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  • 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
  • Aldh1a2 expression in Stage 33 Xenopus laevis embryo: In this lab exercise, students visualize differential gene expression in Xenopus embryos using in situ hybridization.

    Differential Gene Expression during Xenopus laevis Development

    Learning Objectives
    Students will be able to:
    • identify different stages of Xenopus development
    • contrast the strengths and limitations of the Xenopus model organism
    • explain the process and purpose of in situ hybridization
    • compare gene expression patterns from different germ layers or organ domains
    • compare gene expression patterns from different developmental stages
  • 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.