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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.
  • 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.
  • Multiple sequence alignment of homologous cytochrome C protein sequences using Jalview viewer.

    Sequence Similarity: An inquiry based and "under the hood" approach for incorporating molecular sequence...

    Learning Objectives
    At the end of this lesson, students will be able to:
    • Define similarity in a non-biological and biological sense when provided with two strings of letters.
    • Quantify the similarity between two gene/protein sequences.
    • Explain how a substitution matrix is used to quantify similarity.
    • Calculate amino acid similarity scores using a scoring matrix.
    • Demonstrate how to access genomic data (e.g., from NCBI nucleotide and protein databases).
    • Demonstrate how to use bioinformatics tools to analyze genomic data (e.g., BLASTP), explain a simplified BLAST search algorithm including how similarity is used to perform a BLAST search, and how to evaluate the results of a BLAST search.
    • Create a nearest-neighbor distance matrix.
    • Create a multiple sequence alignment using a nearest-neighbor distance matrix and a phylogram based on similarity of amino acid sequences.
    • Use appropriate bioinformatics sequence alignment tools to investigate a biological question.