes of DNA duplication and PCR.
    • Demonstrate the ability to design primers to amplify a nucleotide sequence.
    • Analyze and evaluate the results of DNA agarose gel electrophoresis.
    Module 2
    • Identify the key features in genomic DNA, specifically those required for CRISPR-Cas9 mediated gene edits.
    • Explain how compatible ends of DNA are used to produce recombinant DNA in a ligation reaction.
    • Explain the chemical principles behind plasmid DNA purification from bacterial cultures.
    • Devise a strategy to screen clones based on antibiotic selection and the mechanism of digestion by DNA endonucleases.
    • Predict and evaluate the results of a diagnostic digest.
    Module 3
    • Explain the chemical principles behind DNA purification using phenol-chloroform extraction and ethanol precipitation.
    • Explain the key differences between DNA duplication and transcription.
    • Demonstrate the ability to perform lab work with sterile technique.
    • Compare and contrast the results of a non-denaturing vs. denaturing agarose gel.
    • Evaluate the results of a denaturing agarose gel.
    Module 4
    • Design and implement an experiment that tests the CRISPR-Cas9 principle.
    • Predict the outcome of a successful in vitro Cas9 digest.
    Presentation of Data Post Lesson
    • Summarize important background information on gene of interest from analysis of primary literature.
    • Produce figures and figure legends that clearly indicate results.
    • Organize and construct a poster that clearly and professionally displays the important aspects of the lesson.
    • Demonstrate understanding of the lesson by presenting a poster to an audience in lay terms, mid-level terms, or at an expert level.
    • Demonstrate understanding of procedures by writing a formal materials and methods paper.
  • 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.
  • DNA barcoding research in first-year biology curriculum

    CURE-all: Large Scale Implementation of Authentic DNA Barcoding Research into First-Year Biology Curriculum

    Learning Objectives
    Students will be able to: Week 1-4: Fundamentals of Science and Biology
    • List the major processes involved in scientific discovery
    • List the different types of scientific studies and which types can establish causation
    • Design experiments with appropriate controls
    • Create and evaluate phylogenetic trees
    • Define taxonomy and phylogeny and explain their relationship to each other
    • Explain DNA sequence divergence and how it applies to evolutionary relationships and DNA barcoding
    Week 5-6: Ecology
    • Define and measure biodiversity and explain its importance
    • Catalog organisms using the morphospecies concept
    • Geographically map organisms using smartphones and an online mapping program
    • Calculate metrics of species diversity using spreadsheet software
    • Use spreadsheet software to quantify and graph biodiversity at forest edges vs. interiors
    • Write a formal lab report
    Week 7-11: Cellular and Molecular Biology
    • Extract, amplify, visualize and sequence DNA using standard molecular techniques (PCR, gel electrophoresis, Sanger sequencing)
    • Explain how DNA extraction, PCR, gel electrophoresis, and Sanger sequencing work at the molecular level
    Week 12-13: Bioinformatics
    • Trim and assemble raw DNA sequence data
    • Taxonomically identify DNA sequences isolated from unknown organisms using BLAST
    • Visualize sequence data relationships using sequence alignments and gene-based phylogenetic trees
    • Map and report data in a publicly available online database
    • Share data in a formal scientific poster
  • 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.
  • 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.