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Biochemistry And Molecular Biology
An undergraduate bioinformatics curriculum that teaches eukaryotic gene structureLearning ObjectivesModule 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.
- 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.
- 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:
- 5' capping
- 3' polyadenylation
- 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.
- 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.
- 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.
Sequence Similarity: An inquiry based and "under the hood" approach for incorporating molecular sequence...Learning ObjectivesAt the end of this lesson, students will be able to:
- Define similarity in a non-biological and biological sense when provided with two strings of letters.
- Quantify the similarity between two gene/protein sequences.
- Explain how a substitution matrix is used to quantify similarity.
- Calculate amino acid similarity scores using a scoring matrix.
- Demonstrate how to access genomic data (e.g., from NCBI nucleotide and protein databases).
- Demonstrate how to use bioinformatics tools to analyze genomic data (e.g., BLASTP), explain a simplified BLAST search algorithm including how similarity is used to perform a BLAST search, and how to evaluate the results of a BLAST search.
- Create a nearest-neighbor distance matrix.
- Create a multiple sequence alignment using a nearest-neighbor distance matrix and a phylogram based on similarity of amino acid sequences.
- Use appropriate bioinformatics sequence alignment tools to investigate a biological question.
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.
Understanding Protein Domains: A Modular ApproachLearning Objectives
- Students will be able to compare protein sequences and identify conserved regions and putative domains.
- Students will be able to obtain, examine, and compare structural models of protein domains.
- Students will be able to interpret data on protein interactions (in vitro pull-down and in vitro and in vivo functional assays)
- Students will be able to propose experiments to test protein interactions.
A Close-Up Look at PCRLearning ObjectivesAt the end of this lesson students will be able to...
- Describe the role of a primer in PCR
- Predict sequence and length of PCR product based on primer sequences
- Recognize that primers are incorporated into the final PCR products and explain why
- Identify covalent and hydrogen bonds formed and broken during PCR
- Predict the structure of PCR products after each cycle of the reaction
- Explain why amplification proceeds exponentially
Taking the Hassle out of HasselbalchLearning ObjectivesStudents will be able to:
- Characterize an aqueous environment as acidic or basic.
- Explain that pKa is a measure of how easy it is to remove a proton from a molecule.
- Predict ionization state of a molecule at a particular pH based on its pKa (qualitative use of the Henderson-Hasselbalch equation).
- Calculate the ratio of protonated/unprotonated forms of ionizable groups depending on chemical characteristics and /or environment pH (quantitative use of the Henderson-Hasselbalch equation).
- Apply this knowledge in a medical context.
The Avocado Lab: An Inquiry-Driven Exploration of an Enzymatic Browning ReactionLearning ObjectivesStudents will be able to:
- develop a testable research question and supportive hypothesis regarding the browning of damaged avocado flesh caused by the activity of avocado polyphenol oxidase (aPPO).
- design and execute a well-controlled experiment to test aPPO hypotheses.
- evaluate qualitative enzyme activity data.
- create a figure and legend to present qualitative data that tests multiple hypotheses and variables.
- search for and correctly cite primary literature to support or refute hypotheses.
- know the role of reducing reagents, pH, chelators, and temperature in reactions catalyzed by aPPO.
- explain why the effects of salt and detergent differ for aPPO experiments conducted in situ
- (in mashed avocado flesh) as compared to in vitro (on purified protein).
- discuss how substrate and cofactor availability affect aPPO reactions.
- describe how endogenous subcellular organization restricts aPPO reactions in a healthy avocado.
- evaluate food handling practices for fruits expressing PPO.
Lights, Camera, Acting Transport! Using role-play to teach membrane transportLearning ObjectivesAt the end of this activity, students should be able to:
- Compare and contrast the mechanisms of simple diffusion, facilitated diffusion, and active transport (both primary and secondary).
- Identify, and provide a rationale for, the mechanism(s) by which various substances cross the plasma membrane.
- Describe the steps involved in the transport of ions by the Na+/K+ pump, and explain the importance of electrogenic pumps to the generation and maintenance of membrane potentials.
- Explain the function of electrochemical gradients as potential energy sources specifically used in secondary active transport.
- Relate each molecule or ion transported by the Na+/glucose cotransporter (SGLT1) to its own concentration or electrochemical gradient, and describe which molecules travel with and against these gradients.
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.