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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.
  • ACTN3 from https://upload.wikimedia.org/wikipedia/commons/3/33/Protein_ACTN3_PDB_1tjt.png

    The Science Behind the ACTN3 Polymorphism

    Learning Objectives
    This article accompanies the lesson "The ACTN3 Polymorphism: Applications in Genetics and Physiology Teaching Laboratories." Learning objectives for the lesson include:
    1. Test hypotheses related to the role of ACTN3 in skeletal muscle function.
    2. Explain how polymorphic variants of the ACTN3 gene affect protein structure and function.
    3. List and explain the differences between fast twitch and slow twitch muscle fibers.
    4. List and explain possible roles of the ACTN3 protein in skeletal muscle function.
    5. Find and analyze relevant scientific publications about the relationship between ACTN3 genotype and muscle function.
    6. Formulate hypotheses related to the relationship between ACTN3 genotype and skeletal muscle function.
    7. Design experiments to test hypotheses about the role of ACTN3 in skeletal muscle function.
    8. Statistically analyze experimental results using relevant software.
    9. Present experimental results in writing.
  • Fully annotated mitochondrial genome of a lichenized fungal species (Cladonia subtenuis).  This represents a visual representation of the final project result of the lesson plan. Students will submit their annotation to NCBI (GenBank) and upon acceptance of their annotation, they typically add this publicly available resource into their resume.

    A CURE-based approach to teaching genomics using mitochondrial genomes

    Learning Objectives
    • Install the appropriate programs such as Putty and WinSCP.
    • Navigate NCBI's website including their different BLAST programs (e.g., blastn, tblastx, blastp and blastx)
    • Use command-line BLAST to identify mitochondrial contigs within a whole genome assembly
    • Filter the desired sequence (using grep) and move the assembled mitochondrial genome onto your own computer (using FTP or SCP)
    • Error-correct contigs (bwa mem, samtools tview), connect and circularize organellar contigs (extending from filtered reads)
    • Transform assembled sequences into annotated genomes
    • Orient to canonical start locations in the mitochondrial genome (cox1)
    • Identify the boundaries of all coding components of the mitochondrial genome using BLAST, including: Protein coding genes (BLASTx and tBLASTX), tRNAs (proprietary programs such as tRNAscan), rRNAs (BLASTn, Chlorobox), ORFs (NCBI's ORFFinder)
    • Deposit annotation onto genome repository (NCBI)
    • Update CV/resume to reflect bioinformatics skills learned in this lesson
  • Image from http://www.epa.gov/airdata/ad_maps.html

    Air Quality Data Mining: Mining the US EPA AirData website for student-led evaluation of air quality issues

    Learning Objectives
    Students will be able to:
    • Describe various parameters of air quality that can negatively impact human health, list priority air pollutants, and interpret the EPA Air Quality Index as it relates to human health.
    • Identify an air quality problem that varies on spatial and/or temporal scales that can be addressed using publicly available U.S. EPA air data.
    • Collect appropriate U.S. EPA Airdata information needed to answer that/those questions, using the U.S. EPA Airdata website data mining tools.
    • Analyze the data as needed to address or answer their question(s).
    • Interpret data and draw conclusions regarding air quality levels and/or impacts on human and public health.
    • Communicate results in the form of a scientific paper.
  • Possible implementations of a short research module

    A Short Laboratory Module to Help Infuse Metacognition during an Introductory Course-based Research Experience

    Learning Objectives
    • Students will be able to evaluate the strengths and weaknesses of data.
    • Students will be able to employ prior knowledge in formulating a biological research question or hypothesis.
    • Students will be able to distinguish a research question from a testable hypothesis.
    • Students will recognize that the following are essential elements in experimental design: identifying gaps in prior knowledge, picking an appropriate approach (ex. experimental tools and controls) for testing a hypothesis, and reproducibility and repeatability.
    • Students will be able to identify appropriate experimental tools, approaches and controls to use in testing a hypothesis.
    • Students will be able to accurately explain why an experimental approach they have selected is a good choice for testing a particular hypothesis.
    • Students will be able to discuss whether experimental outcomes support or fail to support a particular hypothesis, and in the case of the latter, discuss possible reasons why.
  • Set Up Fly Traps: The photo is of the fly traps after being set up for the experiment

    Gotcha! Which fly trap is the best? An introduction to experimental data collection and analysis

    Learning Objectives
    Students will:
    • design and execute an experiment
    • collect, organize, and summarize data
    • analyze and interpret data and make inferences
  • 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.
  • Students participating in the peer review process. Practicing the writing of scientific manuscripts prepares students to understand and engage in the primary literature they encounter.
  • Summary diagram of the Pipeline CURE. A diagram describing how undergraduates, faculty, and research trainees progress through a sequence of guided research activities that develop student independence.
  • Image of a writing center

    Visits to the writing center and office hours provide students structured reflection and low-stakes feedback on...

    Learning Objectives
    • Students will be able to write a lab report that contains a descriptive title, complete and concise abstract, substantive and relevant introduction that includes a testable hypothesis, descriptive methods, description and comparison of results of various testable groups, biological explanation of the results that reflect the testable hypothesis, a conclusion that contains societal implications or scientific impact, and references cited in the document.
    • Students will be able to self-identify weaknesses and strengths of their writing.
    • Students will understand how to utilize office hours and the writing center to receive feedback on their lab reports.
  • ACTN3 from https://upload.wikimedia.org/wikipedia/commons/3/33/Protein_ACTN3_PDB_1tjt.png

    The ACTN3 Polymorphism: Applications in Genetics and Physiology Teaching Laboratories

    Learning Objectives
    1. Test hypotheses related to the role of ACTN3 in skeletal muscle function.
    2. Explain how polymorphic variants of the ACTN3 gene affect protein structure and function.
    3. List and explain the differences between fast twitch and slow twitch muscle fibers.
    4. List and explain possible roles of the ACTN3 protein in skeletal muscle function.
    5. Find and analyze relevant scientific publications about the relationship between ACTN3 genotype and muscle function.
    6. Formulate hypotheses related to the relationship between ACTN3 genotype and skeletal muscle function.
    7. Design experiments to test hypotheses about the role of ACTN3 in skeletal muscle function.
    8. Statistically analyze experimental results using relevant software.
    9. Present experimental results in writing.
  • pClone Red Makes Research Look Easy

    Using Synthetic Biology and pClone Red for Authentic Research on Promoter Function: Genetics (analyzing mutant...

    Learning Objectives
    • Describe how cells can produce proteins at the right time and correct amount. 
    • Diagram a bacterial promoter with −35 and −10 elements and the transcription start site.
    • Describe how mutational analysis can be used to study promoter sequence requirements.
    • Develop a promoter mutation hypothesis and design an experiment to test it.
    • Successfully and safely manipulate DNA and Escherichia coli for ligation and transformation experiments. 
    • Design an experiment to verify a mutated promoter has been cloned into a destination vector. 
    • Design an experiment to measure the strength of a promoter. 
    • Analyze data showing reporter protein produced and use the data to assess promoter strength. 
    • Define type IIs restriction enzymes.
    • Distinguish between type II and type IIs restriction enzymes.
    • Explain how Golden Gate Assembly (GGA) works.
    • Measure the relative strength of a promoter compared to a standard promoter.  
  • pClone Red Makes Research Look Easy

    Using Synthetic Biology and pClone Red for Authentic Research on Promoter Function: Introductory Biology (identifying...

    Learning Objectives
    • Describe how cells can produce proteins at the right time and correct amount.
    • Diagram how a repressor works to reduce transcription.
    • Diagram how an activator works to increase transcription.
    • Identify a new promoter from literature and design a method to clone it and test its function.
    • Successfully and safely manipulate DNA and Escherichia coli for ligation and transformation experiments.
    • Design an experiment to verify a new promoter has been cloned into a destination vector.
    • Design an experiment to measure the strength of a promoter.
    • Analyze data showing reporter protein produced and use the data to assess promoter strength.
    • Define type IIs restriction enzymes.
    • Distinguish between type II and type IIs restriction enzymes.
    • Explain how Golden Gate Assembly (GGA) works.
    • Measure the relative strength of a promoter compared to a standard promoter.
  • SNP model by David Eccles (gringer) [GFDL (http://www.gnu.org/copyleft/fdl.html) or CC BY 4.0 (http://creativecommons.org/licenses/by/4.0)], via Wikimedia Commons

    Exploration of the Human Genome by Investigation of Personalized SNPs

    Learning Objectives
    Students successfully completing this lesson will be able to:
    • Effectively use the bioinformatics databases (SNPedia, the UCSC Genome Browser, and NCBI) to explore SNPs of interest within the human genome.
    • Identify three health-related SNPs of personal interest and use the UCSC Genome Browser to define their precise chromosomal locations and determine whether they lie within a gene or are intergenic.
    • Establish a list of all genome-wide association studies correlated with a particular health-related SNP.
    • Predict which model organism would be most appropriate for conducting further research on a human disease.
  • 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.
  • 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.
  • 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.
  • Structure of protein ADA2

    Understanding Protein Domains: A Modular Approach

    Learning Objectives
    • Students will be able to compare protein sequences and identify conserved regions and putative domains.
    • Students will be able to obtain, examine, and compare structural models of protein domains.
    • Students will be able to interpret data on protein interactions (in vitro pull-down and in vitro and in vivo functional assays)
    • Students will be able to propose experiments to test protein interactions.
  • 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.
  • 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.