Describe the principles behind the blue/white color screening method in the identification of the transformants of...

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.

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Expert Solution

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.

gladiator answered 1 year ago

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