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