The pCM999 plasmid shown on the previous page normally expresses the b-gal gene.

Question

The pCM999 plasmid shown on the previous page normally expresses the b-gal gene. The enzyme b- galactosidase normally breaks the disasaccharide lactose into the monosaccharides galactose and glucose. If the artificial substrate X-gal is provided instead of lactose, a blue product will be formed when b-galactosidase attacks it. Thus, if you grow E. coli cells on a plate containing X-gal, the cells expressing a functional version of b-galactosidase will appear blue. Cells not expressing functional b-galactosidase will appear white. This is called “blue-white screening.” With all of that as a preamble, how can you use blue-white screening to help you determine which E. coli cells contain copies of pCM99 with purP?

Explanation / Answer

Firstly, the multiple cloning site can be designed to lie within the b-gal gene of the plasmid. Now, if purP is successfully cloned/ inserted, the b-gal gene will get disrupted and the E. coli cells will not produce blue colonies on X-gal containing media, thus, appearing white in color. E. coli cells with intact b-gal gene, without the purP insert, will still form the functional beta-galactosidase enzyme, producing blue colonies on X-gal containing media.

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