Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Screening Bacterial Colonies Using X-Gal and IPTG: -Complementation

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Many plasmid vectors (e.g., the pUC series, Bluescript, pGem, and their derivatives) carry a short segment of E. coli DNA containing the regulatory sequences and the coding information for the first 146 amino acids of -galactosidase. Embedded in the coding region is a polycloning site that maintains the reading frame and results in the harmless interpolation of a small number of amino acids into the amino-terminal fragment of -galactosidase. Vectors of this type are used in host cells that express the carboxy-terminal portion of -galactosidase. Although neither the host-encoded fragments nor the plasmid-encoded fragments of -galactosidase are themselves active, they can associate to form an enzymatically active protein. This type of complementation, in which deletion mutants of the operator-proximal segment of the lacZ gene are complemented by -galactosidase-negative mutants that have the operator-proximal region intact, is called -complementation (Ullmann et al. 1967) (for more information, see the information panel -Complementation in Chapter 1). The lac bacteria that result from -complementation are easily recognized because they form blue colonies in the presence of the chromogenic substrate X-Gal (Horwitz et al. 1964; Davies and Jacob 1968). However, insertion of a fragment of foreign DNA into the polycloning site of the plasmid almost invariably results in production of an amino-terminal fragment that is no longer capable of -complementation. Bacteria carrying recombinant plasmids therefore form white colonies. The development of this simple color test has greatly simplified the identification of recombinants constructed in plasmid vectors. It is easy to screen many thousands of transformed colonies and to recognize from their white appearance those that carry putative recombinant plasmids. The structure of these recombinants can then be verified by restriction analysis of minipreparations of vector DNA or by other diagnostic criteria.


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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