The purpose of this laboratory manual is to introduce undergraduate students to techniques used in biochemistry and molecular biology laboratories and ensure that they master the lab skills necessary to be competitive in the job market. We present a collection of fifteen experiments that teach students sterile techniques, accurate pipetting, centrifuge usage, PCR, DNA purification, protein expression and purification, HPLC, enzyme kinetics, equilibrium binding assays and introduction to bioinformatics.
The novelty of this manual is the incorporation of a student-driven real real-life research project into the undergraduate curriculum. Since students test their own mutant design, even the most experienced students remain engaged with the process, while the less experienced ones get their first taste of biochemistry research. Inclusion of a research project does not entail a limitation: this manual includes all classic biochemistry techniques such as HPLC or enzyme kinetics and is complete with numerous problem sets relating to each topic.
This course includes several chapters on the latest advancements in bioinformatics: how to access genome databank, perform sequence alignments, design primers, to predict secondary and tertiary structure and to use protein visualization tools.
This course material is based on the textbook written by Gerczei and Pattison under Creative Commons 4.0 license.
|1. Introducing the Bacterial Antibiotic Sensor Mini Project
|What are Antibiotics?
|What is Bacterial Antibiotic Resistance?
|How Do the Bacteria Detect Antibiotics In Its Environment?
|How Does the ykkCD Sensor Exert Its Function?
|What Do We Do During the Mini Project?
|2. Identifying Conserved Elements in the Toxin Sensor and Designing Mutants to Test Whether They are Important for Function
|Mini Project Flowchart
|Why is Sequence Conservation Important for Macromolecule Function, and How Do We Determine This?
|Review of Nucleic Acid Properties
|Identifying Conserved Sequence Elements
|3. Designing Primers for Site-Directed Mutagenesis
|Basics of Site-directed Mutagenesis
|Quickchange Site-Directed Mutagenesis
|4. Performing Site-Directed Mutagenesis
|Performing Site-Directed Mutagenesis
|5. Purifying Mutant Toxin Sensor DNA from Bacterial Cells and Evaluating its Quality Using Agarose Gel Electrophoresis and UV Spectroscopy
|Purifying Mutant Toxin Sensor DNA from Bacterial Cells and Evaluating its Quality Using Agarose Gel Electrophoresis and UV Spectroscopy
|6. Preparing DNA Template for Mutant RNA Sensor Synthesis Using a Restriction Endonuclease
|Preparing DNA Template for Mutant RNA Sensor Synthesis Using a Restriction Endonuclease
|7. Synthesizing the ykkCD Mutant Toxin Sensor RNA in vitro
|Synthesizing the ykkCD Mutant Toxin Sensor RNA in vitro
|8. Purifying the ykkCD Mutant Toxin Sensor RNA and Evaluating its Purity Using Denaturing PAGE and UV spectrometry
|Purifying the ykkCD Mutant Toxin Sensor RNA and Evaluating its Purity
|9. Evaluating the Ability of the ykkCD Toxin Sensor to Recognize the Antibiotic Tetracycline Using Fluorescent Quenching
|Evaluating the Ability of the ykkCD Toxin Sensor
|10. Evaluating Antibiotic Binding to Blood Serum Albumin Using Fluorescence Spectroscopy
|Evaluating Antibiotic Binding to Blood Serum Albumin
|11. Understanding the Importance of Buffers in Biological Systems
|Importance of Buffers in Biological Systems
|12. Molecular Visualization of an Enzyme, Acetylcholinesterase
|Molecular Visualization of an Enzyme, Acetylcholinesterase
|13. Determining the Efficiency of the Enzyme Acetylcholine Esterase Using Steady-State Kinetic Experiment
|Determining the Efficiency of the Enzyme Acetylcholine Esterase
|14. Separation of the Phosphatidylcholines Using Reverse Phase HPLC
|Separation of the Phosphatidylcholines