Each chapter in this book corresponds to a lab in the CHEM 3753: Introduction to Biochemical Methods course at the University of Oklahoma. All of the materials you will need for each lab can be found within its respective chapter. Each chapter will contain a brief introduction; a set of learning objectives; a slide presentation, screencast, or lab demonstration video; the protocol to be followed during your time in the lab; and a set of interactive quiz questions to help you check you understanding as you go. Other interactive features include photos from the lab, links to safety data sheets (SDS), and 3D chemical structures. Components of the book are elaborated upon below. We hope you find this book to be an all-in-one, fun, and engaging learning tool for the biochemical methods course.
Course Description: Biochemistry 551 is an integrated lecture, lab and seminar course that covers biochemistry-centered theory and techniques. The course is designed for upper-level undergraduate students majoring in Biochemistry. Students learn how to apply a broad range of biochemical, genetic, and physical techniques to modern biochemical research. Students also learn how to analyze and interpret the primary scientific literature, develop an understanding of the communication of data, and connect biochemical techniques to basic research.
Lectures introduce concepts and theory that are subsequently explored in detail in experiments. The virtual labs are designed to provide experience with techniques that are used in modern biochemical research through interactive online activities. The curriculum incorporates a research project beginning with the PCR amplification and cloning of the HCAII gene, which codes for the enzyme human carbonic anhydrase II (HCAII). As the semester progresses, students explore how to overexpress, purify and assay wild type and mutant HCAII protein. Experiments covered include PCR, spectrophotometry, gel electrophoresis, protein overexpression and purification, enzyme assays and fluorescence spectroscopy. Several times during the semester, at-home lab experiments are incorporated to provide hands-on experience to supplement student understanding of the virtual labs.
This lab manual contains detailed descriptions of each online laboratory exercise in this course. Please see the course Canvas site for other course information, such as: a syllabus, schedule, assignment guidelines, and lecture and seminar materials.
Table of Contents:
Lab 1: Structural analysis of HCAII using PyMOL
Lab 2: PCR Amplification of HCAII and pETblue2
Lab 3: Lab-at-home: Introduction to Biotechnology Methods
Lab 4: Gibson assembly of HCAII into pETblue2 vector and transformation of E. coli with the reactions
Lab 5: Screening for pETblue2-HCAII clones
Lab 6: Mutant Exploration
Lab 7: Protein Expression and Purification in E. coli
Lab 8: Analysis of His-tagged HCAII Expression and Purification
Lab 9: Determination of Protein Stability using Chemical Denaturation and Intrinsic Fluorescence
Lab 10: Investigation of wild type and mutant HCAII enzyme activity
Lab 11: Forster Resonance Energy Transfer (FRET) to detect ligand-binding to HCAII
Six case studies, case study keys, and instructor notes were developed for this grant project. A brief description of the studies is as follows:
Blood Clotting- This case study discusses the causes, symptoms, and possible treatments for blood clots. I chose this study because the story is about my brother who was misdiagnosed with a clot and almost died. I felt it was a study that included the importance of proper diagnosis in a medical situation.
Immunization-This case study includes a brief history of immunization, how vaccines work, what type of vaccines are available, what chemicals can be found in vaccines, and why people may choose not to be vaccinated. This study was written before the COVID-19 pandemic, but more information can be added to it concerning a possible vaccination for the COVID-19 virus.
The Stereochemistry of Ephedrine- This case study centers around the drug ephedrine. The study discusses how ephedrine binds to adrenergic receptors. Ephedrine is a chiral molecule which means it has stereoisomers. This study focuses on stereochemistry and guides students on how stereoisomers bind to specific receptors. The way an isomer binds to a receptor affects how a drug interacts with our body.
Understanding Solutions- This case study connects the concepts of concentration and molarity in chemistry terms to terms used in a medical field. Students will study the concepts of osmolarity, molarity, hyper and hypotonic solutions, and salt solutions. The study involves the story of a young nurse learning to understand the important terms and solutions in a medical situation.
Red Blood Cell Alloimmunization- This case study discusses the differences of blood types and blood type groups (ABO and Rh). The study focuses on the possibility of complications due to allergic reactions to red blood cell antigens (alloimmunization). Alloimmunization is especially harmful for patients needing blood transfusions or women and fetuses during pregnancy.
Radioactivity- This case study discusses thyroid hormones and how problems with these hormones can be treated with radiation. Students learn about the function of the thyroid and causes of hypo and hyperthyroidism. Students also learn about radioactive treatment, half lives of radiation, and types of radiation.
We are happy to welcome you to our second Open Educational Resource (OER) textbook, Biochemistry Free For All. Biochemistry is a relatively young science, but its rate of growth has been truly impressive. The rapid pace of discoveries, which shows no sign of slowing, is reflected in the steady increase in the size of biochemistry textbooks. Growing faster than the size of biochemistry books have been the skyrocketing costs of higher education and the even faster rising costs of college textbooks. These unfortunate realities have created a situation where the costs of going to college are beyond the means of increasing numbers of students.
Table of Contents
Water and Buffers
Structure and Function of Nucleic Acids
Structure and Function of Carbohydrates
Structure and Function of Lipids
Membranes: Basic Concepts
Membranes: Other Considerations
Catalysis: Basic Principles
Catalysis: Control of Activity
Electron Transport and Oxidative Phosphorylation
Metabolism of Sugars
Metabolism of Polysaccharides
Citric Acid Cycle
Metabolism of Fats and Fatty Acids
Metabolism of Other Lipids
Metabolis of Amino Acids and the Urea Cycle
Metabolism of Nucleotides
Genes and Genomes
Regulation of Gene Expression
Point by Point: In the Beginning
Point by Point: Structure and Function
Point by Point: Membranes
Point by Point: Catalysis
Point by Point: Energy
Point by Point: Metabolism
Point by Point: Information Processing
Point by Point: Techniques
Table of Contents:
Chapter 1: Foundations of Biochemistry
Chapter 2: Protein Structure
Chapter 3: Investigating Proteins
Chapter 4: DNA, RNA and the Human Genome
Chapter 5: Investigating DNA
Chapter 6: Enzyme Principles and Biotechnological Applications
Chapter 7: Catalytic Mechanisms of Enzymes
Chapter 8: Protein Regulation and Degradation
Chapter 9: DNA Replication
Chapter 10: Transcription and RNA Processing
Chapter 11: Translation
Chapter 12: DNA Damage and Repair
Chapter 13: Transcriptional Control and Epigenetics
Table of Contents
2. Overview of Laboratory Responsibilities Tentative Schedule
3. Objectives for Week 1
4. Concepts for Choosing and Expressing Mutants
5. Objectives for Week 2
6. Concepts for Protein Expression
7. Methods for Protein Expression
8. Concepts for SDS-PAGE
9. Methods for SDS-PAGE
10. Objectives for Week 3
11. Concepts for Protein Purification and Desalting
12. Methods for Protein Purification and Desalting
13. Microfluidizer Lysis Method
14. Objectives for Week 4
15. Methods for Protein Standard Curves and SDS-PAGE
16. Objectives for Weeks 5-9
17. Concepts for Enzyme Assays
18. Methods for Enzyme Assays
About the Book
Chemical Biology & Biochemistry Laboratory Using Genetic Code Expansion Manual