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Biochemistry And Molecular Biology
Follow the Sulfur: Using Yeast Mutants to Study a Metabolic PathwayLearning ObjectivesAt 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.
CURE-all: Large Scale Implementation of Authentic DNA Barcoding Research into First-Year Biology CurriculumLearning ObjectivesStudents will be able to: Week 1-4: Fundamentals of Science and Biology
- List the major processes involved in scientific discovery
- List the different types of scientific studies and which types can establish causation
- Design experiments with appropriate controls
- Create and evaluate phylogenetic trees
- Define taxonomy and phylogeny and explain their relationship to each other
- Explain DNA sequence divergence and how it applies to evolutionary relationships and DNA barcoding
- Define and measure biodiversity and explain its importance
- Catalog organisms using the morphospecies concept
- Geographically map organisms using smartphones and an online mapping program
- Calculate metrics of species diversity using spreadsheet software
- Use spreadsheet software to quantify and graph biodiversity at forest edges vs. interiors
- Write a formal lab report
- Extract, amplify, visualize and sequence DNA using standard molecular techniques (PCR, gel electrophoresis, Sanger sequencing)
- Explain how DNA extraction, PCR, gel electrophoresis, and Sanger sequencing work at the molecular level
- Trim and assemble raw DNA sequence data
- Taxonomically identify DNA sequences isolated from unknown organisms using BLAST
- Visualize sequence data relationships using sequence alignments and gene-based phylogenetic trees
- Map and report data in a publicly available online database
- Share data in a formal scientific poster
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.