Blåvit screening

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Författare/Upphovsman: Tkgd2007, Licens: CC BY-SA 3.0
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Författare/Upphovsman: Stefan Walkowski, Licens: CC BY-SA 4.0
Blue-white screening is a popular and efficient molecular biology technique for the identification of recombinant bacteria in cloning experiments.

It relies on the activity of the enzyme β-galactosidase (β-gal), naturally occurring enzyme in E. coli cells, which cleaves lactose molecules into glucose and galactose. β-gal is encoded by the lacZ gene in the bacterial chromosome. There is also the M15 strain of E. coli with a deletion mutation of a fragment of the Lac gene that produces an inactive β-gal (called ω-peptide). Due to α-complementation process, E. coli strains with lacZΔM15 deletion mutation that carries the plasmid vector containing the lacZα sequence can produce the functional β-gal enzyme. For screening the clones with recombinant DNA, X-gal, Isopropyl β-D-1-thiogalactopyranoside (IPTG) along with an antibiotic (such as ampicillin) is added to the medium. X-gal is a chromogenic substrate, and IPTG is a galactose analog that induces lacZ gene expression. The plasmid vector is designed to contain multiple cloning site (MCS) is present within the lacZ sequence. Consequently, in the plasmid with an insert (foreign DNA), α-complementation does not occur, thus a functional β-galactosidase enzyme is not formed.

Blue colonies contain E. coli producing a functional enzyme that hydrolyzes X-gal, resulting in an insoluble blue pigment. In other words, these are bacteria without the insert that have a a functional LacZ gene.

White colonies are formed when a plasmid vector containing insert with foreign DNA (which disrupts the lacZ gene) is taken up by the E. coli, α-complementation does not occur, and therefore the functional enzyme β-gal is not produced. Next, the desired white recombinant colonies can be easily selected and cultured.

The presence of the antibiotic in the medium ensures that only E.coli containing a plasmid-borne antibiotic-resistance gene (eg. ampR selectable marker) can grow at all.