Biology Reference
In-Depth Information
packaging ingredients are obtained by combining a very concentrated mix-
ture of the lysate from two different
λ
strains that are lysogenic. One mutant
λ
strain can progress no further in the packaging process than the prehead stage
because it carries a mutation in a gene (gene
D
) and therefore accumulates this
precursor. The other mutant
λ
strain is prevented from forming any head struc-
ture by a mutation in a different gene (gene
E
), but it can produce the tail com-
ponent. In the mixed lysate, both head and tail components become available so
that a complete phage can be assembled that contains recombinant DNA.
Transfer of DNA into the
E. coli
host involves adsorption of phage to specific
receptor sites on the outer membrane of the
E. coli
. Because phage will adsorb
to dead cells and debris, only healthy bacterial cultures should be used to reduce
loss of efficiency. Once the
E. coli
have taken up the phage, they are plated out on
nutrient agar and allowed to grow at least overnight at 37 °C. Infected bacterial
colonies grow, but clear areas (
plaques
) consisting of lysed cells will be seen sur-
rounded by an opaque background of unlysed bacteria (
Figure 6.5
). Each plaque
represents an original bacterial cell that was infected, ideally only by a single
λ
.
Thus, each plaque should contain multiple copies of a
single kind
of recombinant
DNA molecule. Even the smallest plaque is likely to contain sufficient phage DNA to
be detectable by plaque hybridization, a probe technique described in Section 6.6.
Figure 6.5
An agar plate with
E. coli
and plaques caused by the bacteriophage
λ
. Each clear area
indicates where a single bacterium initially was infected with
λ
. After replication, the emerging
λ
attacked adjacent
E. coli
and lysed them, resulting in a clear “plaque” of killed
E. coli
and
λ
on the
surface of the agar.
