Gene Cloning

Key Tools for Gene Cloning 55
in the plasmid pUC18 and its many derivatives to allow identification of the
recombinants in a single step. These plasmids make use of a gene called
lacZ, which encodes the enzyme β-galactosidase. The normal substrate for
this enzyme is lactose, which it breaks down into glucose and galactose.
However, it can also break down a compound called 5-bromo-chloro-3-
indolyl-β-D-galactoside or X-gal for short. The useful thing about X-gal is
that when it is broken down it produces a blue color. This means that
clones with a functional lacZ gene can easily be identified because they are
blue in color. The family of plasmids to which pUC18 belongs in fact only
contains a part of the lacZ gene that encodes a fragment of the enzyme
called the α peptide and this partial gene is called lacZ′. These plasmids are
used in conjunction with specially engineered host strains, which supply
Box 3.4 Host Strains and Plasmids for Blue–White
Selection
Blue–white selection is based on the lacZ gene, which encodes β-
galactosidase. The normal function of this enzyme is to break down
lactose into its components, glucose and galactose. It is also capable of
breaking down the artificial substrate X-gal (5-bromo-chloro-3-indolyl-β-
D-galactoside) to produce a product with a blue color. In gene cloning
experiments a gratuitous inducer of the lacZ gene is often included. This
compound, IPTG (isopropylthiogalactopyranoside), switches on expression
of the gene but is not broken down by the β-galactosidase.
Insertional inactivation of lacZ is used in many cloning vectors as a way of
identifying recombinant clones. This selection requires the use of special
E. coli host strains. E. coli are naturally lac + (i.e. they can use lactose as a
sole carbon source) in fact, this is one of the biochemical characteristics
routinely used in the identification of E. coli. For blue–white selection
to work special host strains, which have had the chromosomal β-
galactosidase gene inactivated, are needed. These lac – strains are white
when grown on agar containing X-gal and IPTG.
Incorporating the whole of the rather large lacZ gene into cloning vectors
would be impractical. However, the β-galactosidase protein can be provided
in two parts, which can associate with each other to form a functional β-
galactosidase inside the cell. The part of the gene coding for the smaller N-
terminal part of the β-galactosidase protein, the α peptide, is incorporated
into the cloning vector and the part of the gene encoding the remaining C-
terminal portion of the protein is provided by the host strain, usually encoded
on a large stable plasmid called the F′ plasmid. Host strains used for blue–
white selection therefore have a deletion of their chromosomal lacZ gene but
do carry the part of the gene encoding the C-terminal part of the protein.