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Protocol 1.4: Isolation of Auxotrophs–Replica Plating, Toothpicking, or Screening on 2 EM Plates
TRANSPOSON MUTAGENESIS OF Escherichia coli
9 Mutagenized E. coli cells prepared in Protocol 1.3 that are tetracyclineresistant colonies o n Ltetl 5 agar plates
9 Ltetl 5 agar plates
9 minimaltetl 0 agar plates
9 2 EMtetl 0 agar plates
9 sterile toothpicks (To sterilize toothpicks, autoclave for 20 min at a fast
and dry setting.)
9 sterile velvets" Square pieces of cloth about 89in. larger than the petri
plates with a thick nap, such as velvet or velveteen. (As an alternative
to velvets, sterile circular pieces of filter paper with a diameter about
2 mm less than the inside diameter of a petri dish bottom may be used.)
9 platform to put velvets on for replica plating
9 sterile test tubes and rack
9 lX VBC broth, a minimal broth
1. Select Ltetl 5 plates grown in Protocol 1.3 that have between 50 and 125
2. Carefully replica plate the colonies onto a minimal plate and another
Lte t p l a t e .
a. Place a sterile velvet on the replica plating platform.
b. Press a n Lte t agar plate with colonies onto the platform. Apply gentle,
uniform pressure to the bottom of the petri plate to ensure that all
colonies on the agar surface touch the velvet. Remove the agar plate.
c. Carefully press a minimaltetl 0 agar plate to the velvet. Again, using
gentle pressure, make sure all areas of the agar surface make contact
with the velvet. Mark the top of the plate with a permanent marker.
Remove the agar plate.
d. Carefully press a new Lt~u5 agar plate to the velvet. Mark the top of
the plate with a permanent marker. Remove the agar plate.
3. Incubate the plates at 40~ overnight.
4. The next day, compare the growth of bacteria on both plates. An auxotroph will not grow on the minimal plate, but will grow on the Lte t
5. When an auxotroph is found, locate the corresponding colony on the
Lt~t plate. With a sterile toothpick or an inoculation loop, transfer some
of the bacterial colony to a n e w Ere t plate. Streak for single-colony
isolation. Incubate the plates at 40~ overnight.
PROTOCOL 1.4: ISOLATION OF AUXOTROPHS
6. Retest that the colony isolated is an auxotroph by streaking single
colonies on a minimalt~t~ 0 agar plate.
Note: When replica plating, use agar plates that are thoroughly dry.
If plates are too wet, the colonies can smear together. Do not apply too much
pressure when transferring the colonies or colonies may run together.
1. Choose Ltetl 5 plates grown in Protocol 1.3 that have too many colonies
to use for replica plating, but still have separate, distinct colonies.
2. Using a sterile toothpick, touch a colony.
3. With the same toothpick, make a 88 streak on the surface of a minimaltetl 0 agar plate.
4. Make a second streak with the toothpick on the corresponding place
on a n Ltetl 5 agar plate.
5. With a new sterile toothpick, touch a new colony.
6. Repeat the process.
7. Incubate the plates at 40~ overnight.
8. Identify streaks that fail to grow on minimal agar plates. Find the
corresponding streak on the Ltetl 5 agar plate.
9. Streak some of that colony for single colonies on a n e w Ltetl 5 agar plate.
Once it has grown, retest by streaking on a minimalt~tlO agar plate.
1. Be sure to make the streaks in the corresponding places on the pair of
plates. At least 40 to 50 different colonies can be streaked on an agar
plate. A template can be used if desired. Examples are given of a
template in Appendix 1. Do not try to pick colonies so close together
that it is extremely difficult to pick only one colony with the toothpick.
2. Used toothpicks can be collected, resterilized, and reused.
3. Once an auxotrophic strain has been isolated, be sure to make a permanent stock of that strain (See Appendix 2). Label the strain clearly.
Screening for Auxotrophs on 2 EM Agar (Minimal Medium Supplemented with a Small Amount of Nutrient Broth) Plates. Crowded plates
containing from 500 to 1000 bacterial colonies are used. The basis of this
screen is that in the limiting amount of nutrients on 2 EM plates containing
a large number of colonies, the growth of auxotrophs will be slowed
or stopped as the crowded plate becomes depleted of the nutrients the
auxotrophs require for growth. Colonies that are tiny compared to the
TRANSPOSON MUTAGENESI5 OF Escherichia coil
majority of the colonies may be auxotrophs; such colonies will be picked
1. Remove bacteria from a n Lte t plate containing more than 100 colonies
from the mutagenesis protocol by flooding the plate with 5 ml of l x
VBC minimal broth.
2. With a sterile 5-ml pipet, scrape the bacteria off the agar surface.
Remove the bacteria with a pipet and place bacteria in a centrifuge
3. Centrifuge the bacteria for 5 min at 5000 rpm. Decant the buffer from
the bacterial cell pellet.
4. Resuspend the cells in I ml of minimal medium. This stock of mutagenized bacteria can now be tested on 2 EM plates. The 2 EM plates
should be crowded to enhance selection for potential auxotrophs.
A crowded plate should have about 500-1000 bacterial colonies
5. Make serial dilutions of the mutagenized bacteria in I x VBC minimal
6. Using an ethanol-flamed bent glass rod, spread 0.1 ml of that dilution
estimated to give 500-1000 colonies per plate on a 2 EM plate (minimal medium supplemented with 20 ml of nutrient broth per liter).
Make a series of such plates. Also spread 0.1 ml of bracketing dilutions,
such as 10 times higher and 10 times lower concentrations, on 2 EM
7. Incubate plates overnight at 40~
8. Examine the plates the next day. Growth will be slower on this minimal plate. Examine the plates closely (a dissecting microscope may
be helpful here) for the presence of colonies that are much smaller
than the majority of colonies on the plate. These tiny colonies are
potential auxotrophs. The tiny colonies may also be less opaque than
the average colony because the tiny auxotrophic colonies are not as
thick. The plates may need to be incubated longer before the tiny
colonies are readily visible.
9. On the outside of the petri plate, mark the location of the tiny colonies
with a marking pen.
10. Using sterile toothpicks or an inoculation wire, streak the tiny colonies
onto Lte t plates.
11. Incubate plates overnight at 37~
12. Replica plate or toothpick the colonies onto minimal plates.
Colonies that grow o n Lte t but not on minimal medium are auxotrophs.
These auxotrophs will be tested to characterize their biochemical defects.
PROTOCOL 1.5: IDENTIFICATION OF AUXOTROPHS ON POOL PLATES
Identification of Auxotrophs on Pool Plates
9 auxotrophs isolated in Protocol 1.4 and retested to be auxotrophs,
streaked for single colonies o n Ltetl 5 plates
9 pool plates, one of each of 11 different pools
9 sterile toothpicks or inoculation loop
1. Using sterile toothpicks, transfer a small number of bacteria of each
auxotroph onto each of the 11 pool plates. Many different auxotrophs
can be tested on the same pool plates. Be sure to place each auxotroph
in the same location on each of the pool plates.
2. Also streak a prototroph on each of the pool plates as a positive control.
The prototroph will grow on all of the pool plates.
3. Incubate at 37~ overnight.
4. Observe growth on pool plates the next day. Score the amount of growth
for each auxotroph on each pool plate.
5. Return the plates to 37~ and allow further growth.
6. Score growth again after the second day.
7. By examining the components in the pool plates, identify the type of
Making Pool Plates
The following components should be filter-sterilized because they
may be degraded at the high temperatures used in autoclaving. All other
components may be autoclaved.
TRANSPOSON MUTAGENESIS OF Escherichia coli
Solutions containing tryptophan should be stored in the dark.
The salts of glutamic acid and aspartic acid rather than the free acids
should be used.
The following components dissolve more readily in acidic solutions.
Use the acid strength indicated to dissolve the component.
Adenine, 0.1 N HC1
Adenosine, 0.1 N HC1
Guanosine, 0.3 N HC1
Phenylalanine, 0.01 N HC1
Stock solutions of each of the individual components can be made
and combined into the pool groups.
Stocks of each amino acid can be made at a concentration of 10 mg/
ml of the L form or 20 mg/ml of the DL form. Adenosine, guanosine,
thymine, and uracil stocks can be made at 5 mg/ml. Stocks of vitamins and
other components can be made at the specific concentrations indicated:
thiamine (1/zg/ml), diaminopimelic acid (DAP, 300/zg/ml), pyridoxine
(50 /zg/ml), nicotinic acid (50 /zg/ml), biotin (1 /zg/ml), pantothenate
(50/zg/ml). Note that vitamins are trace growth factors; that is, vitamins are
required at very low concentrations. Sterilize the components as indicated.
To make the stocks for pool plates, using aseptic or sterile technique,
combine the stocks of individual components: Mix equal volumes of the
stocks of individual components indicated for each pool.
Pool stocks may be stored indefinitely at room temperature or at 4~
Tightly close caps or wrap caps with Parafilm. Wrap pool stocks containing
tryptophan with aluminum foil to keep out light.
Components of Pool Plates a
~ The components of pools 1 to 5 are listed vertically; the components of pools 6 to 10 are listed
horizontally. How to use the pool plates: If a mutant grows on two pool plates, the auxotrophic requirement
is for the component in common to both pools. For example, a mutant which grows only on pools 3 and
9 is an auxotroph for uracil. If a mutant grows only on one pool plate of plates I to 10, the mutant must
require more than one component from that pool. If a mutant grows only on pool 11, each component
of pool 11 must be tested individually for growth to identify the auxotrophy.