In: Biology
Describe the principles behind the blue/white color screening method in the identification of the transformants of the pCR 2.1 with or without a PCR insertion.
The success of genetic recombination depends on the efficiency
of detection of recombination. In blue/white screening the
recombinant clones are detected by the colour of the colonies,
whether blue or white.
The core principle is the b-gal enzyme of the lac operon. This
enzyme can convert the lactose into galactose and glucose. In the
plasmid, the half of the enzyme genes are encoded. In the host, E.
coli specially designed for this purpose contains the other half of
the b-gal coding sequences. So, when the plasmid enters the host
bacteria, the complementation occurs to give you the functional
copy of the enzyme b-gal.
The plasmid has an MCS site within the b-gal coding region. So when
foreign DNA is inserted into the plasmid at MCS site, the b-gal
gene is damaged and when cloned into the host, complementation
cannot occur.
In the test media, X-gal is used to give you the colour
appearances. IPTG is another chemical used to induce the expression
of b-gal. IPTG is a non-metabolizable inducer of the lac operon.
So, upon successful recombination, the b-gal is disrupted and b-gal
is not functioning. So, it cannot convert the X-gal into the blue
coloured compound and hence the colonies appear colourless. When
the recombination is not successful, the b-gal is intact and upon
successful complementation in the host, the enzyme will be
functional converting the X-gal into a blue coloured
compound.
Thus, blue/white screening helps to detect the recombinant
clones.