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  • 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.
  • A A student assists Colorado Parks & Wildlife employees spawning greenback cutthroat trout at the Leadville National Fish Hatchery; B greenback cutthroat trout adults in a hatchery raceway; C tissue samples collected by students to be used for genetic analysis (images taken by S. Love Stowell)

    Cutthroat trout in Colorado: A case study connecting evolution and conservation

    Learning Objectives
    Students will be able to:
    • interpret figures such as maps, phylogenies, STRUCTURE plots, and networks for species delimitation
    • identify sources of uncertainty and disagreement in real data sets
    • propose research to address or remedy uncertainty
    • construct an evidence-based argument for the management of a rare taxon
  • Plant ecology students surveying vegetation at Red Hills, CA, spring 2012.  From left to right are G.L, F.D, A.M., and R.P.  Photo used with permission from all students.

    Out of Your Seat and on Your Feet! An adaptable course-based research project in plant ecology for advanced students

    Learning Objectives
    Students will:
    • Articulate testable hypotheses. (Lab 8, final presentation/paper, in-class exercises)
    • Analyze data to determine the level of support for articulated hypotheses. (Labs 4-7, final presentation/paper)
    • Identify multiple species of plants in the field quickly and accurately. (Labs 2-3, field trip)
    • Measure environmental variables and sample vegetation in the field. (Labs 2-3, field trip)
    • Analyze soil samples using a variety of low-tech lab techniques. (Open labs after field trip)
    • Use multiple statistical techniques to analyze data for patterns. (Labs 4-8, final presentation/paper)
    • Interpret statistical analyses to distinguish between strong and weak interactions in a biological system. (Labs 4-7, final presentation/paper)
    • Develop and present a conference-style presentation in a public forum. (Lab 8, final presentation/paper)
    • Write a publication-ready research paper communicating findings and displaying data. (Lab 8, final presentation/paper)
  • Figure 2. ICB-Students come to class prepared to discuss the text
  • Structure of protein ABCB6

    Investigating the Function of a Transport Protein: Where is ABCB6 Located in Human Cells?

    Learning Objectives
    At the end of this activity students will be able to:
    • describe the use of two common research techniques for studying proteins: SDS-PAGE and immunoblot analysis.
    • determine a protein’s subcellular location based on results from: 1) immunoblotting after differential centrifugation, and 2) immunofluorescence microscopy.
    • analyze protein localization data based on the limitations of differential centrifugation and immunofluorescence microscopy.
  • Format of a typical course meeting
  • MA plot of RNA-seq data. An MA plot is a visual summary of gene expression data which identifies genes showing differential expression between two treatments.

    Tackling "Big Data" with Biology Undergrads: A Simple RNA-seq Data Analysis Tutorial Using Galaxy

    Learning Objectives
    • Students will locate and download high-throughput sequence data and genome annotation files from publically available data repositories.
    • Students will use Galaxy to create an automated computational workflow that performs sequence quality assessment, trimming, and mapping of RNA-seq data.
    • Students will analyze and interpret the outputs of RNA-seq analysis programs.
    • Students will identify a group of genes that is differentially expressed between treatment and control samples, and interpret the biological significance of this list of differentially expressed genes.