Selection
scheme for recombinant plasmids
The plasmid vector
includes (1) an ampR gene for
resistance to the antibiotic ampicillin,
(2) a polylinker region
that contains a number of unique restriction endonuclease
recognition sites, located inside a (3) LacZ gene
that allows the plasmid to metabolize the sugar X-galactose (X-Gal) and produce a blue
by-product.
A foreign
DNA is restricted with one of the endonucleases
in the polylinker, and recombined with a linearized
plasmid cut with the same enzyme. The plasmids are allowed
to transform a
population of bacteria. Of the very large number of
bacteria in the experiment (106~9s), only a
small fraction take up a plasmid, and of these only a few
of those plasmid contain recombinant
DNA. Rather than screen millions of bacteria
individually, a two-stage
selection
scheme is employed to screen out, first
untransformed bacteria, and then those without
recombinant plasmids.
The bacteria are grown on a petri dish
with ampicillin and
X-galactose. (1)
Only those cells that have an ampR gene from the plasmid
can grow at all. (2) Of those that took up a plasmid,
those that do not
contain recombinant DNA
have an intact polylinker
in the lacZ gene and are thus
able to metabolize X-Gal,
Alternatively, successful
insertion of recombinant DNA into the polylinker disrupts the
lacZ
gene, such that it is non-functional. Blue and White colonies thus
signal unsuccessful
and successful creation
of recombinant DNA,
respectively.
On the petri plate at right, multiple
millions of bacteria do not grow at all, a few hundred
blue colonies grow without recombinant inserts, and only a
few dozen white colonies indicate successful uptake of
foreign DNA.
Subsequent analysis will focus on these.