A powerful tool for biotechnologists is the lac Z gene. You may recall from an earlier section on the control of gene expression, that lac Z is part of the lac operon of E. coli and encodes the enzyme ß galactosidase. This enzyme catalyzes the hydrolysis of lactose into glucose and galactose, allowing the bacteria to use lactose as an energy source. ß galactosidase can also break down an artificial substrate called X-gal to produce a compound that is blue in color. X-gal can thus be used to test for the presence of active ß galactosidase. With this background, we can now look at how the lac Z gene can be of help to molecular biologists when they create recombinant plasmids. In the example described earlier, the gene for human growth hormone (hGH) was inserted into a plasmid. As we noted, the plasmid, as well as the hGH gene are cut with restriction endonucleases to create compatible DNA ends that can be ligated. While the ends of the hGH gene are, indeed, capable of being ligated to the ends of the plasmid, the two ends of the plasmid could also readily rejoin. In fact, given that the two ends of the plasmid are are on the same molecule, the chances of their finding each other are much higher than of a plasmid end finding an hGH gene. This would mean that many of the ligated molecules would not be recombinants, but simply recircularized plasmids. Five percent of the plasmids having inserts of the hGH gene would be very good. That would mean that 95% of the bacterial colonies arising from transformation would contain the original plasmid rather than the recombinant. To make the process of screening for the relatively rare recombinants simpler, plasmids have been engineered that carry the lac Z gene, modified to contain, with the coding sequence, restriction enzyme recognition sites. If one of these sites is used to cut open the plasmid and a gene of interest is inserted, this disrupts the lac Z gene. If the plasmid simply recircularizes, the lac Z gene will be intact. To find which bacterial colonies carry the recombinant plasmids, X-Gal is provided in the plates. Bacterial colonies containing plasmids with the lac z sequence disrupted by an inserted gene will not produce functional ßgalactosidase. The X-Gal will not be broken down and there will be no blue color. By contrast, bacterial cells with recircularized plasmids having no inserted hGH gene will make functional ß galactosidase, so in the presence of X-Gal and IPTG these colonies will produce a blue color. This is summarized in the figure on the previous page.
Figure 9.14.1: Blue-White Screening Strategy