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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.
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
Cutthroat trout in Colorado: A case study connecting evolution and conservationLearning ObjectivesStudents will be able to:
- interpret figures such as maps, phylogenies, STRUCTURE plots, and networks for species delimitation
- identify sources of uncertainty and disagreement in real data sets
- propose research to address or remedy uncertainty
- construct an evidence-based argument for the management of a rare taxon
Out of Your Seat and on Your Feet! An adaptable course-based research project in plant ecology for advanced studentsLearning ObjectivesStudents will:
- Articulate testable hypotheses. (Lab 8, final presentation/paper, in-class exercises)
- Analyze data to determine the level of support for articulated hypotheses. (Labs 4-7, final presentation/paper)
- Identify multiple species of plants in the field quickly and accurately. (Labs 2-3, field trip)
- Measure environmental variables and sample vegetation in the field. (Labs 2-3, field trip)
- Analyze soil samples using a variety of low-tech lab techniques. (Open labs after field trip)
- Use multiple statistical techniques to analyze data for patterns. (Labs 4-8, final presentation/paper)
- Interpret statistical analyses to distinguish between strong and weak interactions in a biological system. (Labs 4-7, final presentation/paper)
- Develop and present a conference-style presentation in a public forum. (Lab 8, final presentation/paper)
- Write a publication-ready research paper communicating findings and displaying data. (Lab 8, final presentation/paper)
Predicting and classifying effects of insertion and deletion mutations on protein coding regionsLearning ObjectivesStudents will be able to:
- accurately predict effects of frameshift mutations in protein coding regions
- conduct statistical analysis to compare expected and observed values
- become familiar with accessing and using DNA sequence databases and analysis tools
Promoting Climate Change Literacy for Non-majors: Implementation of an atmospheric carbon dioxide modeling activity as...Learning Objectives
- Students will be able to manipulate and produce data and graphs.
- Students will be able to design a simple mathematical model of atmospheric CO2 that can be used to make predictions.
- Students will be able to conduct simulations, analyze, interpret, and draw conclusions about atmospheric CO2 levels from their own computer generated simulated data.
A Short Laboratory Module to Help Infuse Metacognition during an Introductory Course-based Research ExperienceLearning Objectives
- Students will be able to evaluate the strengths and weaknesses of data.
- Students will be able to employ prior knowledge in formulating a biological research question or hypothesis.
- Students will be able to distinguish a research question from a testable hypothesis.
- Students will recognize that the following are essential elements in experimental design: identifying gaps in prior knowledge, picking an appropriate approach (ex. experimental tools and controls) for testing a hypothesis, and reproducibility and repeatability.
- Students will be able to identify appropriate experimental tools, approaches and controls to use in testing a hypothesis.
- Students will be able to accurately explain why an experimental approach they have selected is a good choice for testing a particular hypothesis.
- Students will be able to discuss whether experimental outcomes support or fail to support a particular hypothesis, and in the case of the latter, discuss possible reasons why.
The Inside and Outside the BodyLearning ObjectivesStudents will be able to:
- correctly identify when a substance (e.g. fetus, bacteria, toxins) is inside or outside the body.
- recognize the point at which a substance transitions from the inside to the outside of the body and vice versa.
- apply the concept of inside and outside the body to both normal events, such as the movement of oxygen from the alveolus to the blood, and abnormal events, such as the presence of blood in the urine.
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.
Teaching the Biological Relevance of Chemical Kinetics Using Cold-Blooded Animal BiologyLearning ObjectivesStudents will be able to:
- Predict the effect of reaction temperature on the rate of a chemical reaction
- Interpret a graph plotted between rate of a chemical reaction and temperature
- Discuss chemical kinetics utilizing case studies of cold-blooded animals