Table Of ContentBiotechnology
A Laboratory Course
Jeffrey M. Becker
Department of Microbiology
Graduate Program in Cellular, Molecular, and
Developmental Biology
The University of Tennessee
Knoxville, Tennessee
Guy A. Caldwell
Graduate Program in Cellular, Molecular, and
Developmental Biology
The University of Tennessee
Knoxville, Tennessee
Eve Ann Zachgo
Microbial Products and Processes Department
BioTechnica International
Cambridge, Massachusetts
Academic Press, Inc.
Harcourt Brace Jovanovich, Publishers
San Diego New York Berkeley Boston London Sydney Tokyo Toronto
This book is printed on acid-free paper. @
Copyright © 1990 by Academic Press, 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 photocopy, recording, or any information
storage and retrieval system, without permission in writing from the publisher.
Academic Press, Inc.
San Diego, California 92101
United Kingdom Edition published by
Academic Press Limited
24-28 Oval Road, London NW1 7DX
Library of Congress Cataloging-in-Publication Data
Becker, Jeffrey M.
Biotechnology : a laboratory course / Jeffrey M. Becker, GuyA.
Caldwell, Eve Ann Zachgo.
p. cm.
ISBN 0-12-084560-1 (pbk. : alk. paper)
1. Biotechnology-Laboratory manuals. I. Caldwell, Guy A.
II. Zachgo, Eve Ann. III. Title.
[DNLM: 1. Biotechnology-laboratory manuals. TP 248.24 B395b]
TP248.2.B374 1990
660\6--dc20
DNLM/DLC
for Library of Congress 89-17915
CIP
Printed in the United States of America
90 91 92 93 9 8 7 6 5 4 3 2 1
To the parents of Jeff Becker,
Dorothy and Harry Becker
To the parents of Guy Caldwell,
Marc and Victoria Caldwell
To the parents of Eve Zachgo,
Frank and Corinne Zachgo
and to her sister,
Emily Zachgo
Preface
This book is different. To our knowledge, there is no text designed for
instruction at the introductory or even advanced level that is compa
rable to this manual. We have attempted to create a text that consists
of a series of laboratory exercises, beginning with basic techniques
and culminating in the utilization of previously acquired technical
experience and experimental material. Thus, this manual provides
a continuum of experiments using the same biological material
throughout, which is beneficial in understanding a common sequence
of technical stages used in biotechnology in a framework which is
suited to instructional methodologies.
This manual is an outgrowth of an introductory laboratory
course for senior undergraduate and first year graduate students in the
biological sciences at The University of Tennessee, which was de
signed to provide students with an in-depth experience and under
standing of selected methods, techniques, and instrumentation used in
modern biotechnology. A continuum was established by using two
principal organisms: the bacterium Escherichia coli and the yeast
Saccharomyces cerevisiae. In addition, a single plasmid (pRY121) and
a single enzyme (β-galactosidase) for demonstrating various bio-
technological manipulations were utilized. Because our main objec
tive was to provide a step-by-step laboratory course for students,
there are aspects of molecular biology and biotechnology which are
not included.
xiii
x/V Preface
To facilitate the use of this manual by diverse undergraduate
institutions, we have provided a suggested weekly schedule based on a
fifteen-week format, which we have found useful at The University of
Tennessee; ensured ready availability of the few microbial cultures
(Escherichia coli and Saccharomyces cerevisiae) necessary for this
course; attempted to design the course for a minimum number of
reagents and supplies; and do not propose work with radioactivity.
The continued impact of recombinant DNA technology in both
academic and industrial research has resulted in a substantial change
in the curriculum of graduate and undergraduate biology programs.
This increased emphasis on new technology required a novel and
contemporary method of instruction. It is our hope that this manual
will aid academic institutions in their efforts to establish a laboratory
course in molecular biology and biotechnology.
Jeffrey M. Becker
Guy A. Caldwell
Eve Ann Zachgo
Acknowledgments
We would like to acknowledge the following individuals and organi
zations for their time, effort, and support in the preparation of this
manual: Paula Keaton and Marty Danilchuk for their dedicated ser
vice in typing the manuscript; Kim Neifer, Wendy Schilling, and
Kumar Alagramam for their useful comments and advice on the
experimental protocols; Gary Sayler, Frank Larimer, Don Dougall,
Chuck Pettigrew, Min Park, Robert Kreisberg, and Pat Rayman for
their cumulative efforts in the instruction and design of the course; R.
Rogers Yocum for use of plasmid pRY121; David Miller for photog
raphy services; and the Science Alliance, State of Tennessee Center for
Excellence in the Life Sciences, for the laboratory facilities and fund
ing of the biotechnology laboratory at The University of Tennessee,
Knoxville.
xv
Suggested Schedule for Exercises
Week Day 1 Day 2 Day 3 Day 4
1 Introduction/ Exercise 1
Orientation Exercise 2
2 Exercise 3 Exercise 5
Exercise 4
3 Exercise 6* Exercise 7
4 Exercise 8, Exercise 8,
Parts A and Β PartC
5 Exercise 9,f Exercise 9, Exercise 9,
Day 1 Day 2 Day 4
6 Exercise 9, Exercise 10
Day 7
7 Exercise 11, Exercise 11, Exercise 11,
Day 1 Day 2 Day 3
* Exercise 6A (Isolation and Characterization of Auxotrophic Yeast Mutants) is an additional
exercise. (See Appendix 1.)
f Exercise 9A (Large-Scale Isolation of Plasmid DNA by Column Chromatography) is an
alternative procedure which takes 2 days. (See Appendix 1.)
xvii
XVIII Suggested Schedule for Exercises
Week Day 1 Day 2 Day 3 Day 4
8 Exercise 12, Exercise 12,
Day 1 Day 2
9 Exercise 12, Exercise 12, Exercise 12,
Day 3 Day 4 Day 5
10 Exercise 13 Exercise 14, Exercise 14,
Day 1 Day 2
11 Exercise 15 Exercise 16
12 Exercise 17 Exercise 18
13 Exercise 19, Exercise 19,
Part A, Day 1 Part A, Day 2
14 Exercise 19, Exercise 19, Exercise 19,
Part B, Day 1 Part B, Day 2 Part B, Day 3
15 Exercise 19, Exercise 19,
Part C, Day 1 Part C, Day 2
* Exercise 12A (Colony Hybridization) is an additional exercise which takes 2 days more. (See
Appendix 1.)
Introductory Notes
Record Keeping and
Safety Rules
Format of Student Laboratory Records
Ten Dos and Don'ts of Record Keeping
Criteria for Grading the Laboratory Notebook
Safety Rules in the Laboratory
Introductory Notes
Format of Student
Laboratory Records
These guidelines should be followed for the correct recording of the
exercises.
A. Purpose/Introduction
Two or three sentences stating the exact purpose(s) and objective(s) of
the exercise; what was done, to what, and why. Do not include, as a
part of this abstract, familiarization with equipment or techniques
unless applicable. A short conclusion giving results/ideas may be
added, if applicable.
B. Materials and Methods
Include, only, any deviations or additional equipment/procedures
from those stated in the laboratory manual. If none, merely note "No
deviations from the planned procedure were performed." If devia
tions were minor they may be ignored. However, accurate and com
plete cross-referencing to any references or procedures is mandatory.
C. Data/Results
All data generated should be recorded during the exercise. This in
cludes recopying any tables, graphs, formulas, etc., from the labora
tory manual and/or drafting new tables, graphs (labeled), as neces
sary, to accurately and neatly represent the data. This section should
also include all calculations, averages, error analyses, and corrections
of recorded data.
3
