1: Labs

Last updated
Save as PDF

Page ID: 49894

Timea Gerczei Fernandez & Scott Pattison
Ball State Univirsity via De Gruyter

\( \newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} } \) \( \newcommand{\vecd}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash {#1}}} \)\(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\) \(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\)\(\newcommand{\AA}{\unicode[.8,0]{x212B}}\)

1.1: Introducing the Bacterial Antibiotic Sensor Mini Project
Antibiotic resistance is an emerging problem in modern medicine: 70% of bacterial strains are resistant to at least one antibiotic, making treatment of common bacterial infections increasingly difficult. As a result, in the United States more people die from bacterial infections than from HIV infection and breast cancer combined. During the course of the mini project, you will learn about this important problem while getting hands-on experience with a wide variety of techniques.
1.2: Identifying Conserved Elements in the Toxin Sensor and Designing Mutants to Test Whether They are Important for Function
In this lab, you will use bioinformatics to learn how to find the DNA sequence of a given macromolecule and use this sequence to uncover evolutionary sequence conservation. You will use these data to identify conserved sequence segments (invariable blocks) in the ykkCD sensor RNA. During the second half of the lab you will identify conserved structural elements within the toxin sensor. These are elements where the sequence may have been altered during evolution, but the structure was retained.
1.3: Designing Primers for Site-Directed Mutagenesis
During the next two labs you will learn the basics of site-directed mutagenesis: you will design primers for the mutants you designed earlier and perform PCR amplification to make that mutant. In this handout you will review the basics of primer design while in the next handout you will learn about PCR amplification in practice.
1.4: Performing Site-Directed Mutagenesis
In this lab you will perform site-directed mutagenesis using the QuickChange mutagenesis kit (Stratagene). You will learn how polymerases work and how to amplify DNA using polymerase chain reaction (PCR).
1.5: Purifying Mutant Toxin Sensor DNA from Bacterial Cells and Evaluating its Quality Using Agarose Gel Electrophoresis and UV Spectroscopy
Last week you generated the plasmid DNA containing the mutant toxin sensor using QuickChange PCR amplification. This week you will learn how to extract and purify this plasmid DNA from E coli cells, and how to check the quality of the plasmid DNA using agarose gel electrophoresis and UV spectroscopy.
1.6: Preparing DNA Template for Mutant RNA Sensor Synthesis Using a Restriction Endonuclease
In this lab, you get our DNA template containing the mutant sensor RNA ready for RNA synthesis. With this process, you will learn how restriction endonucleases – enzymes that cleave DNA at a given sequence – achieve their extraordinary specificity.
1.7: Synthesizing the ykkCD Mutant Toxin Sensor RNA in vitro
In understanding how the ykkCD toxin sensor recognizes the antibiotic tetracycline you thus far designed mutants to alter the sequence of the sensor, and made the plasmid vectors containing the mutant sensor using PCR amplification. You purified these plasmids from bacterial cells and prepared them to be templates for RNA synthesis. In this lab you will learn how RNA polymerases work. You will synthesize the mutant sensor in vitro using the plasmid DNA template and T7 RNA polymerase.
1.8: Purifying the ykkCD Mutant Toxin Sensor RNA and Evaluating its Purity Using Denaturing PAGE and UV spectrometry
The purpose of this lab is to learn how to purify RNA samples and evaluate their purity using a denaturing polyacrylamide gel electrophoresis (urea PAGE). RNA molecules synthesized in vitro transcription are purified to remove the RNA polymerase, the DNA template and unused nucleotides (NTPs).
1.9: Evaluating the Ability of the ykkCD Toxin Sensor to Recognize the Antibiotic Tetracycline Using Fluorescent Quenching
The goal of the mini project was to identify elements in the toxin sensor that are essential to recognize the antibiotic tetracycline. You identified elements in the sensor that did not change throughout evolution (invariable blocks). You subjected them to the site-directed mutagenesis, modified the sequence of the toxin sensor DNA and made the mutated toxin sensor RNAs. During this lab you will evaluate how well this mutant sensor is able to recognize the antibiotic tetracycline.
1.10: Evaluating Antibiotic Binding to Blood Serum Albumin Using Fluorescence Spectroscopy
In this laboratory, you will study one of the most important functions of proteins. Proteins bind specific small molecules in a very selective fashion. This laboratory focuses on the major extracellular protein in the blood stream, human serum albumin. To study binding, we have chosen a sensitive optical measurement, fluorescence. You will use the wealth of data from this sensitive technique to study the details of antibiotic binding to albumin.
1.11: Understanding the Importance of Buffers in Biological Systems
You should be aware that buffers play a critical role in almost all biochemical systems. Biochemical experiments routinely require a buffer. In this laboratory you will cover the basics of buffer preparation and test the buffering capacity of the resulting solution. This buffer will be used in the enzyme kinetics (acetylcholinesterase) lab later in the term.
1.12: Molecular Visualization of an Enzyme, Acetylcholinesterase
The goal of this laboratory is to analyze some of the major structural elements of an important enzyme, acetylcholinesterase (AChE). To do this, you will use a common structural visualization program and correlate AChE structural elements with the enzyme mechanism. You will be using Chimera, a state-of-the-art molecular visualization program provided by the National Science Foundation through the University of California, San Francisco.
1.13: Determining the Efficiency of the Enzyme Acetylcholine Esterase Using Steady-State Kinetic Experiment
This laboratory introduces you to steady-state kinetic analysis, a fundamental tool for studying enzyme mechanisms. The enzyme studied, acetylcholinesterase (AChE), has a well-understood mechanism and carefully examined structure. Additionally, AChE is physiologically very important and is an example of “catalytic perfection.” You will determine Vmax, kcat, and KM and then analyze the catalytic capabilities of AChE.
1.14: Separation of the Phosphatidylcholines Using Reverse Phase HPLC
This laboratory has 2 goals, (1) to learn more of membrane lipid structures by working with phosphatidylcholines and (2) to learn the basics of an especially important high performance liquid chromatography (HPLC) technique, reverse phase HPLC. You should use your knowledge of phosphatidylcholine structures to rationalize the elution pattern from the HPLC.