Molecular Microbiology Laboratory: A Writing-Intensive Course

By Bruce Geller and Katherine Field

Molecular Microbiology Laboratory, Second Edition, is designed to teach essential principles and techniques of molecular biology and microbial ecology to upper-level undergraduates majoring in the life sciences and to develop students' scientific writing skills. A detailed lab preparation manual for instructors and teaching assistants accompanies the lab book and contains a general discussion of scientific writing and critical reading as well as detailed instructions for preparation and peer review of lab reports. Each experimental unit is accompanied by a number of additional writing exercises based upon primary journal articles.

• Exposes students to the new molecular-based techniques
• Provides faculty with an authoritative, accessible resource for teaching protocols
• The only manual to incorporate writing exercises, presentation skills and tools for reading primary literature into the curriculum
• Based on a successful course for which the author won a teaching award

New to this Edition:- Presents a real-world study of bacterial populations in the environment in the final experiment - Provides an overview of molecular biology in a new review chapter- Demonstrates how to design an experiment and how to interpret the results - Covers grant proposal writing and how panels review proposals - Presents guidance on public speaking and preparing PowerPoint presentations - Includes tutorials on three widely used software packages

• Language: English
• Publisher: Academic Press
• Release date: Dec 31, 2012
• ISBN: 9780123977526

Bruce Geller

Walt Ream, Professor of Microbiology, Oregon State University, received the Richard M. Bressler Senior Faculty Teaching Award for developing the course this book supports. In addition to this work, he has written numerous peer reviewed scholarly papers, review articles, book articles and chapters and has co-authored another book entitled, Molecular Biology Techniques: An Intensive Laboratory Course. His research led to a discovery for which a patent was issued in 2004. He regularly reviews grant proposals for NSF, USDA, DOE, NIH, American Cancer Society, NSERC-Canada, Lindbergh Foundation, Corporation for Plant Biotechnology Research and he reviews papers for Journal of Bacteriology, Plant Molecular Biology, PNAS, Science, Molecular & General Genetics, Microbiological Reviews, Molecular Plant-Microbe Interactions, Biochemistry, Journal of Biological Chemistry, Plasmid, Gene, Plant Physiology, Journal of American Horticulture Society, and Molecular Microbiology. He is a member of Genetics Society of America, American Society for Microbiology, and American Association for the Advancement of Science.

Book preview

Molecular Microbiology Laboratory

A Writing-Intensive Course

Second Edition

Walt Ream

Bruce Geller

Janine Trempy

Katharine Field

Table of Contents

Cover image

Title page

Copyright

Introduction

I Writing-Intensive Course

II Schedule

III Attendance and Grading Policies

IV Laboratory Rules

V Decorum

VI Flowcharts

VII Laboratory Reports

VIII How to Evaluate Laboratory Reports

IX Proposals to Study Environmental Bacterial Populations: Writing Assignment (Class 6)

X How to Read a Journal Article

XI Writing Tips

Acknowledgments

Experiment 1. Plasmid Purification and Restriction Mapping

Abstract

I Introduction

II Background: Plasmid DNA Preparation

III Procedure

IV Laboratory Report [Class 4 (Draft) and Class 5]

V Questions (Class 5)

VI In-Class Writing Exercise (Class 2)

VII Restriction Mapping Exercises (Class 3)

VIII In-Class Writing Exercise (Class 4)

Experiment 2. Affinity Purification of Histidine-Tagged FnbA Protein

Abstract

I Introduction

II Background

III Procedure

IV Laboratory Report

V Critique of Paper by Bush et al.

Experiment 3. Polymerase Chain Reaction and DNA Sequence Analysis of Bacterial Ribosomal RNA Genes

Abstract

I Introduction

II Background

III Procedure

IV Laboratory Report [Class 16 (Draft) and Class 17]

V Questions (Class 17)

VI In-Class Writing Exercise (Class 10)

Experiment 4. Terminal Restriction Fragment Length Polymorphism Analysis of 16S Ribosomal RNA Genes from Uncultured Bacterial Communities

Abstract

I Introduction

II Background

III Procedure

IV Laboratory Report

V Questions (Class 20)

VI Writing Exercise: Editorial on Genetically Modified Crops [Class 14 (Draft) and Class 15]

VII Writing Assignment: Literature Search Problem [Class 14 (Begin) and Class 20]

VIII In-Class Writing Exercise (Class 15)

Appendices

Appendix A. Sample Laboratory Reports

Appendix A.A The additional DNA sequences of the plasmid DNA from T7 promoter primer

Appendix A.B Lengths of the corresponding T-RFLP fragments for each enzyme (MspI and RsaI restriction enzymes) in the additional sequences (S. thermophilus and L. delbrueckii) of the molecular clone library

Appendix B. Sample Proposals

An Outstanding Proposal

A Poor Proposal

References

Appendix C. Sample Tests

Sample Test for this Book

Study Questions from Day and Gastel

Appendix D. Guidance for Presentations

Appendix E. Revised Paragraph from J. Biol. Chem. 266: 3811–3814 (1991)

Appendix F. Abstract from Appl. Environ. Microbiol. 63: 2647–2653 (1997)

Appendix G. Sequence Editing Tutorial

Sequence from the M13r Primer

Steps in Analysis

Analysis of Sequences that Contain the Reverse Complement of the 8–27F Primer

Sequence from the T7 Primer

Steps in Analysis

Appendix H. Peak Scanner Tutorial

Appendix I. Adding Size Standards to Peak Scanner

Appendix J. PRIMER Tutorial

Nonmetric Multidimensional Scaling Using Primer

Appendix K. Checklist for Grading Laboratory Reports

Appendix L. Peer Review Checklist

Appendix M. Career Advice

Appendix N. Molecular Microbiology Laboratory Preparation Manual

Media Room Preparation Manual

Teaching Assistant Preparation Manual

Strain Curator Preparation Manual

Bibliography

Index

Copyright

Academic Press is an imprint of Elsevier

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© 2013, 2004 Elsevier Inc. All rights reserved

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at ourwebsite: www.elsevier.com/permissions

This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

Library of Congress Cataloging-in-Publication Data

Application submitted

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library

ISBN: 978-0-12-397044-2

For information on all Academic Press publications, visit our website at: store.elsevier.com

Printed in the United States of America

12 13 14 9 8 7 6 5 4 3 2 1

Introduction

I Writing-Intensive Course

Students enrolled in this course complete a variety of formal written assignments that develop scientific writing, editing, and reading skills. In completing these assignments, students review one another’s writing and revise drafts. Students use sources outside this manual to complete laboratory reports, prepare a research proposal, write an editorial, and solve a thought problem.

A Goals

This ten-week course teaches undergraduate students molecular biology techniques commonly used in the life sciences and develops the students’ scientific writing skills.

B Means

The course contains four units that introduce procedures most life scientists will encounter during their careers. In the first unit, students prepare plasmid DNA, construct a restriction map of the plasmid, and transform it into E. coli. The plasmid contains a luciferase reporter gene, which introduces the concept of reporter genes through first-hand experience. In the second unit, students express, purify, and analyze an affinity-tagged protein. The third and fourth units require intellectual input from students, who isolate bacteria from environments that they choose. Each student selects one unknown bacterium to culture, examine by light microscopy, and identify by DNA sequence analysis. During this experiment, students learn to isolate genomic DNA, perform polymerase chain reaction (PCR), purify PCR products, and analyze DNA sequences. In the fourth unit, students use terminal restriction fragment polymorphism (T-RFLP) analyses to characterize DNA isolated from uncultured microbial communities. The methods in this course are common techniques that introduce the fundamental principles of molecular biology and microbial ecology.

This is also a writing-intensive course. This manual contains a general discussion of scientific writing and critical reading, and it includes detailed instructions for preparation and peer review of laboratory reports. Additional writing exercises based on journal articles accompany each unit. The studies in these articles employ techniques used in the laboratory exercises. By evaluating these papers, students reinforce their understanding of the technology. Students see how diverse authors report their findings and how formats differ from one journal to another. They also discover that all scientific papers share several essential components. Lectures based on the book How to Write and Publish a Scientific Paper by Robert Day and Barbara Gastel discuss each section of a scientific paper in detail. To build their writing skills and enhance their understanding of molecular microbiology, students compose and revise laboratory reports, edit their peers’ reports, critique journal articles, and study the writing manual by Day and Gastel.

II Schedule

Notes:

Manual=Bruslind, L., Burke, M., and Ream, W., 2001, Scientific Writing for Microbiology Majors.

Day=Robert Day and Barbara Gastel, 2011, How to Write and Publish a Scientific Paper (Santa Barbara, CA: Greenwood).

AEM=Applied and Environmental Microbiology

Notes

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III Attendance and Grading Policies

A Attendance

Attendance is mandatory. Each unexcused absence reduces your final grade 5%. Three absences result in an Incomplete. Arrival more than 15 minutes late counts as half an absence. Requests for an excused absence are considered on a case-by-case basis. Acceptable reasons for an excused absence include illness, a family emergency, or interviews for admission to graduate or professional schools. Please do not attend class if you have influenza or another contagious illness. To request an excused absence, contact the instructor before class. Students with an excused absence must complete all missed assignments.

B Grading

Final Grade

A =94–100% of top score

A− =90–93%

B+ =87–89%

B =83–86%

B− =80–82%

C+ =76–79%

C =70–75%

C− =65–69%

D =50–65%

F =<50%

Lab reports=20% each×4=80%

Test=20%

C Partial Credit

Do not expect partial credit for incorrect answers to questions that involve calculations. The book provides examples of all the test questions that require calculations, along with the solutions. These simple arithmetic problems teach you to prepare reagent solutions correctly.

Attention to detail is important in science and medicine. A careless error when converting units can cause a 1000-fold error in the concentration of a solution. In the laboratory, an error of this magnitude ruins an experiment. In medicine, an error of this magnitude kills a patient. In this class, you learn that careless errors have consequences.

Submit laboratory reports by e-mail before the start of class on the date indicated on the schedule. Do not expect credit for laboratory reports submitted after the deadline.

Notes

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IV Laboratory Rules

You must prepare a flowchart prior to each experiment. You may not begin an experiment without a flowchart. The flowchart is due at the start of class. Ask questions when you do not understand the instructions or the principles involved.

Observe the following safety rules at all times:

1. Do not pipette by mouth. You must have a rubber pipette bulb or another mechanical pipette.

2. Proper attire is required. Wear a laboratory coat, safety glasses, and closed shoes. Do not wear shorts or sandals. Lab coats and protective eyewear are required for the experiments that use phenol, which causes severe burns when it contacts skin. Wash with water to remove phenol.

3. Do not eat, drink, or chew gum in the laboratory.

4. Disinfect your bench surface before and after you work.

5. Insert pipettes into the rubber bulb gently to avoid breaking the pipette and cutting your hand.

6. Assume that all bacteria you use may cause disease.

7. Disinfect contaminated equipment and surfaces.

8. Place used liquid cultures, supernatant solutions, and glassware in autoclave containers. Discard contaminated plates, pipette tips, and plastic tubes in autoclave bags. Discard phenol and chloroform in designated waste containers.

9. Wash your hands after you finish working.

Notes

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V Decorum

This course will prepare students for careers in medicine, industry, or academic science. Decorum is important in these professions. Appropriate behavior is required in this class. When the class period begins, please take your seat, end personal conversations, and turn your attention to the instructor. After the introductory lecture, begin your lab work quietly. During class, limit conversations to work-related matters and discussion of in-class assignments. Idle chatter is distracting and leads to careless errors. Employers do not tolerate idle chatter in the workplace.

Notes

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VI Flowcharts

A How to Prepare a Flowchart

Prepare a flowchart prior to each experiment. You may not participate in the laboratory exercise without a flowchart.

A flowchart outlines each step of the procedure and guides you through the experiment. If you modify a procedure during the course of an experiment, note these changes on the flowchart. Record observations on a separate page as you work.

Flowcharts contain words, symbols, diagrams, and arrows. Begin your flowchart by listing the first step of the procedure. Use an arrow to connect the first step to the second, and so forth. The arrows indicate major procedural steps and direct your attention to the next task. List the steps to proceed from one intermediate to the next beside each arrow. As you complete each step of the procedure, put a check mark on your flowchart beside that step. A sample flowchart follows.

B Sample Flowchart

Notes

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VII Laboratory Reports

A Preparing a Laboratory Report

You must prepare a report for each experiment. In class, record data directly in a bound notebook using a pen. Date each entry in your notebook. Document your work carefully. Complete, accurate records are crucial in research and medicine; patents worth billions of dollars may depend on notebooks that document the underlying research.

Use a word processor to type the text of your report. Insert graphs, tables, figures, and legends into the report immediately after their first citation in the text. Word processors have spell check tools. Spelling errors that your word processor can detect are inexcusable. Some typographical errors, such as form in place of from, can evade detection by your computer, so proofreading is necessary.

Peer review is essential for high-quality writing. Poor organization or awkward sentences are obvious to most readers. Two peers will review a draft of your report during class, so bring two copies. Due dates for drafts and finished reports are in the schedule. Write the draft as carefully and completely as possible. Do not submit a first draft or a rough draft for review. Peers cannot evaluate sections that are missing or incomplete.

Submit the final version of your report by e-mail as an attached Word or PDF file. Name the file as follows: seat number (space) last name (space) first name (space) report number. For example, if I sit in seat 8, I would name the file containing my first report 8 Ream Walt report 1.doc.

B Sections of a Laboratory Report

Laboratory reports must include the following sections (see Appendix A for sample reports):

1. Name.

2. Title. Make the title concise, specific, and informative. Tell the reader what you did.

3. Date. List the dates you performed the experiment.

4. Purpose. State the purpose of the experiment in a few sentences. Be specific.

5. Methods. Briefly describe the procedures used, and reference the appropriate pages in the manual. Do not copy the methods from the manual. Note modifications to the procedure.

6. Results. Introduce this section with one or two sentences that describe the experiment. State the rationale for the experiment, and indicate what you hoped to learn. Describe the experiments you did to answer the question. Present the observations you made during the experiment, not what you think should have happened. Do not repeat the methods section.

Include figures, graphs, and tables in this section. Each figure and table must have a title, a number, and a legend that contains all the information needed to interpret the data. Specify units on the axes of graphs. Label columns and rows in tables. Number each lane on photographs of gels, and indicate the contents of each lane in the figure legend. Place each figure immediately after the paragraph in which you first cite it.

7. Discussion. Offer your interpretation of the data presented in the results section. Begin with a brief introduction that makes the purpose of the experiment clear, then discuss the meaning of your observations. If you can interpret your data in several ways, mention all the possibilities and indicate which alternative you think is correct. Discuss all your results, even if they are unexpected or negative. For example, you should