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#LacZ gene

4 public questions tagged with this topic.

Gene Cloning Process

What is the function of β-galactosidase in blue-white screening?

In blue-white screening, β-galactosidase expressed from plasmid lacZα complements the host ω-fragment to restore active tetrameric enzyme capable of lactose and X-gal metabolism. Under IPTG induction, the functional enzyme hydrolyzes the β-glycosidic bond of the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, X-gal. Cleavage releases 5-bromo-4-chloro-3-hydroxyindole, which spontaneously dimerizes and oxidizes to 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue pigment retained within bacterial colonies. Thus persistent blue color reliably reports intact lacZα activity, while recombinant disruption by insertional inactivation completely prevents color development, enabling clear visual discrimination.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Gene Cloning Process

Insertional inactivation of LacZ gene leads to which of the following on X-gal media?

The lacZ gene encodes β-galactosidase, which hydrolyzes the artificial substrate X-gal into an insoluble blue chromophore. In vectors like pUC19, the multiple cloning site resides within the lacZα fragment encoding the N-terminal α-peptide. Successful ligation of foreign DNA into this site disrupts the open reading frame, preventing formation of active enzyme through insertional inactivation and loss of α-complementation. Without functional β-galactosidase, X-gal remains uncleaved. Consequently, recombinant colonies appear white on indicator plates containing IPTG and X-gal, whereas non-recombinant colonies remain blue.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Gene Cloning Process

In blue-white screening, what do white colonies indicate?

Blue-white screening exploits α-complementation of lacZ encoding β-galactosidase in Escherichia coli. Plasmids carrying lacZα produce functional enzyme when induced by IPTG, cleaving X-gal to 5,5'-dibromo-4,4'-dichloro-indigo yielding intensely blue colonies on indicator plates. Insertion of gene of interest within the multiple cloning site located inside lacZα causes insertional inactivation, abolishing enzyme activity and preventing complementation. No cleavage of X-gal occurs. Therefore white colonies harbor recombinant plasmids with insert, while blue colonies retain empty vector, allowing rapid visual identification of successful cloning events.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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