1‘ 11"..." n

 

vquv

“"1335”
«“31”
'9‘??? vi ,

a
u ..

93.5“ "
1‘ 7‘3’3.

3.4x); - ‘
u an) a v3
(1. B!ar.-:n.nww9:t
\ 4%,: i
‘1 p.

n.
\-

é’ { .‘ | “'3 3‘:
y , 1 VJ fie "
«.312 ‘39:; v

‘ ‘3
"l uh "r
Jfihfi'x ‘ >

It ~ '

In: \I\‘
if $251:
w:

‘3' m‘l‘g‘
M5: (k
\b '
7i

J
Vii

 

 

.<

..- ‘r

L

-.u.- ~

“I.
l ,
”(uh-A
'9
- .

-. . ~m

fifizfiwfi

'1 ' I ‘
.- 1‘
'm‘fa“. Wag".
'N’ '“fv .
a. a

u N
. r I
«:23 mg:
' 15- r-
".e“ Gméfian

c» .
«14‘

a
u .56
n3.

SEC :.

 

, 31L .2“. ,
‘Hx 63.3.1: I"? ‘

m .a '4.

u ‘l‘le

a. ‘raj
.

.‘ . A
v m . I’L‘Vr u-r

iififiy‘

“X“ 37.5%., v I
“ W3?“ ‘
' ’ I. u

'41 ‘fi‘éhu ‘

,‘ r
any 4' "A
”.J .4 x, x .‘L
n 0 ..~
1433;”, cm“; . k

‘33‘13
lb

S, a $5733;
‘ w fi‘ I ‘. l
,. . 3’};
' ‘3 fig“???&- ‘
, W V“
"A t»

ix 3 Y Ma":
““4; “.2- 5; Haj” M:
2? ‘ 311' g . 135;

 

 

mvensnv LIBRARIES

lllllll \lllll \llll‘l l \lllgl \ llll

 

 

 

 

 

 

 

This is to certify that the

thesis entitled

W
“WWW
HAWMBIWCIASS

presented by
CHARLES K. SHARP

has been accepted towards fulfillment
of the requirements for

[taster of Scit-zrx;;=egree in Biological Scierms

 

 

   
  

/

 

MuA/W

Major Wprol’essor

 

/L

DateAugust 14, 1990

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

LIBRARY
Mlchlgen State
Unlverslty

 

r. “w~ _

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due. ‘

DATE DUE DATE DUE DATE DUE I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU In An Affirmative Action/Equal Opportunity Institution '
chS-M

 

 

STRATEGIES
FOR TEACHING DNA EFFECTIVELY
IN A SECONDARY HONORS BIOLOGY CLASS

By

Charles K. Sharp

A THESIS

Submitted to
Michigan State Universtiy
in partial fulflliment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Natural Science

1990

4917- 70:29

ABSTRACT

STRATEGIES
FOR TEACHING DNA EEEECTIVEEY
IN A SECONDARY HONORS BIOLOGY CLASS

by

Charles K. Sharp

Although our understanding of the nature and importance of
DNA began in 1953 (37 years ago). it is only recently that
students in secondary schools have been able to work with the new
biotechnology that has developed around this understanding.

This thesis proposes an approach to teaching an Honors
Biology class that centers as its final goal on a more thorough
comprehension by the student of the nature and role of DNA in the
cell.

Significant laboratory exercises illustrating the new

blotechnologies are included as a central focus to aid in

discussion and conceptualization.

Copyright by
Charles Kirkwood Sharp
1990

This work is dedicated to my wife,

Marilyn Sharp

iv

ACKNOWLEDGEMENTS

I wish to express my deepest appreciation to my wife for her
constant encouragement. and to Dr. Clarence Sueiter for his generous
time and support and advice. I wish to thank Dr. Jon Kaguni for his
helpful suggestions and for some of the laboratory exercises which have
been used. To Dr. Martin Meatherlngton and Dr. Howard Haggerman I wish

to express my gratitude.

TABLE OF CONTENTS

Introduction ...... ..... ..... . ....... ............. 1
Chapter I .......... ..... ....... ...... ... ....... 5
General Statement of a Problem... ..... ... ..... ..5
The App]ication.................................7
Program Goals.... ........... . ................... 9
SubJect 0bJectlves......................... ..... 10

Chapter 2

A Synopsis of the MaJor Historical Scientific
Developments That Led to Our Present Understanding

of DNA ......................................... 16

Reflection ............................... . ...... 24
Chapter 3 ........................ ........... ...... 26

Laboratory Exercise No. 1, Transformation

of E. coil ............................. .. ....... 27

Laboratory Execise No. 2, Colony Trans-

formation .................................... 38

Laboratory Exercise No. 3, DNA Restriction

Analysis ................ .......................46
Summary and Discussion...................... ...... 59
Appendix A

Laboratory Exercise No. 4, The Isolation of

Plasmid DNA from Bacterial Cells................64

Laboratory Exercise No. 5, The Isolation of

Plasmid DNA from Bacterial Cells, An Alternate,

Shorter Method..................................72
Laboratory Exercise No. 6, Method for Determining the
Sensitivity of Methylene Blue for Staining DNA..77
Appendix B.

Honors Biology. Restriction Digest Laboratory

0uestions..... ......... .........................86
Bibiiography........... ........ ... ..... ...........88
General References..... ..... ......................91

TABLE OF FIGURES

Figure No. 1. Restriction Maps of Lambda DNA ......... 48
Figure No. 2, A plot of the marker dyes. (P.50) ...... 58

INTRODUCTION.

There is probably no more important biological knowledge that a
high school student of today must have than a basic understanding of the
DNA molecule, its role in the cell, and its role in making an organism
uniquely what it is.

Recently scientists have begun to sequence the entire human genome
in an effort to understand the complexity of the human organism. This is
a prOJect that. it is estimated. will take fifteen years by a large
number of dedicated scientists working in laboratories throughout the
United States and the rest of the world. (1) The implications for the
human race are so immense as to be unimaginable.

If the human genome proJect is accomplished. it will mean the
probability of not only a complete understanding of human inheritance,
but also a deeper understanding of many of the hereditary diseases of
mankind. Scientists will be able to understand defective enzymes and
polypeptides well enough to be able to treat their adverse effects and
perhaps someday to insert corrective genes into affected tissues and
bring about their expression.

New techniques for the analysis of DNA will be needed; an army of
scientists and peripheral workers will be necessary to accomplish this
task. Our schools will have to provide those young people who have a
basic training in the biotechnology of DNA analysis. They will be
needed to contribute not only to this immense task, but also to the many
applications of genetic manipulation in knowing and understanding the

nature of every kind of living thing.

2

We will need youngrmen and women. too, who will enter the field of
plant and livestock genetics to improve food production. In addition.
scientists in pharmacology and health medicine will require a
disciplined knowledge of DNA technology.

It is important for the teacher to understand that even though
these young men and women may not now choose the field of biotechnology
many of them will be involved directly or indirectly in what is becoming
a maJor commercial. industrial, agricultural and social influence in our
society.

It will be important. as well, to educate young people to the
nature of the genetic options from which they might have to choose as
they raise families and provide for their care (2.3)

The new technology requires that an educated person not only have a
familiarity with the nature of DNA. but also a familiarity with the
techniques used to manipulate and analyze it.

Laboratory exercises involving biotechnology which high school
students can perform have been slow in coming. Few veteran teachers
have a familiarity with the methods and precedires. Beginning teachers
have difficulty introducing what seem to be new and radical concepts
into the high school. Even fewer teachers understand that by using
simple and inexpensive materials, many of which are already in place in
the biology laboratory. students can perform and experience some of the
more sophisticated tasks of biotechnology.

The school in this study. Holy Name High School. is a Catholic
parochial school of about 1300 students in the diocese of Cleveland,
Ohio. Students are drawn from a number of Catholic elementary schools

in Cleveland and surrounding suburbs. Student backgrounds are

3
heterogeneous and repesent all social and intellectual facets of the
community. Students are not selected on the basis of intellectual
superiority. There are special classes for students determined to be
learning disabled and honors classes for those of high ability who
choose to take them. Most of the students would be classified as
'average'.

The classes in which the labs presented in this thesis were used
included forty-three tenth grade secondary students and seven ninth
grade students in two Honors Biology groups. Students were selected on
the basis of their academic grade point average: 3.2 or better. They
were then given an 'lnvitation' to take the Honors Biology class which
required parental permission and a request sutunitted to the counselor
before acceptance into the course.

There is no freshman general science course in this school.

It was known and understood from previous experience as well as a
questionaire given this year that at least 90 per cent of the students
had little or no background in science. Those with some background had,
in elementary school. dissected invertebrates or primitive vertebrates
or had made leaf collections. Some few had studied cells and two or
three had a rudimentary knowledge of DNA.

Because of such varied and, in most cases. limited backgrounds. the
entire first quarter is spent in teaching scientific notation,
dimensional analysis, the metric system, the concept of the atom. and
basic chemistry. So the entire first quarter is spent in preparing the
students for the regular course of studies.

The text book used in the course is Biology by Hessells and Hopson
and is accompanied by a study guide. (See No. 9 in the bibliography.)

4
The classes were taught using what might be called a modified

conceptual method (4. 5), i. e. the students were led to concepts
through questions put forth by the teacher. This was most frequently the
manner in which new subJect matter was introduced. without prior
reference to the text. Text assignments would be given. following the
introduction of the basic concepts, and the learning experience was
subsequently filled in with necessary terminology and more precise
details. Sometimes, but only occasionally, a lecture method was used.

Periods are 40 minutes long five days a week and there are double
periods on Monday and Wednesday that may be used for laboratory work.
Laboratory exercises. however. sometimes ran for three or four
sequential days through both single and double periods.

These would be the kinds of laboratory exercises. for example,
involving measurements in learning the metric system where time was not
a critical factor. Or in sane laboratory exercises where enzymes were
studied or bacteria were transformed, the materials could be placed in
the refrigerator overnight at specific stages and work completed on the
following day. In labs involving the growth of already transformed
bacteria. the bacteria were placed in incubators overnight and observed
the next day. In the case of gel electrophoresis. materials had to be
kept overnight in the refrigerator for completion of the staining
process.

This thesis is an attempt to present understandable and 'do-able'
laboratory exercises. It is the hope that through the use of already
adapted and developed laboratory experiences and through the
modification and development of others that the technology can be

experienced and the biochemistry understood.

 

CHAPTER ONE
GENERAL STATEMENT OF A PROBLEM.

The subJect to be taught. DNA, is really a year long proJect. No
single unit can cover the complexities or the implications of DNA. It
is knowledge that must be introduced with a basic simplicity, reinforced
and reinforced again. and more knowledge added. There are at least four

areas. widely separated, to which the study of and the applications to

DNA apply:

1. In the basic introductory lesson on proteins,
carbohydrates. lipids and nucleic acids.

2. In the study of Prokaryotes including viruses and
plasmids.

3. In the study of Gametogenesis and the Cell Cycle.

4. Finally and most intensively in the study of Molecular

Genetics.

It is the purpose of this thesis not to present a single module or
isolated block of teaching, but rather to show an application to the
four areas of study.

The course that is taught is by design a course in cellular and
subceilular biology and therefore its main concern is for an
understanding of molecular events within the cell. And even though all
aspects will be included in this thesis, it should be pointed out that
the basics of nucleic acids. carbohydrates. proteins and lipids must be
constantly reinforced. Through this continual reinforcement, effective

understanding can result: the students will develop a sense of the cell

5

6
as a highly regulated entity in which are found the biochemical
interactions that cause sickness and health, life and death.

Most recently M. Mitchell Waldrop wrote about 'spontaneous
self-organization': cellular life (Which. after all, is all life.)
defies the Second Law of Thermodynamics. Dynamical systems such as the
cell do evolve from disorder into order, from chaos into organization at
an extremely conplex level. Cellular systems do 'unscranble the egg'.
(6)

Those of us who still cling to the idea of a 'vitallsm' hidden
somewhere in the forces of nature, perceive in this mystery a beauty
which no simple 'mechanistics' can resolve. The paradox is that life,
itself, opposes nature, while obeying all her edicts: energy levels,
universal forces, and all other physical rules which must be obeyed.

And finally before I turn to the business at hand, it must be said
that students need to be reminded that there is a beauty in all of these
events that can be perceived. There is in me a reverential awe that
seems to grow exponentially with time for these highly integrated
cellular events. This thesis describes the manner in which I feel DNA

in its maJor roles is to be taught.

THE APPLICATION

i. The basic studies should begin with an understanding of
biochemical molecules: carbohydrates, lipids, proteins and nucleic
acids. In this area we study the structures of the pentose sugars
through carbohydrates. The primary structure of proteins leads to the
simple statement that genes or DNA determine the primary structure. The
structure of the nucleic acid is determined early on by the sequence of
nucleotides. In this area students first learn of the nitrogenous
bases, the phosphates and the pentose sugars. Here, also, they learn
sane basic differences between DNA and RNA: Thymine in DNA and Uracil in

RNA and the differences in the pentose sugars. (7,8)

2. In the study of Prokaryotes, including the study of viruses,
the students will study plasmids and will be introduced to the concept:
DNA (transcription) ---->RNA(translatlon)---->Protein. There will be a
maJor reinforcement of the previous work on DNA. Plasmids contain the
message for the proteins and enzymes, 1. e. their amino acid sequence
such as for beta lactamase that destroys the antibiotic, ampicillin. At
this point a lab exercise is introduced that demonstrates the transfer
of plasmids and the transformation of a species of E. coli. Specific
plasmids are mentioned: pBR 322, pUC 18, pAMP, and others. Genetic
engineering, restriction enzymes, cloning, DNA replication are maJor

areas of discussion and consideration. (9 - 15)

3. Gametogenesis and the cell cycle require an understanding of
the formation of chrwosomes from chromatin, replication, nucleoscmes.

crossing over, prophase I -which includes the understanding that during

8
this phase transcription stops and translation is reduced to 25 t of its
original activity. Transcription and translation have not as yet been
introduced. Much has been inferred and alluded to, but nothing has
actually been defined in exact terms. We are preparing students through
all of this for the 'final act' which is the full explanation of
transcription and translation. Perhaps the best understanding and
reinforcement can occur when students build models of double stranded
DNA. These models, as part of a lab and take-home exercise demonstrate
3’ and 5’ orientation of the sugars, double and triple hydrogen bonds, a
close approximation of the molecular structures of the bases, the sugars

and the phosphates. <16 - 18)

4. Genetics and Molecular Genetics. After the basics of
inheritance which includes maJor Mendelian principles and principles of
sex influence, sex linked genes, sex limited genes, incomplete and
non-dominance, we can then turn to molecular genetics and all of the
beautiful and highly organized simpliclties of transcription and
translation. Students will construct with construction paper and tag

board models of transcription and translation. (9)

PROGRAM GOALS

Understanding biology in this decade also requires that students
have an awareness and working knowledge of biotechnology. It is
requisite that the teacher also have a background in molecular biology
to teach it. Likewise, it is important for his students to be able to
experience the biotechnology of molecular biology through laboratory
experience. Much of his future is going to be influenced by this new
branch of biology which began about 1939. (19)

The content of this new science has been taught in secondary
schools for the last 20 or so years, but laboratory exercises relating
to it have lagged far behind. Introducing the laboratory experiences
will familiarize students with several aspects of working with DNA in
the laboratory.

This thesis introduces new, simple laboratory exercises with DNA
which will enable both the teacher and the student to develop

self-confidence and understanding.

SUBJECT OBJECTIVES

1. Background preparation for the teaching.

a. It is important for teachers to develop a freedom and an
intimacy in dealing with DNA, restriction enzymes, plasmids, lambda DNA,
and gel electrophoresis.

b. It is important to be able to work with DNA in its many

forms through simple, easily understood lab experiences.
2. SubJect obJectives for the students.

a. Students will appreciate the historical development of
thoughts that lead to scientific truth and discovery.

b. Students will understandthat the scientific world is a
'real' world of people and personalities that are stubborn, selfish,
generous, egotistic, ambitious, thoughtful, religious, atheistic:
containing all the qualities of human beings.

c. Students will describe a DNA molecule, accurately.

d. Students will have an understanding of plasmids,
bacteriophages, restriction enzymes, genetic engineering, transcription
and translation.

e. Students will make, load and run agarose gel to do
electrophoretic studies of lambda DNA, restricted lambda DNA, plasmids.
and restriction digests of plasmids.

f. Students will make methylene blue staining solution and
employ the correct technique for staining gels.

g. Students will understand how to isolate and culture

bacteria for study.

10

11
h. Students will perform the laboratory procedures for the
isolation of plasmids fromlbacterial cultures that are in mid-log
growth.
i. Students will use, with ease, the spectrophotometer, Bausch
and Low - Spec 20, and to use it for determining mid-log bacterial

growth.
3. Concepts to be Presented.

a. Perhaps the most important concept is that the student be
able to visualize the DNA molecule through the construction of a model
of the molecule.

b. The cell is the unit of life for most living things.
(Viruses, viroids, prions, are these exceptions? The existence of these
complex forms of life should be emphasized without providing answers.)

c. DNA is the molecule that provides the information to the
molecules that cause life.

d. Life, sickness, health, death are subcellular events
involving change to the stability of regular molecular structures within
the cell.

e. Subcellular events involve interactions between molecules
within cells. (Examples are the Embden-Meyerhoff pathway, Krebs Cycle
and Oxidative Phosphorylation.)

f. The shape of an organlsn is dependent upon the molecules
produced by the genes (DNA) within the cell.(At this point I initiate
such questions as: Why are dogs, for example, shaped differently from
cows or humans or trees or marigolds? Their cells are much the same and

do most of the same things, i. e. 'e', above, so their enzymes are

12
generally the same (at least have the same active sites, although they
may have evolved slightly different 'non-critical parts'.) The only
thing that is essentially different is their DNA. So the question must
follow: What is different about their DNA? And the answer must be the
sequence of bases in the DNA molecule.)

9. How do we know that there are differences and similarities
in the DNA? We have to be able to look at the DNA of different
organisms and compare the differences in the sequences of the bases.
How are we able to see the differences in the sequencing of bases?

The most commonly used tool is gel electrophoresis.

h. The concept of agarose gel electrophoresis as a tool to
separate different amounts of DNA and to see the presence of DNA by
simple staining of the gel is important. Here a lab involving lambda
DNA can be introduced so students can see the DNA for themselves. Here
is the DNA, now, how do we find its sequence?

i. The sequencing of DNA is too complicated a procedure for
high school or even undergraduate courses in college, but the concept of
restriction enzymes can be introduced and the manner in which they work,
like molecular scissors, to hydrolyze DNA at predictable places into
pieces. Here, too, the nomenclature for restriction enzymes can be
introduced with several examples such as EcoRi, Hind III, and BamHi
along with their cleavage sites.

J. Now the study of viruses, most notably lambda phage must
be introduced. Viruses may be grown as a lab exercise to demonstrate
their existence. Also, the structure of lambda DNA which is understood

can now be stated. And the concept of how viruses attack and invade

13
bacterial cells and are reproduced can be described. This can be
related to the common cold, the flu and other viral diseases.

k. The understanding and study of lambda DNA continues with
the study of the effect of restriction enzymes, EcoRi and Bamfll, on
lambda DNA. A simple laboratory exercise demonstrates this plainly by
having the students prepare restriction digests and electrophoresing
them on agarose gels. The student should learn how to use restriction
digests and to make restriction maps after they have measured the
distances travelled by each of the bands. Here comparisons of bands in
lanes from single and double digests can be shown as well as the
ligated, circular form of the phage lambda DNA.

1. The concept of cloning would comeinext. Teaching this
concept involves an explanation of the use of DNA in cloning experiments
in plants. Here, as well, one might describe the use of the plasmid
from the bacterium Agrobacter tumifaciens (This bacterium causes crown
gall in plants.) as a cloning vector to introduce cloned genes of choice
into plants whose characteristics one wishes to alter through those
genes.

m. To continue the concept of cloning and genetic engineering
I use a laboratory exercise involving 'naturai' genetic engineering.

Two strains of E. cell are grown each of which has a resistance to a
different antibiotic. One of the bacteria possesses the 'P' or
fertility plasnid, the other does not. In this very workable lab
exercise bacteria are produced which have immunities to both
antibiotics. We call these 'mutant' strains.

n. In discussing the events of the aforementioned lab

exercise, students become more familiar with plasmids by reading the

14
the Scientific American article. Plasmids. (15) which clearly states how

plasmids behave in bacterial cells. Using this exercise along with the
previous work gives students a good understanding of these
'endosymbionts'.

0. Now we introduce a simple laboratory exercise in which we
isolate plasmids from bacterial cultures which are in the mid-log stage
of growth. Existence of plasmids can be shown through gel
electrophoresis. Those students who wish to pursue the subJect further,
can restrict the plasmids and subJect them to electrophoresis.

p. There is at least one workable exercise that will enable
us to clone into an ampicillln resistant plasmid a gene for kanamycin
resistance. The opportunity to do this work should be available to
any student who desires to do further laboratory research.

q. To further continue our study of plasmids, students may
introduce ampicillin resistant plasmids containing the Lac 2 gene into
E. coli strains made competent through treatment with cold calcium
chloride and heat shock. Bacteria would be grown on mediumlcontaining
IPTG (isopropylthiogalactoside) and X-Gal
(S-bromo-d-chloro-3-indolyl-betaD galactoside). The colonies can be
easily identified because those containing the plasmid will appear as

blue dots on the agar plates. (20, 21)

4. Applications of Biotechnology of DNA in the World Today

a. Present day genetic engineering of plants and animals:
(1) The development of transgenic mice to study cancer

causing genes (oncogenes) and the aids virus.

15

(2) The development of transgenic strains of cattle,
pigs, sheep, rabbits and chickens in order to produce more desirable
food products. (22)

(3) The development of genetically engineered strains of
various agricultural plants to modify and improve crops. (23)

b. The understanding of, control of and possible alteration of
human gene expression.

(1) This means, someday, being able to introduce healthy
genes into the cells of individuals who have defective genes and causing
those healthy genes to be expressed.

(2) It also means the future possibility of being able to
prevent the expression of genes for degenerative diseases such as
Alzheimer’s and Huntington’s Chorea. ‘

(3) It means being able to control and eventually cure
diseases which are now incurable: the common cold and influenza will
eventually be curable.

d. Through our understanding of viruses and DNA we can now
immunize the population against various diseases:

(1) Immunizations can be obtained against such diseases
as smallpox, measles, three forms of polio, some influenza viruses, and
other diseases. Immnity can be obtained against some new and mutant
forms of influenza viruses almost as soon as they develop.

e. It also means Jobs in the future:

(1) The biotechnology industry has grown extremely

rapidly and is taking all of the trained technicians and scientists

which are available.

CHAPTER TWO

A SYNOPSIS OF THE MAJOR HISTORICAL SCIENTIFIC DEVELOPMENTS THAT LED TO
OUR PRESENT UNDERSTANDING OF DNA.

No work written today better or more clearly documents the history
of the development of our understanding of DNA than Horace Judson’s work
WW. Therefore, apart from past information
obtained fran general references, and in the interest of accuracy, I
have chosen Mr. Judson’s book as a primary source for the chronology,

the events and the quotations.

1868. Johann Friedrich Miescher, with whan the history seriously
begins, went to Tuebingen, to study with. Ernst Felix Hoppe-Seyler (The
man who named hemoglobin.) . Miescher was interested in studying the
nucleus of cells. He began by studying white blood cells which he
collected fran pus from discarded surgical bandages. He discovered in
the isolated nuclei an acidic substance rich in phosphorus, and made up
of very large molecules. He named this substance in 1869 and called it
NUCLEIN.

The following year he began to study salmon sperm for nuclein and
a student of his, Richard Altmann, introduced the term NUCLEIC ACID.

Remarkably in 1892, Miescher pointed out in a letter to an uncle
that 'sane of the large molecules encountered in biology, comosed of a
repetition of a few similar but not identical snail chemical pieces,
could express all the rich variety of the hereditary message, Just as
the words and concepts of all languages can find expression in
twenty-four to thirty letters of the alphabet.”
*JUdson, Horace Freeland, The Eighth.Dgy of creation, flNsw‘Zork: Simon

and SChuster, 1979), p. 28.
16'

 

17
Huch of the elementary chemistry of nucleic acids was done by

Miescher and his students and by other laboratories at that time. From
the discovery of Guanine in 1844, eventually all of the bases were
isolated by 1900 and by the 1920’s. it was known that there were two

kinds of nucleic acids, deoxyribose nucleic acid and ribonucleic acid.

In 1928, Fred Griffith discovered two strains of a bacterium,
Streptococcus pneumoniae, one of which was lethal to mice and another
which was non-virulent. He labelled them type II or R (R stands for
rough surface.) which was non-virulent and type III or S (S stands for
smooth surface...this form was enclosed in a capsule.). Type III was
virulent and caused pneumonia in mice and their subsequent death.

When he inJected mice with live Type II (R form) and dead Type III
(S form), virulent strains of Streptococcus pneumoniae were produced
which killed the mice from pneumonia.

Griffith’s work was really the first time that transformation of
bacterial cells was described. DNA from one form, which in this case
was the dead '8' form, is able to enter a living form which was
non-virulent and transform it into a virulent strain.

The transformation of bacteria was confirmed in 1929.

1944. Oswald Avery, Colin MacLeod, and Maciyn McCarty at the
Rockefeller Institute in New York published on of the first papers
describing the transformation of bacteria. 'R' forms of Streptococcus
pneumoniae which are harmless mutants that do not cause pneumonia, were
transformed into '8' forms, which are virulent strains and cause
pneumonia, by mixing them with the purified DNA extracted from killed

'3' forms. Basing their work on the originial work of Fred Griffith they

18
determined that the “transforming material' ..contalned no demonstrable

protein, lipid or polysaccharide, but was a highly polymerized, viscous
form of DNA. (24)

The work of Avery, MacLeod and McCarty took many years. Avery’s
work was subtle and exhaustive and ingenious. He noted that DNA was
both Autocatalytic and Heterocatalytic. In a letter to a brother he
noted: ' If we are right, and of course that’s not yet proven, then it
means that nucleic acids are not merely structurally important but

functionally active substances in determining the biochemical activities

 

and specific characteristics of cells -& that by means of a known
chemical substance it is possible to induce predictable and hereditary

changes in cells......Sounds like a virus - may be a gene.'*

1951- 52. Alfred Hershey and Martha Chase determined that it was the
DNA of the bacteriophage that contained the genetic component for which

everyone was then searching. They grew viruses on bacterial lawns

containing radioactive phosphorus. 32?. Since phosphorus is present in
DNA and not in proteins, the radioactive phosphorus would indicate the
presence of DNA. They also used radioactive sulfur, 358 as a marker to
detenmine the presence of protein. They infected E. coli with phage
particles grown in bacterial cells containing 355 and 32?.

Since protein contains sulfur and no phosphorus, it then became a
relatively simple matter to determine where the protein was and where
the DNA was. The DNA would be present where the radioactive phosphorus
appeared and the protein would be present where the radioactive sulfur
appeared.

The experiment which they performed is referred to as the “Waring
Blender' experiment. They observed that the electron micrograph of the

*Ibid, P. 39.

19
phages on the outside of the bacterial walls appeared to be empty.

Their problem was to separate the bacteria from the viral fragments on
the outside of the bacterial wall and determine which of the radioactive
isotopes were present and where they were: in the bacteria or in the
viral wall.

They took a chilled suspension of bacterial cells recently infected
with a phage virus and placed it in a Waring blender to separate the
viral shells from the bacterial walls by blending. They then centrifuged
the mixture to pull down the bacterial cells which would be heavier and
separate them from the bacteriophages that were present on the surface
of the bacterial cell. The bacteriophages remained in suspension.

The question had to be, were the DNA and the protein together?
Salvadore Luria had Just proposed that the phage protein had the
genetic role and that the DNA was only added in the last step before the
cell burst.

Hershey and Chase discovered that the radioactive sulfur remained
in the supernatant liquid with the viral shells (protein) and that the
DNA was present in the bacterial cells, which were in the centrifugate.
The virus then attached itself to the outside of the bacterium and
inJected its DNA into the cell. Their modest conclusion inferred that
sulfur containing protein had no function in phage replication
and that nDNA had some function.ll (25)

This proved conclusively the work begun by Avery that the
transforming principle was DNA, that DNA was the actual genetic material

and protein was not.

20
Now the scientific community began to realize that the important

discovery of the future was to be the discovery of the structure of DNA

and eventually the manner in which DNA carried out its genetic activity.

1953. James D. Watson, F. H. C. Crick and Maurice Wilkins determine the
structure of the beta form of the DNA double helix. The paper, a single
page, was published in May of 1953 in Nature magazine.(18) This is the

most revolutionary work of modern science and yet without a lot of luck

and all of the work that preceded it, it would have been impossible.

1940. Now the work of Beadle and Tatum from 10 years previous to the
discovery of Watson and Crick took shape in the minds of the scientific
community. Using a common bread mold, Neurospora crassa, they
determined that it was the undertaking of_a gene to make an enzyme.
Recalling this classical series of experiments made it apparent that the
DNA in some way made enzymes.

It took the work of the 50’s following the discovery of the
structure of DNA by Watson and Crick to discover how the gene produced
enzymes. Even though there were many previous hints even as early as
the early 40’s: the research of Toerorn Caspersson and the Karolinska
Institute in Stockholm and Jean Brachet in Paris. Their work indicated
thatI vigorous protein synthesis was always associated with plentiful
RNA. And further: this RNA was located in the cytoplasm, while DNA was
confined to the cell nucleus.'*

In 1950 it was proposed by two bacteriologists in Strasbourg, Andre
Bolvin and Roger Venrely that DNA makes RNA makes protein.

It must be noted here, too, that a maJor problem was the

development of the energetics to drive the expression of the gene. For

*Ibid, p. 236.

21
this Fritz Lipmann must be credited with his development of the

understanding of the role of ATP in taking the energy out of the
metabolic flow and conducting it to where it was needed to produce
reactions.

Judson’s account of the stimulating and restless period of the 50’s
reveals the names of many scientists who contributed to this exciting
era. Alexander Dounce at the University of Rochester suggested that RNA
ought to be the template for proteins and that the RNA originated on the
tenplate of the DNA gene. George Gamow, the Cosnologist responsible for
the 'Big Bang' theory, proposed a model for DNA synthesis that dominated
the thoughts of many most especially Francis Crick’s for several years.

But, according to Judson, it was Francis Crick, who through
correspondence and conferences and personal diplomacy, directed and
drove the research to discover the mechanisms of genetic action. It was
Crick who deduced the role of transfer RNA; (He spoke at a symposium on
Feb. 18, 1956, and said, 'One possibility is that each amino acid is
combined with its special ’adaptor"...):* it was he who introduced the
term degenerate, a term from quantum physics.

But many, many men published many, many research papers. Fred
Sanger published the amino acid sequence for insulin in 1955. Vernon
Ingram’s chemical analysis of the peptides and eventually of the single
amino acid differences between normal and sickle cell hemoglobin,
developed the understanding of what was the fundamental role of the
gene.

Ribosomes began to appear in electron micrographs. They were
called microsomes and by the end of 1956 it was apparent that soluble
RNA molecules were an intemmediate in protein synthesis, picking up

*Ibid, P. 316.

22
activated amino acids, bringing them to the microsomes and depositing

them there. Robert Holley identified the transfer RNA’s, all of them,
for which he won the Nobel prize in 1968. The structure of ribosmes
were to be determined and proven as the site of protein synthesis.
Richard Roberts named these microsomes, ribosomes, and demonstrated
their activity at the Carnegie Institute of Washington.

But it was at the Fifth International Congress of Biochemistry that

began on the 10th of August 1961 in Moscow that an American scientist
from the National Institute of Health, Marshall Nirenberg, presented

the final answer to the whole equation. His paper, 'The Dependence of
Cell-free Protein Synthesis in E. coli Upon Naturally Occurring or
Synthetic Template RNA.‘ was produced with the collaboration of his
colleague, Heinrich Matthaei. He finally solved the coding problem and
the exact role of messenger RNA. His lecture was poorly attended and
lasted only about 15 minutes: the only scientists in attendance of note
were: Matthew Meselson, Walter Gilbert and Alfred Tlssieres. Nirenberg
and Matthaei had synthesized a long chain of phenylalanine from
polyurldyllc acid and demonstrated that one or more uridylic acid
residues coded for phenylalanine.

Judson states, 'Their lNlrenberg’s and Matthaei’sl aim was Avery’s
too: to understand the specificity of the nucleic acids, by putting in
some known thing to see what came out. Avery’s proof that the
transforming principle was DNA set the agenda for biology. Watson’s and
Crick’s elucidation of the structure of DNA confirmed rather than
concluded that agenda. But the questions that Avery had asked,

Nirenberg and Mathaei began at last to answer.‘ I Nirenberg was asked to

*Ibid, p. 481.

23
give a second reading of his paper in Moscow, this time to hundreds of

scientists.
Following the revelation in Moscow, it was Har Gobind Khorana at
the University of Wisconsin who finally elucidated the entire code.
Nirenberg, Holley and Khorana received the Nobel prize in

physiology and medicine in 1968.

REFLECTION

There is one man who stands out for his imposing mentality as well
as for his contribution to molecular biology. His.name is Erwin
Chargaff. He was an outstanding and highly disciplined scientist, a
polemicist, a moralist, a prophet who forsaw dangers in genetic
engineering, in tampering with nature. His words printed in a letter
to Science magazine, '..the future will curse us for lt'l [He refers to
the manipulation of heredity through transduction of foreign genes into
organisms.l. But his important contribution to science was pivitol:
he noted in 1950 that the ratio of Adenine to Thymine and Cytosine to
Guanine, and purines to pyrimidines was about 1. His work was to shape
the internal structure of the DNA molecule for Watson and Crick.

Francis Crick emerges in Judson’s work as the imposing force, the
scientist who coordinated and directed the efforts of other scientists.

Horace Judson writes of many of the maJor scientists of this period
in molecular biology who, after significant discoveries go other ways,
abandoning research for various things: writing, adnlninstration,
supervision of graduate students and post does. But Francis Crick
continues to do science, albeit a new science.

Judson suggests that after the discovery of Watson and Crick, they
wanted to abandon the proJect. They were llonized, in demand: yet I
somehow they were compelled to continue, to complete the task: to
determine transcription and translation. They became bored with 'DNA'.
Crick seems to have carried on with vigor to the end. Watson’s efforts

and contributions diminish considerably, although Watson now is in

24

25

charge of the Human Genome ProJect. Still, the work is administrative

rather than bench science.

CHAPTER THREE

The laboratory exercises that accmpany this work are presented in

this chapter. These activities form the basis for classroom discussions

from which proper conceptualization and understanding should occur.

26

Laboratory Exercise No. i.

TRANSFORMATION OF E. COLI

Instructor’s Guide.

Target Group.

Entry level, advanced and Honors biology students.

Time Frame.

The time commitment for the instructor is much longer than the
time it takes for the class to do the lab. Preparing the materials is
most time consuming. Student assistants can be used to assist in the
mixing and sterilizing medium.

The time in preparation of the lab is about 3 - 5 hours. The
time spent in actual lab work by the students depends upon how'much time
is allowed to prepare the students to do 'sterile technique“, and actual

student time in doing the lab. Actual student time should be about 20

minutes.
Background Information for the Teacher.

The teacher needs to understand something of plasmids and
plasmid transfer between bacteria. This exercise involves two strains
of E. coli, called J53-R and Ht-99.

There is a plasmid in some bacteria called the 'F' factor

which enables the bacterium to form a conJugation tube (sex pilus) with

27

28
other bacteria in order to transfer plaaiids from the one strain to

another. These bacteria possessing the 'F' factor are termed 'F+':
those not having the fertility factor are termed 'F-'.

In this instance, the teacher will work with a 'male' or donor
or 'F+' strain of bacteria which contains a plasmid for the fertility
factor. This bacterium also has, in addition, a plasmid (designated
pBR322) with the gene that enables the bacterium to produce the enzyme
that will chemically decanpose the antibiotic, chloramphenicoi. The
bacterium is a strain called lit 99, and has this specially "engineered'I
plasnid. It is engineered to be "male'' and thus is able to donate its
plasnid containing the chloranphenicol decomposing gene to another or
recipient bacterium.

The students will also be working with the 'female' or
recipient bacterium, an 'F—' strain, which possesses a plasnid (also
designated pBR322) which enables it to be imune to an antibiotic,
rifampicin. Its plasnid possesses the gene that enables it to produce
the enzyme that will decompose the antibiotic rifampicin. Since it is
an 'F-' strain it does not possess a plasmid for the formation of a
conJugation tube or sex pilus and so is not able to donate its plasnid
to another bacterium.

Both strains of bacteria will be grown separately in a Luria
broth nutrient medium, and when they have reached mid-log growth, a
portion of each will be mixed together in the same kind of sterile
nutrient broth in a separate flask. Some of the 'F+' strains may then
form conJugation tubes with the 'F-' strains when placed together in the
mixed culture and susequently produce a 'mutant' strain with the

characteristics of both parents. The formation of the sex pilus, which

29
will result in the transfer of plasmids, can be stimulated by frequent

agitation or aeration with a simple aquarium pump.

Plasmid, pBR322, is a synthetic plasmid that has been constructed
from several naturally occuring plasmids. It consists of, depending
upon the genes which have been inserted, of about 4400 base pairs.

In this laboratory exercise the two bacterial strains contain the
plasmid, pBR322, but the plasmids in each are engineered slightly
differently. One has the plasmid with the the gene for chioramphenicol

resistance and the other has the gene for rifampicln resistance. (13,

14)

Preparation of Materials.

The teacher should prepare the agar plates beforehand and also

mix the antibiotic solutions of chioramphenicol and rifampicin.
The preparation of the Antibiotics: (14)

1. Chloramphenicol: Dissolve 250 mg Chloramphenlcol in a

mixture of 5 mL of methanol and 5 mL of distilled water.

2. Take 1 mL of the solution in #1. and dissolve in 9 mL of
methanol. THIS IS CALLED SOLUTION A.

3. Add 1 mL of solution A to 9 mL of sterile, distilled
water. This solution, which is solution B, is mixed with cooling agar

before the agar is poured into the pour plates.

The Preparation of leamplcin.

30
1. Dissolve 250 mg of Rifampicin in 25 mL of methanol. THIS

IS CALLED SOLUTION B.

2. Dissolve 1 mL of solution B in 9 mL of sterile distilled
water. This solution, which is solution C, is mixediwith the cooling
agar to be poured into the petri dishes. N.B. (Nota bene, note well)
the agar should be cooled to less than 60 deg. C. before the antibiotics

are mixed with the agar.

Making the Agar Plates:

1. Dissolve three (3) grams of Nutrient Agar in 90 mL of

distilled water and autoclave at 250 F for 15 min.

2. When the agar has cooled to Just below 60 deg. C, add 10
mL of the chioramphenicol, solution B, AND UNDER STERILE CONDITIONS POUR
CAREFULLY INTO PREVIOUSLY STERILIZED PETRI DISHES. (One can use sterile

plastic disposable petri dishes to save time.)

3. The Rifampicin plates are made in the same way as the
chioramphenicol plates, except that rifampicin solution C is poured into

the cooling agar medium.

4. You will need to make plates containing both antibiotics
to determine whether conJugation and transfer of plasmids will have

taken place. Make them in the following manner:

a. One (1) mL of SOLUTION A (containing concentrated
chioramphenicol) and one (1) mL of SOLUTION B (containing rifampicin in

concentrated state.) are mixed with 8 mL of sterile, Dist. water.

31
b. The 10 mL aliquot described in 4a, is mixed with the

cooling agar as previously described and subsequently poured into the

pour plates.

5. Preparation of Bacterial Cultures. Obtain freeze-dried
cultures of E. coil J53-R and Ht-99 from Kemtec.
a. Aseptlcally add approximately 0.5 ml of nutrient
broth to each of the glass vials containing the lyophilized cultures.
Shake the vials gently until the bacteria are suspended and the solution

is cloudy.

b. Label one 250 ml Erlenmeyer flask containing sterile

Luria broth J53-R and another Ht-99.

c. Transfer the contents of each vial, aseptically to

the properly labelled 250 ml Erlenmeyer flask.

d. Incubate overnight in a 37 deg. C. water bath.

Preparation of the Mating Mixture.

1. Transfer, aseptically, 0.1 ml of the overnight culture of
E. coil Ht-99 and 0.9 ml of the overnight culture of J53-R into a 250 ml

Erlenmeyer flask containing sterile Luria broth.

2. Incubate with aeration or in a shaking 37 deg. C water

bath for three to four hours.

3. Maintaining the cultures. The bacterial cultures can be
maintained in liquid suspension in the refrigerator for several days

prior to usage.

 

32

Using one or two student 'volunteer' assistants makes it possible
to complete the preparations described above in a short time. Most of
the time is spent waiting for the autoclave to come to temperature to

complete the sterilization cycle and then cool down.
Problems Encountered.

i. It is necessary to demonstrate sterile technique to students
beforehand.
2. Students may not complete sterile technique well and so some
of the plates may be improperly inoculated.
3. You have to decide whether you want each student to inoculate
the three plates or whether you want them to work in teams. This will

determine how'much medium you prepare and how many plates to pour.
Sources of Materials:

Kemtec has the 'englneered' bacterial strains and 'klts' that
are already prepared. However, one can purchase the materials
separately from them: Kemtec, 9889 Crescent Park Dr., West Chester,

Ohio 45069, (1-513 777 3535) Just outside of Cincinnati.

Background Information for the Student. The students will need to
have the background information on plasmids and the two strains of E.
coli mentioned in the Background Information for the Teacher. It will
be usefull to the student to have available to him the Scientific
American article from December 1980 entitled 'Plasnids'. It will be
usefull to read at least the first four pages in class before doing the

lab.

33

Goals.

The maJor thrust will be to introduce students to the concept of
plasmids: this becomes a good introduction to recombinant DNA

technology.

Performance ObJectives.

1. The student will learn sterile technique in the

inoculation of bacterial plates.

2. The student will observe, indirectly through cause and
effect, the behavior of plasmids in bacteria and will experience the

results of bacterial conJugation.

3. The student will observe the growth of normal and mutant
bacterial strains. He will observe differences in their growth patterns

and in their resistances to antibiotics.

4. The student will experience classic scientific
methodology: simple and direct experimentation with controls and

experimental parameters.

Materials.

Sterile petri dishes (100mm X 10mm or almost any size that is
available.), three per team.

Chloramphenicol

Rifampicin Inoculatlng loops
Nutrient Agar. Luria Broth Agar. Alcohol lamps

Luria Broth

34

Sterile test tubes containing Lurla Broth
Methanol

Sterile distilled water in test tubes.
Incubator, microbiological

Ht-99 bacteria, freeze dried.

J53-R bacteria, freeze dried.

Procedures.

1. Prepare petri dishes as previously indicated so that there
are enough for three for each person or three per team. (It is better if
each person does his own; this procedure provides more data. and gives

better results.

2. Prepare bacterial cultures in Luria Broth at least two
days before doing the lab. Using sterile technique, inoculate sterile
Lurla broth in a 25 ml Erlenmeyer flask with the J53-R strain of E.
coli. Place in an incubator for 24 hrs. Culture the Ht—99 strain in

the same way.

3. Prepare a mixed culture of J53-R and Ht-99 by mixing,
using a sterile pipet and sterile technique, 0.9 mL of the J53-R with

0.1 mL of the Ht-99 culture. Incubate these for 24 hours.

4. It is suggested that you give each student or each team
test tubes containing each of the three cultures, again using previously
prepared sterile test tubes with aluminum foil caps and containing Luria
Broth. These can be inoculated with the original cultures of Ht-99,
J53-R and the mixed culture, respectively, with sterile, disposable

pipets.

35

5. Students should obtain the three cultures and inoculate

them in the following manner:

a. The rifampicin containing pour plates can be
inoculated on the left side of the plate with the J53-R strain, making a
large 'R' with the inoculating loop, being careful not to scratch the

agar in the dish.

b. The same plates can be inoculated on the right side
with the Ht-99 strain, making a large 'H' with the inoculating loop,

again being careful not to scratch the agar in the dish.

c. The same should be done with the chioramphenicol
plates and in the same manner by dividing the plate in half and

inoculating each side with a different strain using the same letters,

'R' and 'H'.

d. The plates containing the mixed antibiotics, l. e.
both the chloramphenicol and rifampicin, should be divided into thirds.
One of the three sections should be inoculated with the J53-R strain
making a large 'R', another of the three sections with the Ht-99, making
a large 'H', the third section of the dish should be inoculated with the

mixed strain using a large 'M'.

6. The petri dishes are now placed upside-down in an

incubator overnight.

Results.

1. Each student or team reads his own plates and records his

data on a chart on the board. He should indicate with a (+) where there

36
is growth of bacteria and what kind of bacteria. and with a (-) where

there is no growth. Each student copies down the class results into his

lab book.
2. The class should then discuss and evaluate their results.
The Preparation of the Laboratory Report.

The student will prepare a laboratory report which will

contain the following:

1. Purpose. The student will prepare a single sentence

statement describing the purpose of the laboratory exercise.

2. Introduction. He will draw his introductory material from
the December 1980 Scientific American article by Robert Novick on
Plasmids and from.other background material supplied by the teacher..

He will write a thorough explanation of plasmids concentrating on

conJugation and the transfer of plasnids fran one bacterium to another.

3. Procedures. The students will indicate the followed steps

in carrying out the experiment.

4. Data. The student will prepare a data table for his

results and for the class results.

5. Graph. In this lab the student will prepare a bar graph
of the per cent of positive growth obtained in the cultures. The graph

is to be done as both hand drawn and computer drawn.

6. Results and Conclusion. The student will be asked to draw

conclusions from the experiment. He will answer such questions as: Was

37
the experiment successful? What did it prove? Did the bacterial growth
demonstrate the presence of plasmids? How? Were there errors or
mistakes in the experiment? What were they? Why do you think they

occurred? Were the errors sufficient to say the experiment did not

succeed in proving what it set out to do?

LABORATORY EXERCISE N0. 2.

COLONY TRANSFORMATION

Target Group.

This laboratory exercise is designed for advanced placement or

honors biology students.
Instructors Guide.
1. Background Information.

The purpose of this laboratory exercise will be to
transform bacteria, which would be killed by the antibiotic amplcillin,
to amplcillin resistant strains. Students will attempt to do this by
making bacteria 'competent' to receive plasmid and subsequently
introducing those plasmids into bacterial suspensions allowing the
bacteria present to Itake them ln'. These suspensions will be
inoculated onto agar plates containing amplcillin and should produce

visible colony growth.

The control will be bacterial suspensions which have not
been inoculated with plasmids. These are 'untransformed' strains that
also will be inoculated on agar plates containing amplcillin: these
should not grow.

The instructor will work with strains of E. coli that
have no plasmids in them. In this instance this instructor used strains
of E. coli designated MM294 which were obtained from Carolina

Biologicals and are plasmid free.

38

39
Plasmids must also be obtained that will be introduced
into the bacterium. This instructor used two different plasmids, both
having an amplcillin resistant gene. One plasmid, pUCiB, was obtained
from Modern Biology, Inc. (Dayton, Indiana) and the other plasmid used

was pAMP which was obtained from Carolina Biologicals.

Preparation of Materials.

Agar plates have to be prepared or obtained already prepared

for use in the laboratory exercise.
The Preparation of the Agar Plates.

1. It will be necessary to have four plates per team of

students with 2 to 4 students per team.‘

2. The petri dishes (plates) will have to be sterilized

before hand.

3. Two of the plates will contain Luria Broth Agar: 25 grams

per liter of distilled water.

a. The Luria Broth Agar is placed in the distilled water
in a beaker and heated until it is dissolved. It is then poured, 100
mi, into 150 ml Erlenmeyer flasks which are covered with aluminum foil
that is held in place with rubber bands and the flasks are autoclaved
(sterilized) at 250 deg. F (121 deg. C) for 15 minutes.

b. After the flasks are removed from the autoclave or
sterilizer, they are allowed to cool to about 100 deg. F (about 40 deg.
C). To half of the flasks a preparation of i.ml of amplcillin is added.

The amplcillin is dispensed with sterile. 1 ml pipets.

 

40

The Preparation of Ampicillln.

1. Add 1.06 g Ampicillin (sodium salt) to 100 ml of sterile
distilled water, mix thoroughly with a clean stirring rod or a magnetic
spin bar or by swirling the solution. Pour into sterile, 10 ml test
tubes previously covered with aluminum foil and freeze or keep cold, if

the amplcillin is to be used immediately, until usage.

Pouring the Plates.

1. Add the agar preparation (6 - 10 ml) minus the amplcillin
to each of two plates. One plate is marked Agar '+' and the other is
marked Agar '-'.

2. To each of the remaining two plates the agar preparation
containing the amplcillin. One plate is to be marked Agar/Amp + and the

other is to be marked Agar/Amp -.

There are new four petri dishes containing Luria Broth Agar or

Lurla Broth Agar/Ampicillin. Each plate is marked as indicated.

Additional Materials Needed.

1. It will also be necessary to prepare a solution of
ice-cold 0.05 M Calcium chloride. This solution is prepared by mixing
5.55 9 Calcium chloride in 1 liter of distilled water. It is to be kept

cold.

2. A water bath is needed that will enable the students to

heat-shock their sample at 42 deg. C. for 90 seconds.

41

3. A small, 10 ml test tube is prepared for each team
containing about 5 ml of Luria broth. The test tube is covered with

foil and placed in the autoclave and sterilized as before.

4. Students will also need two foil covered, 10 ml test tubes

that are sterilized.

Time Frame.

The lab itself takes about 40 minutes to complete.
Considerably more time is required beforehand for preparation. Plates
pre-prepared and marked as indicated can be obtained from Carolina
Biologicals so the time spent in preparing plates can be reduced to
nothing. About three hours of time is needed to prepare the plates for
two classes. Preparing Ampicillin requires about 15 minutes. Host of
the time is spent autoclaving petri dishes and Lurla Broth agar and in
pouring the plates. The preparation and sterilization of test tubes

with and without Lurla broth takes another 45 minutes.

Content Skills Required of the Teacher.

1. The use of a micropipet.

2. To prepare and pour the plates using sterile technique.

Problems Students Might Encounter.

1. The cultures could become contaminated if students do not
inoculate the cultures carefully and handle all materials using sterile

technique.

42

Student Goals.

1. The student will develop greater skill in using sterile

technique.

2. The student will observe the function of plasmids.

3. The student will become familiar with techniques used in

making competent cells.

4. The student will observe the transformation of bacteria.

Background Information for the Student.

1. The student will need to know how to use a micropipet or

its equivalent.

2. Everything is to be kept on ice when not being

manipulated.

3. The student will have to understand and use sterile

technique.
Procedures for the Student. (26)

1. Using transfer pipet add 25 microliters of cold calcium
chloride solution to each of two 10 ml test tubes. Label one tube '+'

and the other tube '-'. Place the test tubes in a beaker of ice.

2. Using sterile technique, transfer a cell mass from the
cell culture into the '+' tube. (Be careful not to transfer agar with

the cells.) Vigorously tap the loop to dislodge the cells from the loop

43
and get them into the solution. Flick the tube until the cell mass has

disappeared and the solution is cloudy. Place the tube on ice.

3. Using sterile technique transfer a cell mass from the cell
culture into the '-' tube, and proceed as in No. 2, above, to suspend

the cells in the solution, placing them on ice.

4. A sterile inoculating loop is now used to add about 10
microliters of either pAMP or pUCIB to the '+' tube. If the loop has a
film of liquid covering the inside of the loop, that will be about 10
microliters. If you can’t get a film filling the loop, the smallest
visible drop will probably be sufficient. Tap the tube to release the
plasnids from the loop and swirl to mix the plasnids with the bacterial

suspension. Place the tube back on ice.

5. Incubate the '+' and '-' tubes for 15 minutes on ice.

6. Obtain four petri dishes containing medium: two should be
labelled LB and two should be labelled LB/AMP. Label one LB plate '+'
and the other '-'. Do the same with the LB/AMP plates.

7. After the test tubes have incubated for 15 minutes on ice
remove the tubes and 'heat shock'l them in a 42 deg. C. water bath for
exactly 90 seconds. The 90 second period of time is most critical.
After the tubes have been I'heat shocked' return the tubes to the ice for

one or more minutes.

8. With a transfer pipet, add 250 microliters of Luria broth
to each tube. Mix well by tapping with your finger and set the tubes at

room temperature.

44

9. Take the '-' tube and add 100 microliters of cell
suspension to the center of the LB '-' plate and another 100 microliters
to the center of the LB/AMP '-' plate. Using a sterile inoculating loop
spread the cells over the surface of each plate being careful not to
scratch the agar surface. Be careful to sterilize the inoculating loop

after each use.

10. Do the same with the '+' tube and spread the cell

suspensions over the surface of the plates.

11. Allow the plates to sit for a minute, then label them
with your team He. or name and place them together upside down in an

incubator for from 12 to 24 hours.

12. Examine your plates the following day and draw and record

the sizes and shapes of the bacterial colonies that grow.
The Preparation of the Laboratory Report.

I. Purpose. A brief single sentence statement of the reason

for doing the laboratory exercise.

II. Introduction. This introduction should contain
information about specific plasmids, notably pUCi8. The teacher may
supply the students with information concerning the size of the plasmid
and the genes it contains. The student should do additional research.
Students need to note the purpose of the calcium chloride in making
cells competent. And they should note the purpose of the “heat shock“.
The teacher may supplement their knowledge where they are unable to

obtain it on their own.

 

45

III. Procedures. The student will note what he has done to

perform the experiment .

IV. Data. The student will prepare a data table of the

results of his own experiment and a data table of class results.

V. Graph. The student will prepare a bar graph indicating
the class results: per cent of Luria broth agar and Luria broth
agar/amplcillin plates showing positive growth of plasmid '+' strains
and plasmid '_' strains of bacteria growing on each of the plates

prepared and used by the class.

VI. Results and Conclusion. The student will determine the
success of the experiment, individually and for the class. He will
present his results and draw conclusions concerning the growth or lack
of growth of plasmid and non plasmid containing bacteria on Luria broth

agar and Lurla broth agar/amplcillin plates.

LABORATORY EXERCISE NO. 3

DNA RESTRICTION ANALYSIS

Background Information for the Teacher.

We will restrict (cut) the DNA from bacteriophage lambda with the
restriction enzymes EcoRI and/or BamHI. There will be three
preparations by the student: one test tube containing the lambda DNA
and the restriction endonuclease EcoRI. a second test tube with lambda
DNA and two restriction enzymes, EcoRI and BamHI, and a third test tube,
the control, with Just lambda DNA and no restriction enzyme.

Phage lambda DNA is viral DNA and consists of 48,502 base pairs.

It is made up of about 50 genes: genes for head and tail proteins of the
virus, genes for attachment, integration, and excision and a gene for
lyslng the cell after sufficient viruses have been produced by the cell.

There are five specific sites where it can be cut with the
restriction enzyme EcoRI. Each site contains the DNA base sequence:

6 A A T T C and attached to this sequence is the complementary strand
C T T A A G. The restriction enzyme cuts between the two complementary
strands cutting each end first between the G and the A.

The restriction cuts occur at 21,226 base pairs (bp), at 26,104 bp, at
31,747 bp, at 39,168 bp, and at 44,972 bp. If it cuts in five places
there are six pieces of lambda DNA that result from the cuts. (Figure
1.) Students will have to know the exact number of base pairs for each
fragment of DNA, so it will be necessary to subtract two adJacent

numbers to determine the number of base pairs between them.

46

47

The DNA fragments will migrate during electrophoresis at a speed
which is inversely proportional to size. The smallest will move fastest
and farthest. Students can then determine which segnent is which by its
relationship to other fragments of larger and smaller sizes.

Fran this information students should, using two cycle semilog
graph paper, produce a standard curve for the EcoRI digest.

BamHI is a restriction enzyme that also cuts lambda DNA in five
places. It cuts, a staggered cleavage as in EcoRI. between the

sequences 6 G A T C C and its complement C C T A G G. The cut is

 

between the first and second 6 and the G A T C. BamHI restricts at
5,505 bp, at 22,346 bp, at 27,972 bp, at 34,499 bp, and at 41,732 bp.
(Figure 1) (21, 27 - 32)

When the mixed digest is produced there should be 11 pieces of
lambda DNA. Although because of the smallness of the size of some of
the bands, they may not be totally visible. And because of the
closeness of the sizes of some of the bands there may be double bands

which appear as one.

Preparation of Materials.

1. The teacher will need to have available a buffer for the
enzymes which will be added to the reaction tube before the enzyme.
These buffers are usually included separately by the supplier when the
restriction enzymes are sent. Boehringer Mannheim gives the following
recipe for the storage and dilution buffer which they send with the
restriction enzyme:

a. For EcoRi: TrisHCl, 10 mmole/llter (1.58 g) : NaCl,
200 mmole/l (11.7 9): EDTA, 1 mmole/l (0.37 g): dithioerythritol, 0.5

' 48
PROPERTIES OF'TWDlRESTRDZEEJNliflnflflfiiHEIDI;BHHEITEEIR.RESTRICTIGNWHAPS.

Restriction.Ehzyme Bacterial Source ' Reoognition.Sequenoe
‘EboRl Becherichia.ooli G»A.A'Tlr(2
CT T'A.AwG
//’\1\1iT?TTCI
C'T'T.A.A

Ikfiflnjxflfiktiflhmiof Lambda.DNA.- Ebde

Arrows indicate the position.of the recognition.sites where EboRl restricts.
Sites are.in.numberwoflbase pairs fromlthe left.end.of Lambda.DNAuwhidh is
48,502 base pairs long.

ii i i i

21,226 44,972
26,104 39,168
31,747
Restriction.Enzyme Bacterial Source: Recognition.Sequenoe

Esmiffl. lixnllus amyloliquefac1ens (3(31113C2C
(:C:1?11(;(;
Gr . GlA.T'C C
(2C11?A.G

IEsNJjLerxiiEm>cfifIambda.DNA.- Bam Bl

Arrows indicate thejposition.of the recognition.sites where Bam.H1 restricts.
Sites are in.numberwof base pairs fromthe left.end.of Lambda.DNA.

i lei/1L

5,505 22,346 34,499
27,972 41,732

 

Figure NO. 1. FEEERECTHJNIQUEBCE'IAHEDB.DNB.

 

49
mmole/l (0.08 9): Triton x-ioo (a polyoxyethylene ether, a surfactant),
0.2% (v/v) (2ml): glycerol, 50* (v/v) (500 ml): pH ca 7.

b. Hacket, Fuchs and Messing (20) offer the following
recipe in their lab manual, An Introduction to Recombinant DNA
Techniques,: for one liter of a 10X EcoRi buffer (pH7.2): 0.5 M Tris (pH
7.2) (70.2 g Tris NO] + 5.45 g Tris base): 0.1 M Magnesium chloride
(9.5 g): 0.5 M NaCl (29.25 g): 0.02 M dithiothreitol (3.09 g): and store

at -20 deg. Ce

Electrophoresis gels will need to be prepared either in class or

prior to class before the lab can be completed.

1. Preparation of concentrated TBE buffer (Tris - borate -
EDTA): Trimma base - 54 grams, Boric Acid - 27.5 grams, EDTA
(Ethylenedlamlnetetracetic acid) - .05 M at pH 8.0 - 20 ml of this to

the above.

2. Dissolve Tris (Trlzma) base and boric acid in a 500 or 800
ml beaker with stirring, pour this into a 1 liter volumetric flask or 1
liter graduated cylinder, add 20 ml of EDTA at pH 8.0 and add water to
make 1 liter. This solution is a concentrated solution: it will need to
be diluted: 1 part concentrated solution to 4 parts distilled water
to make the running buffer and the buffer in which you will mix the

agarose to make the gel.

a. To make EDTA: dissolve 0.93 grams EDTA in 50 ml of
distilled water. Use 1 N NaOH or 1 N HCl to adJust the EDTA solution to
pH 8.0. Then add 20 ml of this to the TBE buffer, as above.

3. To prepare the gels:

 

50

a. Make 500 ml of diluted buffer by taking 100 ml of

concentrated buffer and adding 400 mi of distilled‘water.

b. To make enough medium for one gel: take 0.6 grams of
agarose and add to 50 ml of dilute buffer. Heat until boiling and the
solution clears. Pour the dissolved agarose into the mold for the gel
and place the comb which will form the wells about 65 mn fran one end of

the mold.

4. Methylene Blue Stain. The gel will be stained in a

solution of methylene blue: 0.25 grams/liter of distilled water. (33)

5. A two liter beaker of ice should be obtained before hand
since students and the teacher will need to have beakers of ice

available in which to keep their reaction tubes.

6. The restriction enzymes and lambda DNA should be thawed
and kept cold on ice throughout their usage. They may be refrozen

afterwards with little loss of activity.

Time Frame.

For this laboratory exercise, about two hours of preparation
time before hand if one makes one’s own buffer and makes and pours the
gels before class. If the buffer is already on hand and is ready for
use, there is essentially no preparation time for the buffer.

About 30 minutes to one hour is needed to make the TBE buffer.

With a microwave oven each student can prepare his own gel in
about 3-4 minutes. If one prepares seven gels for a normal class. it

would take about 40 minutes to heat and pour the TBE - agarose mixture

 

51
and into the already prepared form. About 20 minutes or more is needed

for cooling each of the gels and for removing the tape and the combs

from the gels.

About 15 minutes is needed to make the methylene blue stain.
One needs to take the materials out of the freezing

compartment of the refrigerator and thaw them on ice before starting the

lab.

Problems Students Might Encounter.

If the forms for the gel are not properly taped the hot,
liquifled gel will flow out of the form. The form needs to be double
taped with ordinary masking tape, if one is using the homemade variety
of electrophoresis chamber. The cats need to be straiait up and down
and straight across in the gel, and should not be removed until

completely cool. (When the gel is cool, it will be translucent.)

Student Goals.
1. The student will make his own gel.

2. He will prepare the restriction digest and load the

restriction digest into the wells.

3. The student will connect his gel to a power source and

electrophorese his samples.

4. The student will know and understand the action of

restriction enzymes on Lambda DNA.

 

52
5. The student will relate this laboratory exercise to an
understanding of how the sequence DNA is detenmined and of the methods

used.

Background Information for the Student.

In this laboratory exercise, you will digest the DNA from the
Bacteriophage Lambda with one and then with two restriction
endonucleases. You will separate the fragnents that you digest by gel
electrophoresis.

Both restriction enzymes cut the lambda DNA in five places.
You will need to determine the number of bands of DNA which will appear
on the gel for the single digest and then for the double digest. You
will need to measure the distance each band travels from the center of

the well.
Procedures for Preparing the Restriction Digest.

1. Obtain 3 - 1.5 ml sterile micro-test tubes and label them
as follows: 'A' (EcoRi): 'B' (EcoRi/Bam H1): and 'C' (Lambda DNA).

Test tube '0' will be the control.

ALL REAGENTS MUST BE STORED ON ICE THROUGHOUT THE FOLLOWING STEPS.

2. Set up the following matrix to use as a checklist as you
add reagents with micropipetors to each reaction tube. (To reaction

tube C, 1.5 microliters of water are also added.)

BUFFER MUST BE ADDED BEFORE THE ENZYME/ENZYMES.

 

53
USE A FRESH, STERILE MICROPIPET TIP FOR EACH REAGENT.

THE FOLLOHING AMOUNTS ARE IN MICROLITER MEASUREMENTS:

A 6 6 1 5 0
B 6 6 0 1 5
C 6 6 O O

3. Pulse the micro-test tubes for 1 - 2 seconds in the

centrifuge.

4. Tap the tubes gently to mix the reactants.

5. Place the tubes in a 37 deg. C. water bath and incubate

them for 20 minutes.

AT THIS POINT THE MICRO-TEST TUBES CAN BE FROZEN AND HELD UNTIL
ELECTROPHORESIS.

6. Remove the tubes fran the 37 deg. C. water bath.

7. Using fresh, sterile micropipet tips load the entire
contents of each reaction tube into separate wells in the gel. Since we
are only loading three wells with reaction mixture, load only every

other well.

a. In loading the well, steady the pipet with both hands

resting your elbows on the table for additional support.

54

b. Be careful to get all of the contents into the well

and not to push the pipet tip through the bottom of the well.

c. Depress the pipet plunger slowly when the pipet tip
is in the well and continue to depress the pipet plunger until after the

tip has been removed from the well.

d. Use a fresh, sterile tip for each reaction mixture.

8. Connect the gels to the power source with the wells
farthest away from the positive electrode. (Migration of the DNA will
be from the negative side toward the positive side.) If you use 100
volts at about 1.5 amps, the migration will take about 1 hr. Weaker

power sources will require a longer period of time.

9. Load the marker dye mixture in the center well. This will
indicate when the electrophoresing is done. (The marker dyes are:
Xylene Cyanol which moves with the equivalency of 2800 base pairs: this
dye is an aqua- marine color. Bromophenol blue moves with the weight
equivalency of 250 base pairs: this dye is a purple color. Orange G
moves with the equivalency of 70 base pairs and is a yellowish - orange

color. (21,28)

10. After the gels have been electrophoresed, they will need

to be stained and destained. (33)

a. Making sure the power source is disconnected.

carefully remove the gel from the form in the electrophoresis chamber.

55

b. Place the gel in a container suitable for staining:

the gel should remain flat.

c. Pour the methylene blue stain over the gel and stain

for 15 minutes.

d. After 15 minutes carefully pour the stain into a
beaker (It can be reused.), and cover the gel with distilled or tap

water and allow the gel to destain for two minutes.

e. Discard the destain and destain again for another two

minutes. And then destain a third time for two minutes.

f. Carefully wrap the gel in plastic wrap or seal the
staining chamber with plastic wrap and place in the refrigerator for 24

hours.

11. Remove the gel fran its container and examine over a
light box. If no light box is available, holding the gel carefully to
the window will enable students to see and measure the distance the

bands have moved.

a. Measure the distance each DNA band has moved from the
center of the well and record the distances. Record the distances in

millimeters.

b. Sketch the gel on plain, white paper with a pencil
and try to indicate the relative heaviness of the bands by making them

darker or lighter with the pencil.

56
12. Wrap the gel and place it back in the refrigerator for
future reference after you have made the drawings and measured the

distances the bands have moved.

i3. Obtain a sheet of two cycle semi-log graph paper (Figure
No. 2) and plot the size of the bands in base pairs (Y axis) against the

distance in mm moved (X axis)..

a. Plot the distance moved by the bands in the EcoRi
lane of the gel against the number of base pairs. (Remember that the
smallest number of base pairs will be the band farthest away from the
center of the well.) This line you draw between the points will
constitute your standard curve. You can use this curve to determine
base pair sizes of other bands that have moved known distances, but

whose sizes you may not know.

Questions for the Student.

1. What is the size of each of the fragnents of the EcoRi

restriction?

2. What is the size of each of the fragments from the

EcoRi/Bam H1 restriction?

3. Plot the points on the standard curve which represent the
number of base pairs of each of the bands of the mixed digest. Read
down to the 'X' axis and determine the distance each of them should have
moved. Do the distances on the graph coincide with the distances
actually moved? If there is an error calculate the per cent error for

each band.

57

4. Draw a straight line to a scale of 1 cm a 4 kllobases
(4000 base pairs). Indicate with arrows, as precisely as possible, the
location of the recognition sites (restriction digest sites) for EcoRi.

Indicate the size in base pairs of each of the fragments.

5. Do the same thing for the mixed digest.

6. Explain how you might make a restriction map of Lambda DNA

from these two pieces of information.

 

 

Dmmmmm.
Fiqureib. 2. Aplotofthemarkerdves.

SUMMARY AND DISCUSSION

The laboratory exercises that were used in the classroan were:
Laboratory Exercise No. 1, Transformation of E. coli: Laboratory
Exercise No. 2, Colony Transformation: and Laboratory Exercise No. 3,
DNA Restriction Analysis.

During the laboratory exercises, students worked in pairs. Each
student was required to write a laboratory report. Students wrote
laboratory reports on Laboratory Exercises 1 and 2, and were asked to
answer scaffolded type questions following Laboratory Exercise No. 3.
See Appendix B for the questions.

The answers to the questions were such that 80 t of the students
showed a clear understanding of what had been done in the lab i.e.
received a grade of 21 or better out of 25 possible points.

For all other laboratory exercises a laboratory report was required
in which the student wrote an Introduction, with a Purpose, a set of
Procedures, a Data Table or Tables, and a Graph or Graphs and included
some sort of Conclusion.

Procedures from these laboratory exercises and Laboratory Exercise
No. 4 as well as additional methods were used by students who completed
science fair proJects for the Northeast Ohio Regional Science Fair held
at Case Western Reserve University in March of this year.

Laboratory Exercise No. 1 was done as it was written with small
revisions by a senior student, Kimberley Anne 8.: she received a first
place for her work and her explanation which had to include a complete

understanding.

59

60

Another science fair proJect, done by two students, Thomas P. and
Anne Marie P., a senior and a Junior, respectively, involved procedures
from laboratory exercises 2, 3, and 4 and the use of agar plates with
I.P.T.G. and X-Gal. The title of this more elaborate proJect was: Gene
Fragnent Insertion in Plasnid Puc 18.

For this proJect they received first place also. They were invited
to make a presentation at the Cleveland convention of the American
Society of Medical Technologists. They received cash prizes from the
ASMT and a number of items of equipment were donated to our school by
the members of the convention who rewarded them with a standing ovation.

In Thanas P.’s case, his work with the science fair proJect has
influenced his choice of career. Ann Marie P. is doing research now,
under my supervision, on the Human Genome ProJect and hopes to be a
Westinghouse Scholar applicant for next year.

As a final note to the year, thirteen students received
questionnaires to be filled out after they had cmpleted their second
semester final exam. (34) They were told to conplete the questionnaire
to the best of their ability.

All students, without exception, were able to demonstrate a
satisfactory understanding of the chemical composition of DNA. That is.
they knew the names of the bases, the base pairs, the location of the
linkage bonds to the sugar and the location of the phosphate to
deoxyribose sugar bonds.

To the question, 'Did the construction of the cardboard model of
MA help in your understanding? (Students were asked to construct a DNA
model showing adenine, thymine, cytosine and guanine on one side with

their conplements on the other side with 3’ to 5’ bonding on one side

61

and 5’ to 3’ bonding on the other.) eleven students answered that it
helped them to understand the 3’ and 5’ bonding of the phosphates to the
sugars, and the bonding of the bases to each other. Two students felt
that the construction of the model was confusing to them and not
helpful.

To the question, 'We did two labs involving plasmids. Do you
recall what happened in each of them? Were they successful?

The general response was: 'We inserted plasnids into bacteria to
make them immune to antibiobics.‘ Students used terms such as 'inJected
plasmids into bacteria", "plasmids can transform bacteria“ and I'lf
successful plasmids that are placed in a cell will express genes
contained on the DNA'

All students agreed that the labs were successful, and they were.
Two of the students showed confusion between names of plasmids and
strains of bacteria: one student recalled that 'we inserted a plasmid
into agar that was inoculated with amplcillin to see if it was immune to
amplcillin.‘l

To the question, 'We did a lab involving the cutting of a piece of
DNA with restriction enzymes. Do you recall what was cut? where was it
cut? with what was it cut? where did the restriction enzyme cane fran?
why restrict a piece of DNA?

The responses were not as complete as I would have liked.
Nevertheless they showed an acceptable level of comprehension. 'Lambda
DNA was cut with a restriction enzyme called EcoRi. We cut up the DNA
to help determine the lengths of the seanents..' All thirteen students
stated that we cut the DNA with EcoRi. Nine students noted with clear
understanding that Lambda DNA was 'cut'. Eight of the students noted

62
that the restriction enzyme came from Escherichia coli. Nine students
noted that the reason for cutting the DNA was to 'help map the
chromosome“ or 'to determine where nucleotide sequences were located" or
'to study the fragnents easier‘ or 'to discover the lengths of the
fragnents' or 'to gain a further understanding of each particular band'
or similar answers. Five students remenbered that we also used the
restriction enzyme, BamHi, although none could state that it came from
Bacillus amyloliquifaceans - one student did mention
'Bacilles' Three students were able to remember that EcoRi cut at the
sequence G A A T T C and its compliment and indicated the correct 'cut'I
'wlth a sketch. One student noted, “Bacteriophage lambda DNA which is
48,502 base pairs. The cutter was pUCiB (restriction enzyme) (to) study

and differentiate base pair length.‘

It has been the purpose of this thesis to present laboratory
exercises that would be invocative and provocative and 'do-able'. The
degree of understanding that might grow from these labs is indicated by
the answers to the questionaire presented above.

At no time did I feel that I had achieved 100% success in my
teaching. But I am heartened by what the students did learn, as of June
8, 1990.

Two of these labs were done for the first time this year as
classroom activities: the amplcillin transformation lab with bacterial
strain mm294, and the lab involving restriction digests. Laboratory
exercise No. 1, has been done for at least four years and with
increasing success each time. The success is related to, in part, the
deeper understanding of the lab experience that I, myself, develop from

study and working with the students. The two, new introduced labs will,

63
in time, increase in adaptability to student ability to comprehend and
to use the knowledge in the real world.

These laboratory exercises_dig stimulate discussion and motivate
classroom work. They_did familiarize the student with the basics of
biotechnology. They_di_d begin to answer the questions, 'How are these
things done?':'How do these things work?‘ and 'How do we obtain meaning
from this technology?"

These exercises, I believe, bridge the gap between knowing and
doing, between knowledge and understanding, between imagination and
reality.

There is a serious need for students to understand the knowledge
which they obtain. Most of them do not. Even in an Honors Biology
class it is impossible to have complete understanding of any concept.

The most important tasks in Education are for the teacher to
motivate, to stimulate and to create an attitude for a desire to learn.
The appreciation for the seriousness of the learning concept is
sometimes, but certainly not always felt by the student. There is
always the one or two and sometimes three or four students who respond
to the teaching concept with enthusiasm and desire. These are the
students for whom the teaching may provide the inspiration for a future
career.

For the other students, it can only be hoped that enough of an
intelligent understanding may result so that future decisions may be

directed by reasonable choices.

APPENDIX A

APPENDIX A

LABORATORY EXERCISE NO. 4.

THE ISOLATION OF PLASMID DNA
FROM BACTERIAL CELLS

(E.COLI . STRAIN MC 1061, CONTAINING THE SYNTHETIC PLASMID.
pBR 322.)

BACKGROUND INFORMATION.

A plasmid is a piece of double stranded DNA that occurs
naturally in the form of a ring or circle. It exists, mostly, in
bacterial cells, and is considered an endosymbiont. It possesses the
genes that control its own replication number. In other words it
replicates using the bacterial enzymes and metabolic systems. It can
also be cut into pieces with special enzymes called restriction enzymes.

If the plasmid ring is cut in one place the ends can be
'engineered' to be 'sticky' and a gene with compatible 'sticky' ends can
be mixed with the plasmid culture and will attach itself chemically to
the cut ends. The circle will close and the plasmid, now a vector
carrying a gene, can be put back into the same bacterial cell or into
another one.

There are a number of reasons for isolating plasmids from
bacterial cells. Perhaps the most cannon use of plaanids is as cloning

vectors. ThE technology for isolating plasmids and inserting genes into

64

65

the piasnids has been available for some time. Once the plasmids have
been reintroduced into the bacterium. the genes inserted into them can
then be cloned by the bacterium as it makes multiple copies of the
plasmid.

The plasmids from the bacterial cell are first restricted with
a restriction enzyme. Next a gene or piece of DNA of choice is allowed
to interact with the restricted filament and ligated with DNA ligase.
The plasmid can then be put back into the bacterial cell. The bacterial
cells will be cultured and grown to produce the product of the gene that
was inserted into the plasmid which is replicated many times. This is
cloning.

The plasmids will be isolated in one laboratory period and
subJected to electrophoresis analysis in the second period.

In this laboratory exercise we are shall isolate the synthetic
plasmid, pDR 322, from a strain of bacterial cells called 801061. We
shall use gel electrophoresis to demonstrate its isolation.

The plasmid will then be subJected to digestion with the
restriction enzyme, EcoRI; the cut and uncut plasmids will be
differentiated by gel electrophoresis.

Plasmid pBR322 is a circular, double stranded DNA. It was
constructed fran parts of several other naturally occurring plasnids.

It consists of 4363 base pairs with one EcoRI restriction site. It has
an amplcillin resistant gene that produces the enzyme beta lactamase
that destroys ampicillin and also a tetracycline resistant gene. (13.20)

One of the first products to be produced commercially in this

way was the human growth hormone. It was produced from a gene cloned

into a strain of E.coli through the use of a plasmid vector.

66

Perhaps the first maJor product having a truly momentous
impact was the cloning of the human insulin gene. This was available
for the first time commercially in December of 1983. The product is
called Numuiin and it’s availability abrogates the necessity of using

bovine (from cows) and/or porcine (from pigs) insulin.

Before piaanid vectors can be used, their structures have to
be known. One has to know where to cut the circular plasmid so as not
to interfere with its ability to replicate itself, mobilizing the
enzymes of the bacteria.

An additional note: the first living organism was patented by
a research scientist by the name of Ananda Chakrabarty. On June 16,
1980 the Supreme Court of the United States granted Dr. Chakrabarty and
his colleagues the right to patent a form a soil bacterium called
Pseudomonas. This bacterium was especially selected for the plasnid
which enabled the bacterium to use a maJor environmental poison, 2.4,5-T
(2.4,5-trichlorophenoxyacetic acid) the principle ingredient in Agent
Orange, as a source of food.

In this case, the plasmid occurred naturally. It was not
engineered. Dr. Chakrabarty and others simply had to grow enough
cultures of these bacteria until they found the one that had the I'right'
plasmid. Then it was Just a matter of growing the bacteria in large
quantities.

In this way the Love Canal in Niagara Falls, New York, a
neighborhood that had to be evacuated because of the environmental
toxins that were buried near the homes of the residents, has been

restored to normal use.

67
BEHAVIORAL OBJECTIVES:

i. The student will learn the use of the clinical centrifuge

in biotechnology.

2. The student will reinforce his previous learning about

plasmids.

3. The student will learn how plasmids are extracted for use

in genetic engineering.

4. The student will reinforce his learning and practice of

sterile technique.

5. The student will be able to determine the maJor events in

each step of the lab procedure and their purposes.

6. The student will become familiar with and practice genetic

engineering techniques.

7. The student will reinforce and develop a deeper

understanding of the relationship between bacteria and plasmids.

8. The student will have a deeper understanding of genetic

engineering.

TIME REQUIRED:

Teacher Preparation: If the teacher uses freeze dried or
lyophilized cultures of bacteria, it will involve activating the
bacterial culture and allowing it to incubate for about 12 hours.

Fifteen minutes are required for inoculation.

 

68
Preparation of the STET (10% sucrose. 5% Triton X-100, 50 mN
Tris-HCl pH 7.5. 50 mN EDTA) solution and Storage Buffer solutions takes
about 30 minutes.
Student Lab Time: The student can stop at the end of each
centrifugation, store the contents in the refrigerator and proceed the

following day to the next stage. About 40 minutes each day for three

days.

Material Preparation:

1. STET: To prepare 50 ml: 0.33 grams TrisHCl: .05 grams
Tris (base): 0.93 grams EDTA: 5 grams sucrose: and 2.5 ml of Triton

X-100. AdJust PH 7.5 with 0.1 N NaOH.

2. Storage Buffer: TE buffer for storage: 0.13? grams of
Tris 861: 0.016 grams of Tris (base): 0.037 grams of EDTA. Dissolve in
sterile distilled water and dilute to 100 ml. AdJust to pH 7.4 with 0.1
N NaOH. (21)

Activation of the Bacterial Cultures.

1. Prepare one liter of Luria broth and put 200 ml of broth

in five 250 ml Erlenmeyer flasks.

2. Cover each flask with a sponge stopper such as those used

in culturing fruit files.

3. Sterilize at 121 deg. C for 15 minutes.

4. Cool the flasks and using sterile technique inoculate

bacterial cultures into one or more of the flasks.

69

5. Insert a sterile 1 ml pipet through the sponge stapper and

into the inoculated Luria broth solution.

6. Connect the pipet to an air pump. using sterile neoprene
tubing. Place cotton in the tubing to trap any particles that might be
pumped into the culture from the air pump. (This is a substitute for a
shaking water bath usually required when growing bacterial cells to

their most positively growth state.)

7. Place the flask in a water bath at 37 deg. C. and allow to
incubate with the bubbling of the air through the culture until the
bacterial culture reaches mid-log growth. Mid-log growth occurs when
the optical density in a spectrophotometer at 550 nanometers reaches

between 0.35 and 0.55.
Procedures: (35)

1. Carefully withdraw 100 microliters of plasmid-harboring

bacterial cells from the bacterial cultures.

2. Suspend the cells in 50 microliters of cold STET in a 1.5
nulllliter microfuge tube. Tap the tube with your finger until all of

the cells are suspended and the solution becomes cloudy.

3. Add 4 microliters of a fresh lysozyme solution. (Ten

mg/ml in water.)

4. Suspend the microfuge tube in a boiling water bath for 40

- 60 seconds.

70
5. Centrifuge the microfuge tube for 10 minutes at room

temperature.

6. Remove the supernatant with a micropipet and place in a

clean microfuge tube.

7. Add 40 microliters of isopropanol to the supernatant.

8. Chill for 5 minutes in a dry ice-ethanol bath.
re
i
9. Centrifuge in the refrigerator for 5 minutes to collect %
the precipitate (This should be the plasmids). up]

 

10. Remove the supernatant and dry the pellet under vacuum.

11. Resuspend the pellet in 20 microliters of sterile

distilled water.

12. The pellet may then be restricted with the restriction

enzyme EcoRi and electrophoresed in an agarose gel.

Questions and Answers.

Question 1. How can we determine whether or not we have

plasmids? Name two ways.

Answer 1. We can determine whether or not we have plasmids by
electrophoresis or by taking bacterial cells that lack the plasmid.
making them competent and putting the plasmids into them. Then grow

these bacteria on plates containing amplcillin and tetracycline.

71
Question 2. What formed when the isopropyl alcohol was added

to the solution? What was the precipitate?

Answer 2. A cloudy, gelatinous, white precipitate formed.

The precipitate was DNA.

Queston 3. What was the first centrifugate?

Answer 3. The first centrifugate was bacterial cells.

Question 4. What should the final centrifugate have been?

Answer 4. The final centrifugate should have been plasmids

and perhaps some RNA.

Question 5. Why do we use sUch rigid sterile technique?

Answer 5. The use of sterile technique is to prevent the

destruction of the DNA by nucleases.

SOURCES OF MATERIALS.

Sigma Chemical Co.
P.0. Box 14508

St. Louis, Mo. 63178
1-800-325-3010

Boehringer Mannheim Biochemicals
P.0. Box 50414

Indianapolis, Ind. 46250
1-800-262-1640

LABORATORY EXERCISE NO. 5.

THE ISOLATION OF PLASMID DNA
FROM BACTERIAL CELLS
(E. COLI, STRAIN MC1061, CONTAINING THE PLASMID PBR322)
An Alternate. Shorter hethod

INTRODUCTION TO THE LAB.
See introduction to Laboratory Exercise No. 4.
PITFALLS AND CAUTIONS.

Precise timing of centrifugation will be important. Therefore
it is important to know the speed of the centrifuge. For example if the
lab procedure calls for centrifuging the sample at 15,000 rpm’s for 10
minutes, that would mean centrifuging for 15 minutes at 10.000 rpm’s or
50 minutes at 3000 rpm’s.

Dry ice should be obtained ahead of time: it is important that
the plasmids and other DNA be quickly frozen before the final
centrifugation.

Always, when dealing with DNA, absolute sterile technique must
be used. The work area should be thoroughly washed with soap and water
and alcohol, gloves should be worn and frequently replaced or frequently
washed. Anything that touches the bacterial culture or DNA should not

touch anything else beforehand.

MATERIALS.

72

 

73
i. A bacterial culture of cells at mid-log growth that has

been stimulated to produce multiple copies of plasmids.

2. Centrifuge tubes. sterilized and with tops covered with
aluminum foil, or otherwise sealed. Depending upon the size you will
need about 12 of these. Microfuge tubes are best, but only if you have

a microfuge.

3. Clinical centrifuge or faster centrifuge. if possible.

4. STET. A solution of 10% sucrose. 5 2 Triton X-100 (a
detergent), 50 mh Tris HCl pH 7.5 and 50 mN EDTA. (AdJust the pH with a
0.1 N NaOH solution.) Fifty milliliters of this should be enough for

several teams.

5. Dry ice.

6. Two ten milliliter sterile test tubes in which to pour and

mix the products.

7. A vortexing machine, if one is available. Otherwise,

mixing can be done by flicking with the fingers.

8. Ordinary ice in a beaker.

9. A boiling water bath in a small beaker: this comes later
on in the procedure so it can be prepared as the materials are being

centrifuged.

10. Lyophillzed lysozyme which is suspended in the STET

solution at a concentration of 10 milligrams/milliliter. One milliliter

74
of this should be enough because you will use only 200 microliters or

0.2 ml of this for each team.

11. Hicropipets and their plunger: these will need to have
been sterilized beforehand. The sizes to be used varies from 20
Inicroiiters to 200 microliters. If a pipetman is available, use it with

sterilized tips.

12. Isopropyl alcohol. You will use 3 mls of this per team.

13. A dessicating chamber or vacuum chamber.

14. 12 Sterile 15 mi centrifuge tubes.

PROCEDURES: (35)

1. Obtain a bacterial culture that has been grown to mid—log
stage in a medium treated to stimulate rapid plaanid replication. You

will need about 60 ml.

2. Place the culture medium into 4 - 15 ml sterile centrifuge
tubes and centrifuge the cells for 5 minutes at 10,000 rpm. This should
be done at 4 deg. C. The centrifuge can be placed in a refrigerator and

centrifuged inside the refrigerator.

3. Discard the supernatant liquid.

4. Add 1 ml of STET to each of the centrifuge tubes and mix
the centrifugate with the STET by flicking with a finger or vortexing

until the cells are resuspended.

 

7S

5. Pour the contents of all four centrifuge tubes into a
small. 10 ml sterile test tube and add 200 microliters of lysozyme

suspended in STET. Mix by flicking with your finger.

6. Incubate on ice for 5 minutes.

7. Transfer the tube to a boiling water bath and incubate for

exactly 1 minute.

8. Put the tube back in the ice and cool the sample.

9. Place the sample in a sterile centrifuge tube, balance the

 

tube in the centrifuge with a centrifuge tube on the opposite side of
the centrifuge that has a volume of water equal to that of the material
in the sample tube. centrifuge the sample at 10,000 rpm for 15 min, or

preferably at 15,000 rpm for 10 minute.

10. Carefully pour the supernatant liquid into a clean.
sterile test tube. DO NOT TRANSFER ANY OF THE GELATINOUS MATERIAL AT
THE BOTTOM TO THE NEXT TUBE.

11. To the supernatant in the clean. sterile test tube add 3

ml of isopropyl alcohol. Mix by flicking.

12. Incubate the sample on Dry Ice for 15 minutes or until

the sample is completely frozen.

13. Centrifuge the sample after pouring back into a clean.
sterile centrifuge tube at 15,000 rpm for 15 minutes or at 10,000 rpm

for 22.5 minutes, or at 3000 rpm for 60 minutes.(21)

76
14. Carefully remove the supernatant with a micropipet. Try
not to disturb the centrifugate at the bottom...it contains the

plasmids.

15. Dry the pellet in a vacuum chamber or in a dessicating

chamber or air dry the sample.

16. Resuspend the pellet in 300 microliters of TE buffer or
sane other storage buffer as indicated for the particular type of

plasmid you are isolating.

17. Store the plaanid in the storage buffer at 4 deg. C.

FOR FURTHER INVESTIGATION.

1. Continue this lab by electrophoresing the sample and see

if you really successfully isolated plasmids.

2. How would you determine the concentration of the plasnids

in your sample?

3. What would be your next steps in preparing a sample for

cloning a gene into the plaanid?

 

LABORATORY EXERCISE NO. 6

METHOD FOR DETERMINING
THE SENSITIVITY OF METHYLENE BLUE
FOR STAINING DNA

Background Information for the Teacher:

Lambda DNA is the 'guts' of Bacteriophage lambda. It is one
of the best understood of the viruses. There are many electron
photomicrographs of this phage so its appearance is clearly understood.

The entire sequence of nucleotides of the DNA is known and it
contains 48,502 base pairs having a molecular weight of 3 X 10E?
Daitons. (36) The 4.8 kilobase structure contains about 50 genes.

These genes direct the bacterial cell to make: head proteins, tail
proteins, attachment, integration, and excision proteins as well as
lysis proteins.

The lambda DNA integrates itself into the bacterial genome
(chromosome) and then directs the rest of the formation of complete
viruses finally producing the enzymes that lyse the cell releasing about
a hundred completed viruses into the environment to attack other
bacterial cells.

When you observe the results of the experiment, you should
detect a single band. If you do observe two bands. then there should be
a smaller. fainter band farthest from the well and a larger. heavier
band closer to the wells. The smaller band will be a supercoiled form

of the viral DNA. It takes the shape of a 'T' and flows through the gel

 

78
pores faster than the circular form. Most of the lambda DNA will be of

the circular form which will appear as a denser band.

Before the gels are fixed and stained, the marker dyes should
be measured in terms of how far they have moved from the wells. Plot
the distance the bromophenol blue and xylene cyanol have moved on two
cycle semilog graph paper: the first section for hundreds and the
second for thousands. You will be graphing distance on the “X' or
horizontal axis and base pairs on the 'Y' or vertical axis. (Figure No.
2) If you extrapolate the line, i. e. extend it beyond the points you
have graphed you will be able to determine the equivalent base pair
values for lambda DNA. All you will need to do is measure the distance
the lambda band has moved from the well. Find that place on the line
you have drawn on the graph and then read straight over to the 'Y' axis.
This should tell you within reasonable accuracy the number of base pairs

in the viral DNA.

Target Group:

This lab is designed for an Honors Biology or an Advanced
Placement class in biology. It can be used in either a first or a

second year class.

Course and Topic the Lab Would Reinforce:

1. This lab would be suitable for a module on molecular
genetics because it deals with DNA.
2. It would be suitable for a module on viruses or

microbiology because it deals with the DNA of the lambda phage.

79
3. It would be a suitable module as an introduction to the
biochemistry of DNA.
4. It would be suitable to reinforce learning in the metric
system because it deals with nanograms. micrograms. milliliters, and

microliters.

Purposes of the Lab:

1. The purpose of the lab is to familiarize students with
electrophoresis and to determine the sensitivity of DNA to methylene

blue stain.
Special Equipment and Supplies Needed:

1. One needs electrophoresis equipment, whether the home made
variety or the more sophisticated equipment such as Gelteach. The source
of these could be the kinds of kits made up at the Michigan State
University Molecular Biology Institute. Modern Biology in Dayton,
Indiana produces good. simple and practical electrophoresis equipment.
as does Geltech of Salem, Ohio.

2. One needs lambda DNA and the chemicals for buffers:
Agarose, Boric Acid. Tris and Tris-HCl. EDTA, Methylene Blue. Sources
of these could be Sigma Chemical Co., or Boehringer Mannheim or other
chemical companies.

3. One needs pipettlng devices and small sterile test tubes

or centrifuge tubes.

Time Frame:

 

80
i. The gels and buffer need to be prepared beforehand: this
would take about 40 minutes. (This could be done before or after
school.)
2. At least one period of preparation and explanation and

demonstration of technique before the lab period.
3. Then about 40 minutes to prepare the dilutions and load

the gels for electrophoresing. (The teacher would have to take
responsibility for turning off the apparatus when the electrophoresis is
finished.)

4. The gel may then be put into a fixative overnight and
stained and destained on the following day.

5. Staining and destaining take about 30 minutes. The gels
are then wrapped in Saran and placed in a refrigerator until the
following day.

6. They are then measured with a metric ruler and the
distances from the starting point noted. This three day long proJect
includes instruction and preparation. (If the teacher decides to do his
own dilutions, etc., a two successive lab period interval is

sufficient.)

Content Skills and Knowledge Required of the Teacher.

1. The teacher needs to understand the use of the equipment.
2. There would need to be some knowledge of lanbda DNA.
3. Their would need to be sane understanding of marking dyes

and how they work.

4. The teacher would need to know how to make and

electrophorese gels.

81
5. It would be necessary to have the ability to graph with
semilog graph paper.
6. The teacher would also have to have a good understanding

of I'sterile technique'.

Problems Students Might Have and Suggestions for Overcoming Thmm.

1. Mixing solutions correctly, if they are to mix them. The
teacher might have to watch over their procedures and correct mistakes

as they are made.

2. Loading the wells carefully so as not to overload or
dispense the materials into the buffer instead of the well. The teacher
might demonstrate good technique and again watch them as they inoculate

the wells.

3. Using sterile technique and avoiding contamination.
Encourage students to wash frequently, to wear rubber gloves and to
avoid touching contaminating surfaces with the equipment. DNA
nucleases are everywhere. on skin, table tops on any surface that cmes
human in contact with our bodies. They will catalyze the hydrolysis of
DNA so that bands will not appear on the gels: you will get nothing that

is useful.

4. Following the proper lengths of time in staining and
destaining and handling the gels properly. Oversee this part of the lab

diligently.

Guide for Preparation of Materials:

82
1. Glycerlne-sterile distilled water mix (20% v/v): Place
0.4 ml of glycerlne in 1.6 ml of sterile distilled water. Mix this into

sterile test tube. Prepare one 2 ml test tube per team.

2. Electrophoresis gels will need to be prepared either in

class or prior to class before the lab can be completed.

3. Preparation of 1 liter of concentrated TBE buffer (Tris -
borate - EDTA): Trlzma base - 54 grams, Boric Acid - 27.5 grams. EDTA
(Ethylenediaminetetracetic acid) - .05 M at pH 8.0 - 20 ml of this to
the above.

Dissolve Tris (Trizma) base and boric acid in a 500 or 800 ml
beaker with stirring, pour this into a 1 liter volumetric flask or 1
liter graduated cylinder. add 20 ml of EDTA at pH 8.0 and add water to
make 1 liter. This solution is a concentrated solution: it will need to
be diluted: 1 part concentrated solution to 4 parts distilled water
to make the running buffer and the buffer in which you will mix the

agarose to make the gel.

4. To make EDTA: dissolve 0.93 grams EDTA in 50 ml of
distilled water. Use 1 N NaON or 1 N HCl to adUust the EDTA solution to
pH 8.0. Then add 20 ml of this to the TBE buffer. as above.

5. To prepare the gels:

a. Make 500 ml of diluted buffer by taking 100 ml of
concentrated buffer and adding 400 ml of distilled water.
b. To make enough medium for one gel: take 0.6 grams of

agarose and add to 50 ml of dilute buffer. Neat until boiling and the

 

83

solution clears. Pour the dissolved agarose into the mold for the gel
and place the comb which will form the wells about 65 mm from.one end of

the mold.

Alternatives for Organizing, Dispensing and Removal of Materials.

The students can do as little or as much of the work as the
teacher feels necessary. The teacher can do all of the preparations.

if he or she desires it.
Disposal of Used Material:

The electrophoresis buffer can be saved to be reused.
(Buffers have been used three times with no loss of effectiveness.) The
gels can eventually be discarded in the waste basket after they have

been used.
Procedure:

1. Obtain a fresh sample of lambda DNA of known quantity. (An
example might be a sample that is obtained from.Boehringer Mannheim that
contains 0.25 mlcrograms/microllter in a quantity of 100 microliters of
storage buffer. The amounts will be clearly marked on the container
which contains the lambda DNA.)

Since we are going to be measuring amounts of DNA in
nanograme. it is worthwhile to note that that there are 1000 nanograms

per microgram. Thus 0.25 micrograms is 250 nanograms.

2. Dilute the sample so that small enough quantities of

lambda DNA can be carried in larger quantities of solution.

84
a. The manner of diluting a sample containing 0.25
mlcrograms/ microliter would be: Take 5 microliters (This will contain
1.25 micrograms or 1250 nanograms of lambda DNA.) of the sample
indicated above and dilute to 1 milliliter (1000 microliters) in a

mixture of sterile distilled water and glycerine. (36) Now each

microliter of sample will contain 1.25 nanograms of lambda DNA. (The
glycerine will add to the specific gravity of the water and enable the
sample to sink to the bottom of the well and stay there. For the
preparation of the glycerine. see the Preparation of Materials, No. i.
as indicated previously in this exercise.)

3. Set up the following matrix to follow in placing the

samples in the wells of the gel:

Well No. Microliters Glycerine- Quantity

of lambda water of Lamda
DNA dilution Diluent DNA in ng.

1 24 6 30

2 5 25 6.25

3 10 20 12.5

4 2 microliters of dye mixture.

5 12 18 15

6 14 16 17.5

7 20 10 25

8 22 8 27.5

85

4. The gel should be electrophoresed at 100 volts for
approximately 1 hr. [The marker, Orange G. will indicate when the
electrophoresis is complete (equivalent to 70 bp) when the dye has

reached the end of the gel.)

5. The gel will be removed from the electrophoresis chamber
(Disconnect the power source first!) and placed in a container for

staining.

6. Pour methylene blue stain over the gel and allow to stain

with gentle agitation for 15 minutes.

7. Pour off the stain (It may be reused.) and destain with

tap or distilled water at 2 minute intervals each three times.

8. Wrap the gel carefully in plastic wrap and place in the
refrigerator overnight to allow the stain to concentrate in the DNA
bands.

Results and Conclusion.

A class discussion can provide an effective conclusion to this
laboratory exercise. The background information can be discussed and
reviewed. The student can be quizzed on the background information and

the purposes and results of the lab.

APPENDIX B

 

APPENDIX B

HONORS BIOLOGY
RESTRICTION DIGEST LABORATORY
QUESTIONS

Write the answers in complete sentences skipping a line after each
answer. Incorporate the meaning of the question into the answer and

whereever appropriate underline the words that constitute the answer.

1. What is EcoRI?

2. What is phage lambda DNA?

2a. What is the size of lambda DNA?

2b. Draw a picture of a bacteriophage virus. labelling all the parts.
(Text reference is a good picture of bacteriophage lambda.)

3. What does a restriction enzyme do?

4. How many EcoRI restriction sites are there on Lambda DNA?

4a. Describe the nucleotide sequence where EcoRI cuts. (Text Reference.)
4b. Where does EcoRI cut Lambda DNA? (Draw a straight line to a scale
of 1 Om = 4 kllobases and indicate with arrows as precisely as possible
the places where the restriction enzyme cuts the line.)

5. What is the size of each of the fragnents of an EcoRI cut of lambda
DNA? (Notes)

6. Where does EcoRI get its name?

7. Where does BamNI get its name?

7a. Now'many BamHI sites are there on lambda DNA?

86

87
8. Where does BamNI cut Lambda DNA? (Answer this in the same way as

question 4b.) (Notes.)
9. Describe the nucelotide sequence where BamHI cuts. (Text Reference)
10. How many pieces would there be in a mixed digest (EcoRI and BamHI)?

11. Draw a straight line map of lambda DNA, to the same scale as

indicated above, and indicate with an arrow where each cut would be in
a mixed digest. On the bottom of the line map indicate the total length
from the start of the lambda DNA of each cut. Indicate above the line
the actual size in base pairs of each of the fragments. (3 points)

12. From the semilog graph that you have already made of your own
individual EcoRI digest and the distance moved by each of the bands
(your standard curve) determine the distances that the bands of of
digest of BamNI should move. (Use red dots and red circles around the
dots and indicate the size in kllobases, example: 3,600 bp = 3.6 Kb.
Round the numbers off to the nearest 0.1) (3 points)

13. Determine the distances that bands would move in a mixed digest of
EcoRI and BamHI. (Use green dots and green circles around the dots. and

indicate the size in kllobases.) (5 points)

BIBLIOGRAPHY

BIBLIOGRAPHY

1. Watson. James D.. “The Human Genome ProJect:
Past. Present. and Future“, Science. April 6. 1990.

2. Consumer Reports. The telltale gene.
Consumer Reports. 55: No. 7, pp. 483 -488. July 1990.

3. Winter. Ruth. New miracle drugs. Family
Circle. September 4. 1990. pp.51 - 55.

4. Anderson, Charles W.. “Teaching for meaningful
and self regulated learning of science.", produced for the
Institute on Research on Teaching. College of Education.

Michigan State University, March 1988.

5. Anderson. Charles W.. “Combining research
and practice to improve science teaching.“ Lecture
presented as part of Frontiers of Science Program.
November, 1988.

6. Waldrop, M. Mitchell. "Spontaneous order.
evolution and life. Science. March 30. 1990.

7. Felsenfeld. G. ”DNA.". Scientific American.
October. 1985. pp. 58-68.

8. Darnell. J. E. Jr.. "RNA.". Scientific
American. October. 1985, pp. 68 -88.

9. Wessells. N. and Hopson, J. Biology, Random
House. Inc.. New York. 1988.

10. Watson. J.. Tooze. J. and Kurtz. D.

W. by w. B. Freeman 00.. New
York. 1983.

11. Stryer. Lubert, Biochemistry. Third Edition.
W. H. Freeman Co., New York, 1988

12. Champe and Harvey._B_i_g_gn_emi_s_t_Ly_._ Lippincott’s
Illustrated Reviews. J. B. Lippincott. Philadelphia, 1987.

88~

89

13. Balbas. Paulina. Xavier Soberon. Enrique
Merino. Mario Zurita. Hilda Lomeli. Fernando Valle. Noemi
Flores and Francisco Bolivar. Plasmid vector pBR322 and its
special-purpose derivatives - a review. (1986) Gene 50.3-40.

14. Kemtech Educational Corporation. Introduction
to Natural Genetic Engineering. Kemtech Educational
Corporation. 1980. Clncinatti.

15. Novick. R., “Plasmids.“ Scientific American.
December 1980. pp. 102-27.

16. McIntosh. J. R. and Koonce. M. P.. “Mitosis“.
Science. November 3, 1989.

17. Laskey. Ronald A.. Fairman. Michaela P.. and
Blow. J. Julian. “S Phase of the cell cycle“. Science.
November 3. 1989.

18. Watson. J. and Crick. F. H. C. “Molecular
structure of nucleic acids, a structure for deoxyribose
nucleic acid.“ Nature. April 25. 1953.

19. Judson. H. P.. Ibe_Eigb1b_Dax_o£_§neaLLQn.
Simon and Schuster. New York. 1979.

20. Hackett. Fuchs and Messing. An_1ntngdu§tign_tg
. BenJamin/ Cummings Pub.. Menlo

Recombinant_DNA_Ie§bnigues
Park. Ca. 198?. (A laboratory manual.)

21. Anderson. John N...A.Labonatonx.§ounse_in

CollesezLexei_Bioiogx. Modern Biology. Inc.. Dayton. Ind..
1989.

22. Pursel. V. G.. et al. “Genetic engineering
of livestock.“ Science. June 16. 1989.

23. Gasser. Charles S. and Fraley. Robert T.
“Genetically engineering plants for crop improvement.“
Science. June 16. 1989.

24. Avery. O. T.. C. M. MacLeod and M. MacCarty.
“Studies on the chemical nature of the substance inducing
transformation of pneumococcal types." J. Exp. Med.. 79:
137-158 (1944).

25. Hershey. A. D. and M. Chase. "Independent
functions of viral protein and nucleic acid in growth of
bacteriophage.“ J. Gen. Physiol.. 36: 39-56 (1952).

26. Micklos. David. _DNA_SeLence‘_A_Einst
Labonaton1_Course_in_Recombinant_DNA_Ieehnoiosx. Cold

90
Spring Harbor Laboratory and Carolina Biological Supply Co.,
1987.

27. Freifelder. David. _Mglegglan_higlggy. Jones

and Bartlett Publishers. Inc.. Portola Valley.
CA. 1983.

28. Sueiter. Clarence. WW
. Michigan State

University. 1989.

29. Hedgepeth. Joe. Howard M. Goodman. and Herbert
W. Boyer. DNA nucleotide sequence restricted by R1
endonuclease. Proceedings of the National Academy of
Science. Vol. 72. No. 9. pp 3310-3314. Sept. 1975.

30. Polisky. Barry. Patricia Greene. David E.
Garfin. Brian J. McCarthy. Howard M. Goodman. and Herbert W.
Boyer. Specificity of substrate recognition by the EcoR1
restriction endonuclease. Proceedings of the National
Academy of Science. Vol. 69. No. 11. pp. 3448-3452. Nov.
1972.

31. Kessler. Christoph. Peter S. Neumaier and
Werner Wolf. Recognition sequences of restriction
endonucleases and methylases - a review, Gene, 33 (1985) pp.
1-102.

32. Tikchonenko. T. I.. E. V. Karamov. B. A.
Zavizion and B. S. Naroditsky. Eco R1 activity: enzyme
modification or activation of accompanying endonuclease?.
Gene, Vol. 4. pp. 195-212. 1978.

33. Sharp. Charles R., Procedures for staining DNA
in agarose gels with methylene blue. High School Biology and
Chemistry Teachers Research Manual. Michigan State
University. East Lansing. Michigan. July 1989.

34. Sharp. Charles K.. Unpublished questionnaire
to students. June 1990.

35. Kaguni. John. Personal communication, Michigan
State University. East Lansing. Michigan. June-July 1989.

36. Sueiter. Clarence. Personal communication,
Michigan State University. East Lansing, Michigan. July
1989.

GENERAL REFERENCES

1. Meselson, M. and F. W. Stahl. “The replication of DNA in
Escherichia coll." Proc. Natl. Acad. Sci. USA. 44: 671-682 (1958).

2. Wilkins. M. H. F.. Stokes. A. R., Wilson. H. R. 'Molecular
Structure of Deoxypentose Nucleic Acids.‘l Nature, April 25. 1953.

3. Franklin. R. and Gosling. R. 'Molecular Structure in Sodium
Thymonucleate.‘ Nature, April 25. 1953.

4. Cantor. Charles R., “Orchestrating the Human Genome ProJect“.
Science. April 6. 1990.

5. Anderson. Charles W.. 'Assessing Student
Understanding of Biological Concepts.‘ presented at the
national invitational conference, High School Biology Today and
Tomorrow. Washington. D. 0., October 10-12. 1988.

91