WHOLE EVOLUTION ALMANAC
DO-IT-YOURSELF MUTATION KIT
ORCHESTRATING EVOLUTION AT YOUR KITCHEN
TABLE
The idea of orchestrating evolution yourself seems like heady stuff indeed. But
with
our Do-It-Yourself Mutation Kit, described in the pages that follow, you
should be able to
observe the process at your kitchen table. We'd like to start
you off with some basic
biology: Individual organisms and entire species change
as a result of mutations in genes.
You might think of a genetic mutation as a
spelling mistake within the subunits that
compose the gene. Since most mutant
organisms have trouble adjusting to the environment,
it's a good thing that
mutations are rare. Of course, every so often a mutant is better
suited to a
given habitat than its ancestors. When that happens, the mutant organism will
have more offspring than its nonmutant siblings. As evolution takes its course,
the
mutants will thrive.
To see the process in action, all you've got to do is follow the
instructions
below. Your guide for this journey of exploration will be Saccharomyces
cerevisiae,
a common yeast. Since the yeast grows rapidly, you will be able to
observe the results of
each experiment in two to three days,
To order the kit, send a $20 check or money order to
molecular biologist Hemant
Chikarmane at the Marine Biological Laboratory, Woods Hole, MA
02543.
INSTRUCTIONS
After you have received your kit, open it up and make sure all the
materials are
there. You should find five petri dishes labeled as follows: ADENINE
NEGATIVE A
and ADENINE NEGATIVE B (these two plates lack adenine, an essential nutrient);
COPPER A and COPPER B (these two plates contain copper salt, which can kill the
yeast); and
MASTER, a master plate that contains a cocktail of nutrients on
which all forms of yeast
can grow. You should also find a tube of yeast
culture, four sterile spreaders, four
sterile pipettes, and 40 sterile
toothpicks. Keep the kit in the refrigerator until you
decide to use it. For
each experiment, take out only those items that you actually need.
To help you with your experiments, get hold of a marker, as well as paper and
pencil to
keep track of your results. We also suggest that you go to the
drugstore and buy some
antibacterial soap, antiseptic, and ordinary bleach (ask
the pharmacist to suggest
appropriate brands). A small container for waste
would help, too.
Before you begin, read
the instructions once through and keep these basic
laboratory principles in mind:
Cleanliness
and sterility. Make sure you work at a clean table with as little
draft as possible. Wipe
the table clean with an antiseptic such as Lysol before
and after you work. Since the air
contains bacteria and fungi that can
contaminate your experiment, be careful not to open
your petri dishes unless you
are working with them. Since your fingers also have all sorts
of bacteria on
them, do not touch the sterile surface of the plate with your fingers.
Transfer
of organisms should be done only with the sterile pipettes or sterile
toothpicks.
After each experiment, wash up with antibacterial soap.
Spreading culture on the plate. To
isolate mutants that have evolved, you must
spread yeast from the culture uniformly on the
petri dish. To do so, place the
petri dish on the table and lift the cover with your left
hand, placing it
beside the dish. Pick up the culture tube of yeast with your left hand
and
shake it (figure 1). Then unscrew the top and place that on the table, too.
Pick up a
sterile pipette, squeeze the top, and dip the tip into the yeast
culture tube (figure 2).
Slowly release the bulb to draw yeast culture into the
pipette. Hold the tip of the
pipette about a quarter inch above the center of
the open plate. Gently squeeze the pipette
bulb so that two to three drops fall
on the surface (figure 3). Discard the pipette into a
waste container filled
with bleach. Cap the tube. Now pick up the spreader and hold it so
that the
short arm gently touches the surface of the petri dish. Spread the liquid
evenly
over the surface (as shown in figure 4) and carefully rotate the plate.
Growing yeast.
After you have spread the culture on your plate, leave it alone
at room temperature (70-75
degrees F or 22-25 degrees C) for one to three days.
(Keeping the petri plate at 85 degrees
F or 30 degrees C will promote faster
growth.) Through the course of these instructions,
this will be referred to as
incubation. Yeast will first appear as pinhead-sized dots and
may ultimately
assume a diameter of an eighth of an inch.
Transferring yeast from plate to
plate. Open a petri dish and, using a sterile
toothpick, remove a yeast sample about the
size of a pinhead (figure 5). Open
the second petri dish and touch the tip of the
toothpick to the surface. Slide
the toothpick gently, making a streak about half an inch
in length. Discard the
toothpick and do not reuse.
THE EXPERIMENTS
1. Selecting for mutants
that regain a lost function. Normal yeast cells have
the ability to make their own
adenine, an essential component of DNA. The yeast
strain in your tube, however, has a
mutation in the gene called ade 1 (for
adenine). As a result of the mutation, an important
enzyme is no longer active
and the yeast cannot make adenine. Without adenine, yeast
cannot replicate
(unless you supply the adenine yourself). However, if you place these
mutants on
your petri dish, they will still produce the cascade of chemicals leading up to
(but not including) the crucial enzyme. The chemical that accumulates when the
enzyme
cannot be produced is red; therefore, the colony that you will plate out
and incubate in
your petri dish will appear red. In this experiment, you will
start with red yeast and
mutate some organisms back to the original form -white
yeast with the ability to produce
adenine. Experimental procedure: Shake up your
tube of yeast and put three drops of yeast
culture on the petri dish marked
ADENINE NEGATIVE A. Spread the drops carefully and
uniformly with the spreader.
Then incubate the plate for two to four days, until white
colonies--the new
mutants--start to grow. The mutants arose spontaneously and, according
to
current evolutionary thought, have been "selected" by the environment to
survive.
Refrigerate this plate and save it for experiment 3.
2. Selecting for mutants that develop
resistance to a Poison. In this
experiment You will isolate mutants that can resist the
lethal power of copper
salt. Compounds of metals like copper, lead, and mercury are toxic
to cells
because they block the action of proteins and enzymes. Some cells randomly
develop
mutations that lead to resistance to metal salts. In the case of yeast,
these mutations
work by overproducing a protein that binds tightly to the metal,
preventing damage to the
cell. Experimental procedure: Spread three drops of
yeast from your culture tube onto the
plate marked COPPER A and incubate for two
to five days, You should see small colonies of
copper-resistant yeast forming.
Wait until the colonies grow to about an eighth of an inch
in diameter. Then
refrigerate the plate and save it for experiment 3.
3. Proving that all
the mutations are transmitted to offsPring whether or not
they are advantageous to-the
organism. In this experiment you will grow your
mutant yeast cells on the MASTER dish,
which will enable all your cells to grow
equally well. Experimental procedure: Gather the
two petri dishes you used in
experiments 1 and 2 and place them beside you on the table.
Take your marker
and, turning over the MASTER dish, draw a line down its center so that you
divide it in half. Mark one area ADENINE and mark the other area copPER. Now
take the
plate labeled ADENINE NEGATIVE A and choose a colony at random. Pick
up that colony on the
tip of a sterilized toothpick and transfer it, in the form
of an individual streak, to the
area marked ADENINE on the MASTER dish. Create
four more adenine streaks on this area,
then store the ADENINE NEGATIVE A plate
in the refrigerator. Now streak the
copper-resistant mutants from the COPPER
plate onto the area marked COPPER in a similar
fashion. Incubate the Plate for
one to two days, and note that the streaks will grow up
into elongated colonies.
These colonies grow even though the mutants no longer have any
particular
survival advantage.
4. Showing that mutations affecting one trait arise
independently of mutations
affecting another. Experimental procedure: Remove from the
refrigerator the
plates labeled ADENINE NEGATIVE B (this plate contains no adenine) and
COPPER B
(this plate contains copper salt). Take your marker, turn the plates over, and
draw a line down the center of each dish, as you did with the MASTER plate in
experiment 3.
As you did in experiment 3, label one half of each plate ADENINE
and label the other half
COPPER. Now place the MASTER dish in front of you.
Using sterile toothpicks, transfer
material from the COPPER section of the
MASTER dish to each of the two areas labeled COPPER
on the two new Plates.
Transfer material from the ADENINE section of the MASTER dish to
each of the two
areas labeled ADENINE. Incubate these two dishes. Note that the mutant
colonies
isolated from the ADENINE segment of the MASTER dish will grow on the second
ADENINE
NEGATIVE plate but not on the COPPER plate. The mutant colonies isolated
from the COPPER
segment of the MASTER dish will grow on the second COPPER plate
but not on the ADENINE
NEGATIVE plate, Consider the history of these yeast
colonies and you will reach one
conclusion: The yeast cells have retained their
mutant traits throughout each and every
transfer. These colonies should grow
quickly, since the mutants adapted to these two
plates have already evolved
during experiments 1 and 2.