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CRISPR/Cas9 in yeast: a multi-week laboratory exercise for undergraduate studentsLearning ObjectivesWeek 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.
- 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.
- 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.
- 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.
- Design an experiment to determine auxotrophic phenotypes.
- Predict the outcome of multi-step experiments.
- 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.
- 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.
Using CRISPR-Cas9 to teach the fundamentals of molecular biology and experimental designLearning ObjectivesModule 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.
- 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.
- 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.
- Design and implement an experiment that tests the CRISPR-Cas9 principle.
- Predict the outcome of a successful in vitro Cas9 digest.
- 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.
Serotonin in the Pocket: Non-covalent interactions and neurotransmitter bindingLearning Objectives
- Students will design a binding site for the neurotransmitter serotonin.
- Students will be able to determine the effect of a change in molecular orientation on the affinity of the molecule for the binding site.
- Students will be able to determine the effect of a change in molecular charge on the affinity of the molecule for the binding site.
- Students will be able to better differentiate between hydrogen bond donors and acceptors.
- Students can use this knowledge to design binding sites for other metabolites.
The Science Behind the ACTN3 PolymorphismLearning ObjectivesThis article accompanies the lesson "The ACTN3 Polymorphism: Applications in Genetics and Physiology Teaching Laboratories." Learning objectives for the lesson include:
- Test hypotheses related to the role of ACTN3 in skeletal muscle function.
- Explain how polymorphic variants of the ACTN3 gene affect protein structure and function.
- List and explain the differences between fast twitch and slow twitch muscle fibers.
- List and explain possible roles of the ACTN3 protein in skeletal muscle function.
- Find and analyze relevant scientific publications about the relationship between ACTN3 genotype and muscle function.
- Formulate hypotheses related to the relationship between ACTN3 genotype and skeletal muscle function.
- Design experiments to test hypotheses about the role of ACTN3 in skeletal muscle function.
- Statistically analyze experimental results using relevant software.
- Present experimental results in writing.
A virtual laboratory on cell division using a publicly-available image databaseLearning Objectives
- Students will name and describe the salient features and cellular tasks for each stage of cell division.
- Students will predict the relative durations of the stages of cell division using prior knowledge and facts from assigned readings.
- Students will describe the relationship between duration of each stage of cell division and the frequency of cells present in each stage of cell division counted in a random sample of images of pluripotent stem cells.
- Students will identify the stages of cell division present in research-quality images of human pluripotent stem cells in various stages of cell division.
- Students will quantify, analyze and summarize data on the prevalence of cells at different stages of cell division in randomly sampled cell populations.
- Students will use data to reflect on and revise predictions.
Plotting Cranial and Spinal Nerve Pathways in a Human Anatomy LabLearning Objectives
- Identify and describe the functions of cranial and spinal nerves
- Identify cranial and spinal nerve origination points and what structures they innervate
- Trace the routes that cranial and spinal nerves take throughout the body