Biology Reference
In-Depth Information
CONJUGATION
Strain
E. coli
K-12 can carry an F plasmid capable of integrating into the
E.
coli
chromosome at various locations in different orientations. They can
transfer their DNA during synthesis, at high frequency into a recipient
E.
coli
through physical contact, and thus designated as Hfr or high frequency
of recombination. The donor-recipient pair can be broken artificially by
high shear rate in a blender, for example, and the transfer of genetic material
is thus interrupted.
A typical experiment consisted of mixing a donor strain with a good gene
and a recipient strain with a defective gene. For example, the recipient
might have a defective mutation of one of the genes for the synthesis of
amino acid threonine, designated as
thr
-
Without providing threonine in the
growth medium, the recipient strain could not grow. On the other hand, the
donor carried the normal gene, designated as
thr
+
.
In order to select
recombinants with
thr
+
gene, no threonine was added to the growth medium
in which the mixture was plated. However, another genetic marker was
required to prevent the growth of the donor strain. Usually, the gene which
provided resistant to streptomycin,
strM
+
,was used. The recipient was
strM
+
while the donor was
strM
-
.
The addition of streptomycin to the
medium without threonine could only support the growth of the desired
recombinant.
At various times after mixing the donor and recipient strains together, the
culture was vigorously blended for a minute before plating. If the
thr
+
gene
from the donor strain had not been transferred into the recipient strain, no
thr
+
recombinants could result, except by reversion of
thr
-
to
thr
+
usually at a
very low frequency around 10
-7
. Therefore, no colony would appear on the
selecting plate. After a certain duration, the
thr
+
gene from the donor would
enter the recipient. By recombination with at least two cross-overs, the
thr
+
gene could replace the
thr
-
gene on the recipient chromosome, to give rise to
the required recombinants. They would form colonies on the selecting
plates. The number of recombinants was plotted against time after mixing.
The intersection of this curve with the time-axis was recorded. That time
would be proportional to the length of the
E. coli
chromosome from the
point of F plasmid integration to the genetic marker
thr.
Using different Hfr's of different orientations and starting points of transfer
in such interrupted mating experiments, many pioneer researchers in this
field have established the approximate locations of numerous genetic
markers on the
E. coli
chromosome map. The chromosome is circular and
