3.12 Colored indicators
Color reactions may be used in several ways in cloning. Usually an enzyme such as beta-galactosidase is detected by using a substrate that gives a colored reaction product after being attacked by the enzyme. E. coli will synthesize b-galactosidase after it runs out of glucose if lactose is present in the medium. An analog of lactose know as IPTG (iso-propyl-thiogalactoside) will induce gene transcription leading to betta-galactosidase synthesis, but the enzyme is unable to hydrolyze the compound.
Another compound known as X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside)
is a substrate for the enzyme and a blue colored product results after galactose
is hydrolyzed off and air oxidation of the indoxyl product takes place.
X-gal is not an inducer of the enzyme. The situation actually is a little
more complicated that this because two gene products are needed to give
an active enzyme and the system is under control of a repressor (see Molecular
Biology section). If we wish to use the beta-galactosidase system in a plasmid,
then the host cells must contain mutations in one or more of the so-called
lacZ sequences of the host genes.
Figure 24. Detection with colored indicators
The beta-galactosidase system is especially useful because it is inducable. That is the promoter is only activated (and the gene transcribed) in the presence of the inducer (which as we have noted could be lactose or IPGT). Thus, if we want to produce large amounts of a new protein in E. coli, a straight-forward procedure is to incorporate the very effective inducable promoter and lacZ' gene into the plasmid directly ahead of our gene of interest. A "fusion" protein consisting of the first part of b-galactosidase and our new gene product will be produced after we have grown a dense culture of transformed E. coli and only after we add IPGT. Examples of this approach are given in the next section.
The reaction is equally useful in detecting successful transformations. If the plasmid has an expressible promoter and lacZ gene region (such as the pUC series of plasmids), any transformed cells will give blue plaques with incubated with IPGT (inducer) and X-gal (chromogenic substrate). If we insert our new gene into the lacZ sequence (interrupting it) then the plaques will be colorless. The pUC plasmid series also have an amphicillin resistance gene for additional selection and a short sequence containing multiple restriction sites to make them highly useful vectors.