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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.

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.