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Bacterial conjugation
Bacterial conjugation is a process by which the genetic
material from one bacterium is transferred to another bacterium
through direct contact. The donor bacterium is the one that
contributes the genetic material and the recipient bacterium is the
one that receives the genetic material. The donor strain is F+,
meaning that it contains the F plasmid. The genes on the F plasmid
encode for proteins that construct the sex pilus. The pilus bridges
the donor strain and the recipient strain and through it the F
plasmid is copied and transferred to the recipient strain. The
recipient does not have an F plasmid before conjugation.
Therefore, the recipient strain is F-. Conjugation is the
phenomenon you will study today.
Pilus
You will work with two strains of Escherichia coli: one donor and one recipient. The F plasmid in
the donor strain also carries a gene lac+ that allows a bacterium to utilize lactose (a kind of sugar) as an
energy source. This gene is normally part of the bacterial chromosome, but in this case, the gene is on the
plasmid. Such a bacterial strain is said to be F’, which indicates that its plasmid carries chromosomal
material. The chromosomal material on F’ can be transferred to the receipt during conjugation. This
donor strain has one other genetic property that is important in the experiment: they are sensitive to the
antibiotic streptomycin. That means they will die in the presence of streptomycin. The genetic features
of this strain can be symbolized as F+ lac+/strs. It is conventional to show the plasmid's genetic features
left of the slash and the chromosome’s features right of the slash. It is also conventional to italicize the
names of the genes.
The recipient strain lacks an F plasmid. It has a defective lac gene so it cannot utilize lactose but it
is resistant to streptomycin because it has a streptomycin-resistant gene on its chromosome. Thus, its
genetics can be characterized by the symbols F- lac-/strr.
If the donor and the recipient strains are mixed and conjugation takes place, the plasmid will
move from donor to recipient, with all of its genes. The gene lac+ on the F plasmid of the donor thus
enters into the recipient, and the recipient now has two copies of lac genes: the lac+ allele on the F
plasmid and the lac- allele on the chromosome. This "partial diploid" cell is called a merozygote.
To test whether a bacterial strain can use lactose, i.e., lac+, the cells are grown on a medium called
MacConkey agar. This agar contains lactose and indicator dyes that turn red in the presence of acids that
are produced when lactose is metabolized. Red colonies mean that lactose is being metabolized, pale
creamy colonies (the normal color of E. coli colonies) mean that lactose is not being metabolized.
Therefore, the donor strain will form red color colonies on MacConkey and the recipient strain will form
creamy color colonies on MacConkey.
Now here is a practical problem: Both the donor cells and the recipient cells that receive the F
plasmid after conjugation, i.e., the resulting new donor cells, can metabolize lactose. How to distinguish
them? Both will be red in MacConkey agar. This problem is solved by the use of streptomycin.
Remember that the donor F+ strain is susceptible to streptomycin, and the recipient F- strain is resistant.
If, after conjugation is completed, all of the cells are grown on MacConkey medium that contains
streptomycin (a selective plate), then the cells that will be both alive (strr) and red (lac+) are the ones that
have received the lac+ allele via conjugation. Because the recipient strain received the entire F plasmid
during conjugation, it becomes F+ and can serve as a donor in future conjugations.
Genetics
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Lab
PROCEDURE
It is essential that you use sterile techniques!
1. Wipe the workspace with disinfectant.
2. When transferring bacterial cultures, use a pipetteman and sterilized pipette tips. Pay
attention to the instructions.
3. Discard pipette tips after use. Do not re-use any pipette tips.
4. When a loop is used to transfer bacteria, be sure to flame it both before and after use.
I. During the morning class period, do these steps to initiate conjugation.
1. Obtain two 2-ml tubes of nutrient broth from a 37° incubator. Label one of them DONOR and
the other one RECIPIENT.
2. Obtain from the same incubator two bacterial cultures, one labeled DONOR and the other
labeled RECIPIENT. (Of course, one is F+ and the other is F-.) Using a pipetteman and pipette
tips, transfer 0.2 ml of each bacterial cell suspension to its appropriately labeled tube of
nutrient broth. Use one pipette tip for each transfer and discard the tip after one use. Have
the tubes uncapped for as short a time as possible.
3. Loosely place the caps on your tubes and tape the caps. This is to make sure the caps stay on
during shaking. Do not seal it, though. Bacteria need oxygen to grow well.
4. Place them in the 37ºC shaking incubator. During the remainder of the period, the
subcultured cells will rapidly reproduce, producing large, actively growing populations.
5. At the end of the period or in the beginning of the laboratory period, mix the two types of
organism together. Obtain a small plate. Using a pipette tip, transfer 1 ml of DONOR
subculture to the interior of this conjugation plate. Using a new pipette tip, transfer 1 ml of
RECIPIENT to the same plate. Gently swirl the plate to ensure mixing of the two cultures. Be
sure to mark your plate to identify it as yours. Place both the conjugation plate (with lid on)
and the two subculture tubes to the incubator. Discard the pipette tips as instructed.
6. Wait at least one hour before proceeding to the remaining steps.
II. During the afternoon lab period, set up conditions to find cells that have received plasmids.
1. Find and remove three agar plates from the incubator: two SELECTIVE plates and one
INDICATOR plate. Selective plates contain streptomycin; indicator plates do not.
2. Using a marking pen or pencil, draw a line down the middle of the indicator plate (on the
bottom of the plate) and do the same thing to one of the selective plates. Label one side
DONOR and the other side RECIPIENT. Label the bottom of the other selective plate
CONJUGATION.
3. Flame to sterilize a loop and transfer a drop of DONOR subculture (from the morning’s work)
to each of the plate areas labeled DONOR. Streak the area as instructed.
4. Flame to sterilize the loop and transfer a drop of RECIEPIENT subculture (from the morning’s
work) to each of the plate areas labeled RECIPIENT. Streak the area.
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Genetics
Steps 3 and 4 have set up your controls for the experiment, since neither donor nor recipient
subcultures should have conjugation within them.
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Lab
5. Flame to sterilize the loop again and transfer a drop of conjugation culture (from the small
plate set up during the morning session) to the remaining SELECTIVE plate. Streak the plate.
6. Place all three plates upside down in the 37ºC incubator overnight.
III.
Examine the resulting growth patterns sometime during the next day.
1. Look carefully at each area of each plate, noting whether there are colonies, and what their
colors are. Which colonies are the result of conjugation and subsequent gene transfer? Did
the control groups behave as expected? Your lab report should clearly describe how you set
up the experiment, why the various tubes and plates contained what they contained, and how
the results showed (or failed to show) recombination by plasmid transfer during conjugation.
Genetics
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Lab