Blue-white screening
Blue-white screening
Blue-white screening (pronunciation: /bluː waɪt ˈskriːnɪŋ/) is a molecular biology technique used to facilitate the identification of recombinant bacteria. It is based on the activity of the enzyme Beta-galactosidase, which can cleave X-gal, a synthetic substrate, leading to the production of a blue precipitate.
Etymology
The term "blue-white screening" is derived from the color change that occurs in the bacterial colonies. Colonies that have taken up a plasmid vector without an insert (non-recombinant) turn blue, while those that have taken up a plasmid with an insert (recombinant) remain white.
Process
The process of blue-white screening involves the use of a plasmid vector that contains a multiple cloning site (MCS) within the LacZ gene, which codes for the enzyme Beta-galactosidase. When a DNA fragment is inserted into the MCS, it disrupts the LacZ gene, preventing the production of functional Beta-galactosidase.
The bacteria are then grown on agar plates containing the substrate X-gal. If Beta-galactosidase is present (indicating no insert in the plasmid), it will cleave X-gal, producing a blue precipitate. If Beta-galactosidase is not present (indicating an insert in the plasmid), X-gal will not be cleaved and the colonies will remain white.
Related Terms
- Beta-galactosidase: An enzyme that cleaves lactose into glucose and galactose. In blue-white screening, it is used to cleave X-gal, producing a blue precipitate.
- X-gal: A synthetic substrate for Beta-galactosidase. When cleaved, it produces a blue precipitate.
- LacZ: A gene that codes for Beta-galactosidase. In blue-white screening, it is disrupted by the insertion of a DNA fragment.
- Multiple cloning site (MCS): A short segment of DNA which contains many (poly) restriction sites - locations that can be cut by restriction enzymes. The MCS is a component of a plasmid vector used in blue-white screening.
External links
- Medical encyclopedia article on Blue-white screening
- Wikipedia's article - Blue-white screening
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