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_Bacterial Conjugation Experiment _
By: Paige Davenport
INTRODUCTION: Bacteria, in general, reproduce asexually, but in order to
increase diversity, they have developed a mechanism for transfer of genetic
material from one bacterium to another. The ability to perform this transfer
is conferred by a set of genes which are called F for 'fertility.' These genes
exist on a small, circular piece of deoxyribonucleic acid (DNA) that
replicates independently from the bacterial chromosome, or they can be
integrated into the chromosome. The bacterium containing this gene (sometimes
referred to as 'male' or F+) extends its pilus to a neighboring bacterium
(sometimes referred to as 'female' or F-), and the two cells are attached.
This process is called conjugation. The third manifestation of the F factor is
Hfr, which is the term for the F element becoming integrated into the genome.
When conjugation occurs, the F genes start travelling across the pilus,
bringing the remainder of the genome behind it. Most often, the entire genome
isn't transferred. The bacterial genome that is delivered can be measured in
minutes from the origin of transfer. That is, the amount of time it takes for
a particular gene to be transferred from one bacterium to another indicated
how far it is from the origin of replication. METHODS AND MATERIALS: Media
Preparation: The starting material was Medium 56-glucose agar (MM560). The
components of the MM56 are found in figure 1. From this, we made two types of
media, complete and selective. The reagents used, along with their stock and
final concentrations are found in figure 2. The formula: Stock volume =
[(Final concentration)(final volume)] / (stock concentration) was used to
calculate the amounts of each reagent added to the complete and selective
media. (The final volume was 600 ml.) These values are also found in figure 2.
To make the complete media, appropriate amounts of each amino acid, glucose,
vitamin B1, and streptomycin were aseptically added to the MM56 media and
poured into sterile petri plates. The selective media was prepared so that 84
plates were made. These plates were made much in the same way as the complete
plates, except that 28 of the plates contained all of the reagents except
proline, another 28 were without histadine, and another 28 were without
threonine. These were labeled with "pro-", "his-", and "thr-". Viable Counts:
The strain used was Escherichia coli K12. The donor and recipient cultures
were kept as much as possible in a water bath at 37ºC. Viable counts of the
donor (Hfr) and recipient were done on complete MM56 and Luria agar. Serial
dilutions were performed to obtain a 10-7 and 10-8 dilutions of the recipient,
and 10-2, 10-7, and 10-8 dilutions of the donor. Two Luria plates were
inoculated with 1 ml each of the 10-7 dilution of the recipient. Two Luria
plates were inoculated with 1 ml each of the 10-8 dilution of the recipient.
Two Luria plates were inoculated with 1 ml each of the 10-7 dilution of the
donor. Two Luria plates were inoculated with 1 ml each of the 10-8 dilution of
the donor. Two complete MM56 plates were inoculated with 1 ml each of the 10-7
dilution of the recipient. Two complete MM56 plates were inoculated with 1 ml
each of the 10-8 dilution of the recipient. One complete MM56 plate was
inoculated with 1 ml of the 10-2 dilution of the donor. Each of these were
inoculated for 48 hours. Conjugation: 30 sterile test tubes were filled with 9
ml each of sterile saline solution. A supply of sterile top agar was kept in a
water bath to keep liquid until needed. One ml of the donor culture was added
to 20 ml of the recipient culture in a flask. This mating mix was kept in the
37ºC water bath. Immediately (0 minutes), 1 ml of the mating mix was removed
from the flask and added to 9 ml of sterile saline, making a 10-1 dilution.
This was placed on the vortex for 60 seconds to interrupt any mating taking
place in the mix. 1 ml of this mixture was added to another tube of 9 ml
sterile saline, making a 10-2 dilution and this solution was vortexed for one
minute. This was repeated twice more to produce a 10-3 and a 10-4 dilution.
For each dilution, three tubes of top agar were inoculated with 1 ml of that
dilution. One each of the agar-dilution mixtures were poured onto a threonine
deficient plate, a proline deficient plate, and a histadine deficient plate.
This procedure was repeated every ten minutes for sixty minutes. The top agar
was allowed to harden and the plates were incubated at 37ºC for 48 hours.
RESULTS: After incubation, the plates were removed and colony counts were
performed (figure 3). The time of entry of each gene was calculated, using
this formula: [(Recipient/ml)/(donor/ml)] * 100. The calculated times of entry
are found in figures 4 and 5. From this information, a map of E.coli K12 was
constructed (figure 6). DISCUSSION: The time of entry of the amino acid
threonine was found to be consistent with that found in the lecture handout
"Bacterial Gene Transfer - Conjugation". The time of entry for proline (10
minutes) was longer that the handout value of about 5 minutes. The actual time
for proline might have been 5 minutes, but the samples were taken at 10 minute
intervals. The time of entry for histadine (10 minutes) differed drastically
than the handout time of about 44 minutes. These discrepancies could be caused
by a variety of errors. The cultures could have been contaminated. The media
could have been prepared incorrectly. The wrong amino acids could have been
added or the plates could have been labeled incorrectly. The amount of time
spent vortexing the mixes (in order to separate the mating pairs) might have
been insufficient. Also, the dilutions were placed on the vortex before making
each dilution, instead of only after taking the sample directly from the flask
in the water bath. Figure 1 - MM56 Components Chemical Amount in 1L of MM56
Na2HPO4 (0.1M) 611 ml KH2PO4 (0.1M) 384 ml MgSO4o7H2O (10%) 2 ml (NH4)2SO4
(10%) 1 ml Cu(NO3)2 (15%) 1 ml FeSO4o7H2O (0.05%) 1 ml Figure 2 - Reagents
used Reagent Stock concentration Final concentration Calculated stock volume
Streptomycin 50 mg/ml 200:g/ml 2.4 ml DL- Threonine 1% in H2O 10 ml/L 6 ml
L-Leucine 2% in H2O 10 ml/L 3 ml L-Proline 2% in H2O 10 ml/L 3 ml
L-Histadine 1% in H2O 5 ml/L 3 ml L-Arginine 6% in H2O 33 ml/L .33 ml Vitamin
B1 0.1% in H2O 0.2 ml/L 1.2 ml Glucose 40% 10 ml/l 1.5 ml Figure 3 - Colony
counts Threonine deficient plates Time 10-1 10-2 10-3 10-4 0
minutes TNTC TNTC 96 17 10 minutes 41 TNTC TNTC 127 20 minutes 38 5 TNTC 148
30 minutes 88 4 TNTC TNTC 40 minutes 216 TNTC TNTC TNTC 50
minutes 34 5 TNTC 42 60 minutes NG 6 TNTC 53 Proline deficient plates
Time 10-1 10-2 10-3 10-4 0 minutes NG NG NG 3 10 minutes 37 NG TNTC 44 20
minutes 49 TNTC TNTC 58 30 minutes 43 TNTC TNTC 40 40 minutes 11 5 TNTC 94 50
minutes 53 TNTC 50 51 60 minutes 14 NG TNTC NG Histadine deficient plates
Time 10-1 10-2 10-3 10-4 0 minutes 10 2 NG NG 10 minutes 46 43 26 3 20
minutes TNTC 136 NG 4 30 minutes TNTC 184 8 3 40 minutes TNTC 340 20 NG 50
minutes 45 TNTC 27 3 60 minutes 41 250 42 NG TNTC = Too numerous to count NG =
No growth Figure 4 - Time of entry table (percentages of recombinants)
Time Thr - Pro - His - 0 minutes .44 0 0 10 minutes 5.8 2 .02 20
minutes 6.7 2.6 .06 30 minutes .004 1.8 .08 40 minutes .0098 4.3 .09 50
minutes 1.9 2.3 .002 60 minutes 2.4 0 .11 Figure 5 - Time of entry graph
(percentages of recombinants) Figure 6 - Map of E. coli K12
Word Count: 1341
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