THE
Transformer
protocol
Student s guide
Inside a living cell, plasmids can be copied, but they rely upon
Bacteria everywhere
the molecular machinery of the cell carrying them to do this
Bacteria are the commonest living things on Earth. Despite the job. So, in the right conditions, a test tube of bacteria will grow
fact that they are microscopic, together they weigh more than all and multiply, but a test tube of plasmids will just sit there. Neither
the planet s plants and animals combined. Because some bacteria plasmids nor the DNA from which they are made are alive.
can cause disease they tend to receive a bad press. However,
most bacteria are harmless and they perform a vital role by Plasmids are duplicated separately from the bacterial
helping to recycle elements, breaking down dead organisms to chromosome, and not just when the cell divides.
simpler materials so that they can be used again. They also make Consequently one bacterial cell may hold many identical
nutrients available to other living things by, for example, extracting copies of plasmids.
nitrogen from the air for protein production by plants. Even
old
some of the bacteria that inhabit our intestines are useful  for
strand
example, Escherichia coli (E. coli for short) provides us with essential
vitamins.
new strand
Cells express themselves
old
Bacterial cells are enclosed by cell membranes containing the
strand
cytoplasm. Most bacteria also have a cell wall surrounding the
membranes. Some have one or more thread-like hairs (flagella)
new strand
Nucleotide
to propel themselves along.
Inside the cell there s just one chromosome, in the form of a
ring. The chromosome is made of DNA, which carries the
instructions needed for making proteins. A set of instructions
for making all or part of a protein is called a gene. The genes in
bacteria are arranged one after the other, like beads on a string,
5'
3'
along the chromosome. The genetic  recipe of E. coli is 4.6 million
base pairs long  that s enough information for making about
Phosphate
3 000 proteins. Fortunately, every E. coli has about 15 000 protein-
making  factories (ribosomes) where the genetic instructions
can be translated and the genes  expressed .
Deoxyribose sugar
Just before bacteria divide to form two new cells (which, in
favourable conditions, can happen every 30 minutes or so),
3'
5'
the whole chromosome is copied so that each new cell ends
Complementary
up with a full set of instructions.
base pairs
Chromosome
DNA structure and function. The structure of DNA can be
likened to that of a twisted rope ladder. At the sides are chains of
Plasmids
sugar and phosphate molecules. In the centre are the bases: adenine
(A), guanine (G), cytosine (C) and thymine (T). Hydrogen bonds
between the bases hold the two helices together A always pairing
with T, and C always pairing with G. Through the double helix, genetic
information is copied from one cell or generation to the next. When
DNA is duplicated, the two complementary strands are untwisted,
cytoplasm
and nucleotides are brought into place alongside each of the old
Ribosomes
sites of protein
strands to form new ones. The new copies are checked to ensure
synthesis
accurate copying. Several different enzymes perform these tasks.
Cell
wall
The order of bases in the DNA specifies the sequence of the
Cell membranes
Not to scale
amino acids in proteins. Three bases in a row (a triplet) specify
each amino acid. A particular gene  a length of DNA 
Some of the main components of a bacterial cell. determines the structure of all or part of a specific protein.
A little extra DNA helps
Bacterial  sex
Sometimes other smaller rings of DNA, called plasmids, are also Plasmids are sometimes transferred between bacterial cells in a
found in bacteria. Plasmids carry just a few genes. They are not essential natural  mating process called conjugation. This is as close as
for the bacteria, but they may help them to survive in some bacteria get to sex (bacteria don t actually reproduce by sexual
environments. For instance, some plasmids help the bacteria that means). Some bacteria can also pick up extra DNA from their
carry them to resist the toxic effects of heavy metals, or to live on surroundings. This is not as easy as it sounds, as DNA molecules
particular nutrients. can be very large and they have to cross the bacterial cell
2
INTRODUCTION
membrane somehow. Special proteins are needed to help ferry
the DNA across and since only a few species have these proteins,
this sort of DNA transfer (called  transformation ) is thought to
be fairly rare in nature.
Entry restrictions
Even if DNA (a plasmid or smaller fragments) makes it across
the cell membrane, many bacteria can destroy the incoming
genetic message. This is particularly important if the DNA is
from a bacteriophage  a type of virus that preys upon bacteria.
The bacterial defence mechanism consists of enzymes 
restriction enzymes  whose job is to  restrict the invasion of
viruses. There are many hundreds of different restriction
enzymes that  recognise particular sequences of DNA and cut
it up, like very precise molecular scissors. The bacteria s own
A computer model of restriction enzyme EcoRI.
DNA escapes damage by  disguising the sites where the
This enzyme is obtained from Escherichia coli, strain RI. The
restriction enzymes cut  by adding methyl ( CH3) groups to
DNA is shown at the top of the picture, as a ball-and-stick
the DNA bases.
model, looking down the axis of the double helix. The enzyme
wraps around the DNA and moves along it,  searching for a site
Bacteria perform useful tasks
where the enzyme can cut. EcoRI cuts at G AATTC.
It s not only bacteria that benefit from restriction enzymes.
Parts of the enzyme s structure are shown here: flat sheets (beta-
They re now an important tool used by medical researchers and
pleated sheets) in yellow; coils (alpha helices) in pink and turns
biologists of all kinds.
in blue and white.
With restriction enzymes almost any section of DNA, and
The data for this image was obtained from the Nucleic Acid
consequently any single gene, may be cut out at will. The end of
Database (http://ndbserver.rutgers.edu/NDB/ndb.html) where
one DNA molecule can be joined to another that s been cut
you can view the computer model in three dimensions. (Search
with the same enzyme. The combined DNA can then be put
for the protein with the ID code PD0055.)
into a cell in which it may be expressed and duplicated so that it
passes from one cell division to the next. For microorganisms,
one of the most successful methods for transferring genes is to
 paste them into plasmids. The result is a ring of  recombinant
A cause for concern?
DNA that can be put into a bacterium. Specialised plasmids can
be used to ferry genes from bacteria into yeast cells or even plants.
Genetic modification of this type has already given us new and
improved medicines and vaccines, and altered crop plants with
In this way, microbes can be  re-programmed to make a wide variety
new characteristics. As a research tool, genetic modification has
of useful substances including vaccines, insulin and other hormones
helped us to study our own genes and those of other species,
and materials such as plastics, helping to reduce the consumption of
and this work will have major consequences for human health.
valuable oil reserves. Microbes can be altered to clean up toxic wastes,
protect us from food poisoning and help in biological research.
However, many people are concerned about the potential dangers
of  meddling with genes and doubts have been raised about the
How bacteria are genetically modified. First, the gene
wider economic, social and environmental impact that such work
of interest is cut out, using carefully selected restriction enzymes.
may have.
Next, some plasmid DNA is cut with the same enzymes. The
two fragments are joined using DNA ligase to form a recombinant
So that you can better understand and assess this technology,
plasmid. The plasmid DNA is put into a suitable host strain of
this kit provides a practical activity which will allow you to
bacteria. The transformed (genetically-modified) cells are selected.
investigate some of the key techniques, coupled with a discussion
The plasmid DNA is copied within the bacteria, and the proteins
task so that you can begin to think about and critically assess
it encodes are produced.
some of the wider implications of genetic modification.
DNA
DNA ligase bonds
DNA fragments
New protein made
by bacteria
Plasmid put into
Restriction
Gene
bacterium
enzyme
isolated Plasmid
Gene spliced
cuts DNA
copied
into plasmid
at specific
sites
Cell divides
Bacterial
chromosome
Transformed cell
3
The plasmid The bacterium
lactase gene
lactase enzyme breaks down X-Gal
(which has a similar structure to
chromosome
lactose sugar), forming blue indigo dye
the arrows show the direction
in which the genes are read
p2k
6.3 kb
kanamycin resistance gene
the enzyme encoded by this Escherichia coli K12 DH5Ä…18
gene stops the antibiotic a feeble, harmless strain that has been
kanamycin from working developed specially for DNA work.
Unlike  normal bacteria, it cannot
origin of replication thrive outside the laboratory. It has
this is needed so that the plasmid can be copied, no plasmids of its own, and cannot
using the bacterium s DNA copying machinery pass on plasmids to other cells.
Chill both tubes on ice for about Add all of the bacteria from one tube to the plasmid DNA.
10 minutes. Close the tube and mix gently by tapping the side of the tube.
3 4
This prepares the cells to take up
plasmid DNA by punching holes
through the cell membrane.
Bacteria
Bacterial cell
membrane
Plasmid
Outer
solution
membrane
10 µL
containing 60 ng
Peptidoglycan
of plasmid DNA
Inner
Channel
membrane
through
membrane
Add 250 µL of recovery broth, warmed to 37 °C, to each tube. Using a new pipette each time, add 120 µL of bacterial cell
suspension to each plate. (One drop from a disposable
Mix it into the bacterial suspension by tapping the tubes gently.
6
7
pipette is exactly 40 µL.)
Incubate the tubes for at least 15 minutes at 37 °C.
This period gives the transformed bacteria time The bacteria will survive better if the plates are pre-warmed to 37 °C.
to express the kanamycin resistance gene on the
plasmid.
When, subsequently, the cells are grown on plates
that contain kanamycin, they are able to thrive.
Squeeze this part
3 drops
of the bulb for
(120 µL)
250 µL
better control
Squeeze the
stem here for
250 µL
250 µL better control
3 drops
(120 µL)
Bacteria
LB agar
+ plasmid
+
kanamycin
Recovery
+
broth Bacteria X-Gal
Chill the transformation buffer on ice for Scrape 2 3 large colonies or an equivalent amount from the
at least 5 minutes before you start. stock plate. Take care not to dig into the agar as you do this.
1 2
Mix the cells into one tube of chilled transformation buffer.
Repeat the process with the second tube.
250 µL
250 µL
Twiddle
vigorously
Sterile
to dislodge the
transformation
Stock bacteria
bacteria and
buffer
These should be grown
disperse them
3 4 days in advance,
at 18 25 °C
Heat shock the bacteria in both tubes for exactly
30 seconds at 40 42 °C, then return them to the
What s going on?
5
ice for 1 2 minutes so that the heat shock stops.
Bacterial cell
Floating
holder
p2k plasmid DNA
The transformation buffer contains positively-charged ions e.g., Ca2+.
These ions bind to the negatively-charged phosphate groups of the
DNA and the phospholipids of the cell membranes, shielding their
negative charges. This allows the DNA to approach the cell
membrane and to pass through channels in it that are formed
where the inner and outer cell membranes meet.
The heat shock is thought to help this process.
Spread the culture all over the plates, using a sterile Colonies transformed with plasmid DNA are blue.
spreader for each one. Let the culture soak into the agar This colour is an indigo-like dye, which is made by linking
8 9
if necessary. Incubate the plates overnight, inverted, at 37 °C. two molecules that result from the breakdown of X-Gal.
BIOHAZARD
TRANSFORMED
CELLS MUST BE
DESTROYED
Rotate the Petri dish as AFTER USE
you move the spreader
back-and-forth
Lactase
Br
splits the
Seal plates
X-Gal here
Cl
with tape
HOCH2
Label the base of each
plate with your initials,
O
O
the date and type of HO
X-Gal is:
bacteria used
NH
5-bromo-
OH
4-chloro-
Two of these
3-indolyl-
units join to form
²-D-galactoside
an indigo-like dye
OH
Transformation
Plasmid
Transformation is the uptake and expression of DNA by cells.
DNA
Although some bacterial species can take up DNA from their
environment naturally, most cannot. Cells that can take up
DNA in this way are termed  competent . However, natural
transformation is a relatively rare event. Most bacteria have
not evolved the membrane proteins that allow foreign DNA
to be  recognised and absorbed. Without these special
mechanisms, gene-sized DNA molecules are too large to
diffuse or be transported through the cell membranes.
Chemical transformation
Escherichia coli (the common gut bacterium) is the best-understood
and most studied organism on Earth. It is not naturally
competent. However, in 1970 a method was developed to
artificially transform this and other bacteria. Today, such
transformation is a key process used in the genetic modification
of microbes.
Plasmid DNA entering a bacterial cell. Both DNA and
The original method involved suspending rapidly-dividing cells
the bacterial cell membrane normally have a negative charge,
in a  transformation buffer of cold calcium chloride solution
so they repel one another. However, solutions of positive ions
then subjecting them to a brief heat shock in the presence of
can be used to neutralise both the DNA and the cell surface,
the DNA to be taken up. Later it was found that other divalent
allowing the plasmid DNA to enter cells through pores in the
cations (such as magnesium, Mg2+, manganese Mn2+, and barium
bacterial membranes. This is a schematic diagram, showing the
Ba2+) all had a similar effect to that of calcium ions. The exact
general principle involved. In reality, the relative sizes of
mechanism of DNA uptake is poorly understood. One
components and the distribution of charges will differ from that
hypothesis is as follows:
shown here.
Cooling the cells to 0 °C stabilizes the normally fluid bacterial
Selectable marker genes
cell membranes.
Positively-charged ions in the transformation buffer (e.g., Ca2+)
The transformation process is very inefficient and only a small
are then able to bind to the negatively-charged phosphate
proportion of the cells treated will take up plasmid DNA.
groups in DNA and the phospholipids of the cell
Therefore a means of selecting those cells that have been
membranes, shielding their negative charges.
transformed is needed. Antibiotic resistance markers are often
This allows the plasmids to approach the membrane and
used for this purpose. The p2k plasmid includes a kanamycin
the channels through it that are formed where the outer and
resistance gene (called APH(3')-I) put there specifically to act as
inner cell membranes meet.
a genetic marker.
The heat shock helps to force the plasmids through the
channels by creating a thermal imbalance on either side of
The strain of Escherichia coli used here is incapable of
the cell membranes.
hydrolysing the sugar lactose, because it lacks the gene for
the enzyme lactase (²-galactosidase). However, the p2k
plasmid carries this gene, and if a bacterium takes up the
Other transformation methods
plasmid it gains the ability to hydrolyse lactose.
In the last decade, other methods of transforming bacteria have been
The colourless compound X-Gal is hydrolysed by lactase,
introduced. These include, for example, subjecting the bacteria to
yielding galactose and an insoluble indigo dye. The dye is
brief pulses of very high voltages. This technique  called
precipitated within the bacteria, enabling X-Gal to be used
electroporation  punches holes in the bacteria through which DNA
as an indicator of lactase activity (transformed cells are blue).
can enter. This is the most efficient method devised so far.
Why can t lactase action and this blue colour alone therefore be
used to select transformed cells?
A procedure called particle bombardment or ballistic impregnation
is also sometimes used to introduce DNA into
Cells with resistance plasmids are normally disadvantaged
cells. With this method, the DNA is first
compared to their neighbours without them. In the presence of
stuck onto minute tungsten or gold
appropriate antibiotics however, such plasmid-bearing cells thrive
beads. Using a  gene gun , the DNA- while their less well-endowed neighbours perish. In this way,
coated particles are fired into the cells.
selection pressure is applied to maintain the plasmid in the
This technique is often used for
population of cells.
transforming plant cells.
Without that pressure, the few transformed cells would be swamped
 Chemical transformation remains popular by their untransformed neighbours, and the plates would be covered
however, because it is simple, inexpensive and does by a uniform  lawn of ordinary bacterial cells rather than individual
not require specialist equipment or materials. blue colonies.
6
ADVANCED INFORMATION
amino
How kanamycin acts on bacteria
acid
Kanamycin kills bacteria by stopping protein synthesis at the Transfer
RNA
ribosomes. Unlike several other antibiotics (e.g., penicillin or
small (30S)
subunit
ampicillin) kanamycin kills all cells, rather than just those that are
actively growing. Consequently, bacteria transformed with p2k
AUG
require a short  recovery period before they are transferred onto
anticodon
CCA
CCA
kanamycin-containing plates. This allows the resistance marker
gene to be expressed, and ensures that the transformed cells are
not killed on contact with kanamycin-containing growth medium.
large (50S)
subunit
While it would be more practical to use a marker (like ampicillin
GGC
GGC
GGC
GGC
resistance) that did not need a recovery period, there are several
AUGCCGGGUUACUUA
AUGCCGGGUUACU
AUGCCGGGUUAC
5' AUGCCGGGUUAC 3'
compelling reasons for using kanamycin and kanamycin resistance
Messenger RNA
r RNA
r RNA
NA
codon
(see below).
Bacterial ribosome
Protein synthesis at a bacterial ribosome. Successive
How the resistance mechanism works
transfer RNA (tRNA) molecules, each carrying an amino acid, are
brought to the ribosome according to the genetic code of the
There are very many different mechanisms that confer resistance
messenger RNA (mRNA). The amino acid residues are strung
to the effects of kanamycin and related antibiotics. At least seven
together to make a protein. Kanamycin interferes with this process
of these mechanisms work by transferring a phosphate group
by binding irreversibly to the 30S sub-unit of the bacterial ribosomes.
onto the kanamycin, altering its structure.
The kanamycin/ribosome complex is able to start protein synthesis
by binding to mRNA and the first tRNA. However, the second
The APH(3')-I gene encodes an enzyme that catalyses the transfer
tRNA cannot bind, and the mRNA/ribosome complex dissociates.
of a phosphate group from ATP onto a hydroxyl ( OH) group
of kanamycin. The modified antibiotic which results is unable
to bind to the bacterial ribosome, so the antibiotic is inactivated.
This particular resistance mechanism has been found to occur
Further investigations
in about 50% of gram-negative bacteria.
There are numerous variations on the technique described
The enzyme which confers the resistance is relatively unstable,
here which can be attempted to try to improve the
and it is inactivated by increased temperatures or pH changes.
efficiency of transformation.
Its requirement for ATP means that this enzyme can only
function in environments where that compound is abundant
You could investigate the effect of changing:
(e.g., inside cells).
" the age of the host cells used;
" the amount of plasmid DNA used;
" the duration of the heat shock;
Use of kanamycin and the resistance gene
" the intensity of the heat shock
(i.e., its temperature);
Many transformation experiments use plasmids that confer
" the duration and/or temperature of the
resistance to the antibiotic ampicillin. However, in the
recovery period.
construction of p2k we have chosen to incorporate a kanamycin
resistance marker for several important reasons:
To determine the effect of altering these factors, it is useful
to calculate the transformation efficiency. This is expressed
unlike ampicillin, kanamycin is very seldom used to treat human
as the number of transformed colonies produced per µg
disease, having been superseded by other drugs;
of plasmid DNA. The transformation efficiency can be
it is needed in small amounts in culture plates (a tenth of the
calculated as follows:
concentration normally used for ampicillin);
unlike ampicillin, kanamycin is not absorbed by the gut (in
1. Calculate the mass, in µg, of plasmid DNA used in Step 4.
clinical use, it has to be injected). Therefore the safety hazard
Concentration of the plasmid DNA x Volume of plasmid
posed by accidental ingestion is reduced;
DNA solution used = Mass of plasmid.
ampicillin resistance enzymes (²-lactamases) often provide
2. Determine how much of the cell suspension you spread,
resistance to many other similar antibiotics whereas this
in µL, onto the LB/antibiotic/X-Gal plate. Volume of
particular kanamycin resistance gene affects a lesser range of
suspension spread / Total volume of suspension = Fraction
antibiotics of limited use. APH(3')-I confers resistance mainly
of cell suspension spread.
to kanamycin and neomycin;
3. Calculate the mass of plasmid contained in the cell
for several other technical reasons, the use of kanamycin
suspension spread onto the LB/antibiotic/X-Gal plate. Mass
resistance markers is now widely accepted as safe, even in food.
of plasmid x Fraction of cell suspension spread = Mass of
Scientists disagree, however, about the wisdom of using
plasmid spread.
ampicillin resistance markers in such products. This is mainly
4. Determine the number of colonies per µg of plasmid
because of the slight risk that the marker gene will pass into
DNA. Colonies counted / Mass of plasmid spread (µg)
other organisms, giving them the ability to withstand not only
= Transformation efficiency.
ampicillin, but other antibiotics too.
7
UAC
Donor
Transfer of antibiotic resistance
F+ strain
1 F plasmid unwound
Resistance to the effects of some important
and replicated
antibiotics is now widespread in several species
of disease-causing microbes. Special steps have
The F plasmid
been taken to ensure that the procedures
has about 30 genes.
followed in this practical exercise do not
Sex Bacterial
These include genes
pilus chromosome
contribute to this problem. for making the pilus
and for a mobility
(mob) protein.
Antibiotic resistance has evolved to give
The mob protein
conducts the plasmid
bacteria the ability to thrive in environments
through the pilus.
2 Single-stranded DNA
containing antibiotics secreted by other
passes into recipient cell
microorganisms. Resistant bacteria often
F plasmid
through pilus
produce proteins that inactivate specific
antibiotics or stop them from working in some
way (e.g., by preventing their transport into
Recipient
bacterial cells). Resistance genes are often
F- strain
carried on plasmids, which can pass from one
bacterial cell to another of the same or a related
3 Complementary
species by a natural  mating process called
DNA synthesised,
conjugation.
forming new F plasmid
During conjugation, a tube or pilus is formed
between adjacent cells, through which the
plasmid passes. The genes required for the
Conjugation between bacteria
formation of the pilus are also carried on a
Conjugation involves one-way transfer of DNA from a donor ( male ) to a recipient
plasmid (an F or fertility plasmid).
( female ) strain, through a tube called a sex pilus. The pilus is made by the donor
cell using genes encoded by a specialised F plasmid. The F plasmid can also
The bacterial strain provided in this kit
temporarily become part of the bacterial chromosome. There it can pick up extra
does not carry an F plasmid. Nor does it
genes that are carried with it when it later passes into another cell by conjugation.
carry any bacteriophages which may pick
F plasmids with these extra genes are called F' plasmids.
up DNA and transfer it to other cells.
Biological containment
Deleted genes
The bacterial strain used in this procedure is non-pathogenic. It has been selected
For a plasmid to travel through a pilus, two
for its suitability for work of this type and its inability to survive outside the
additional requirements must be met. The
laboratory.
plasmid must possess a gene encoding a
mobility protein (mob) and have a nic site. The
Over many years of laboratory use and millions of generations, changes have also
mobility protein nicks the plasmid at the nic
occurred in the lipopolysaccharides on the outer membrane of this bacterial strain.
site, attaches to it there and conducts the
These changes mean that it is not possible for this strain of E. coli to colonise
plasmid through the pilus. p2k has neither a
the mammalian gut.
nic site nor the mob gene.
Physical containment
This means that once it has been
introduced into a bacterial cell by artificial In addition to the biological containment measures described above, the practical
means (transformation) the plasmid procedure requires that good microbiological practice is followed to ensure that
cannot naturally transfer (by conjugation) the microorganisms are physically contained during the investigation and destroyed
into other cells that do not possess it. afterwards.
Mass Volume
1 gram (g) = 1 000 milligrams (mg) 1 litre (L) = 1 000 millilitres (mL)*
1 milligram (mg) = 1 000 micrograms (µg) 1 millilitre (mL) = 1 000 microlitres (µL)
1 microgram (µg) = 1 000 nanograms (ng)
NOTE
Some people prefer to use the cubic decimetre (dm3) and cubic
Nucleic acid
centimetre (cm3) in preference to the litre and millilitre, as S.I.
1 kilobase (kb) = 1 000 bases
units for volume are derived from those for length.
1 megabase (Mb) = 1 000 kilobases (kb)
National Centre for Biotechnology Education, The University of Reading, PO Box 228, Whiteknights, Reading, RG6 6AJ.
8
Telephone: [phone] Fax: [phone] eMail: [e-mail] Web: www.ncbe.reading.ac.uk
Copyright © Dean Madden, 2000 ISBN: 0 7049 1372 0
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