Cloning with LacZ and X-gal plasmids
Plasmids that are commonly used in gene cloning are about 3000 nucleotide pairs in length, have an amphicillin resistance gene and a gene that encodes the enzyme beta galactosidase. The gene encoding this enzyme is called the LacZ or blue color gene. The beta galactosidase enzyme will use galactose sugar as a substrate. If a special version of this sugar called X-gal is placed in the media, a neat thing happens inside the bacteria that have the blue color gene. These bacteria will be expressing the gene, making the beta-galactosidase enzyme and will cleave galactose sugar molecules. The ’X’ in X-gal is a colorless pigment when attached to the galactose sugar but turns blue when cleaved by beta galactosidase. The blue color pigment will form in the cells and blue colonies are formed on the plates. Therefore two traits can be observed in a single plate with Amp antibiotic and X-gal. First, bacteria plated on this media will either grow or not grow. If they grow, they will either form blue or white colonies. Observing these trait in the bacteria will tell the gene cloners which bacteria are cloning genes. We will discuss the steps of the process that leads to the blue or white bacteria colonies.