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Positive selection vectors based on xylose utilization suppression.

P E Stevis, N W Ho

    Gene
    |January 1, 1987
    PubMed
    Summary
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    Researchers developed a novel positive selection system using Escherichia coli (E. coli) and xylose isomerase activity. This system enables efficient cloning by ensuring transformants only grow when a DNA fragment is inserted into the vector.

    Area of Science:

    • Molecular Biology
    • Microbiology
    • Biotechnology

    Background:

    • High xylose isomerase activity in wild-type Escherichia coli (E. coli) leads to a xylose-negative (Xyl-) phenotype.
    • This natural phenomenon presents an opportunity for developing novel selection systems in molecular cloning.

    Purpose of the Study:

    • To engineer a versatile positive selection system for molecular cloning using E. coli.
    • To create a cloning vector that facilitates the identification of successful transformants.

    Main Methods:

    • Deletion of the xylA promoter using exonuclease BAL 31.
    • Insertion of the modified xylA gene under the control of the lac promoter into the pUC9 vector, creating pLX100.
    • Transformation of wild-type E. coli strains with the pLX100 vector.

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    Main Results:

    • The pLX100 vector induces high xylose isomerase activity and a Xyl- phenotype in E. coli transformants.
    • Restoration of the xylose-positive (Xyl+) phenotype occurs when activity drops below a critical threshold (approx. 100 units).
    • The vector contains unique restriction sites (HindIII, PstI, BamHI, XhoI) for cloning applications.

    Conclusions:

    • The engineered plasmid pLX100 functions as an ideal positive-selection cloning vector.
    • E. coli transformants with pLX100 require DNA insertion into unique sites to grow in minimal xylose medium, enabling positive selection.