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Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin
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Screening Bacterial Colonies Using X-Gal and IPTG: α-Complementation.

Michael R Green, Joseph Sambrook

    Cold Spring Harbor Protocols
    |December 4, 2019
    PubMed
    Summary

    Many plasmid vectors utilize alpha-complementation for blue-white screening of recombinant DNA. Insertion of foreign DNA disrupts this process, leading to white colonies, aiding in genetic selection.

    Area of Science:

    • Molecular Biology
    • Genetics
    • Biotechnology

    Background:

    • Plasmid vectors like pUC, Bluescript, and pGem contain segments of Escherichia coli DNA encoding the N-terminal portion of beta-galactosidase.
    • Host cells express the C-terminal portion of beta-galactosidase, enabling alpha-complementation when combined with the plasmid-encoded fragment.
    • Alpha-complementation allows for the formation of enzymatically active beta-galactosidase from non-functional fragments.

    Purpose of the Study:

    • To explain the mechanism of alpha-complementation in molecular cloning.
    • To detail the blue-white screening method for identifying recombinant plasmids using X-Gal and IPTG.
    • To investigate factors influencing bacterial transformation efficiency.

    Main Methods:

    • Utilizing plasmid vectors encoding the N-terminal fragment of beta-galactosidase.

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  • Employing alpha-complementation for blue-white screening in host cells.
  • Assessing transformation efficiency by plating bacteria on agar plates versus in top agar.

    • Main Results:

    • Blue colonies indicate functional alpha-complementation (non-recombinant plasmids).
    • White colonies signify disrupted alpha-complementation due to foreign DNA insertion (recombinant plasmids).
    • Transformation efficiency is slightly enhanced when bacteria are plated in top agar.

    Conclusions:

    • Alpha-complementation serves as a vital screening tool in molecular cloning.
    • The blue-white screening system effectively distinguishes recombinant from non-recombinant bacterial colonies.
    • Plating transformed bacteria in top agar may improve transformation efficiency.