Question

To study the lac operon in E. coli, you added IPTG in the growth media, which is another analog of allolactose that is an inducer of the lac operon but is not metabolized by β-gal. You measured the activity of the enzyme β- galactosidase (β-gal) by X-gal. After mutagenesis (induce mutations) you find mutants that never turn blue, which DNA component(s) could produce such phenotype?

Answer 1

IPTG is also known as Isopropyl β-D-1 thiogalactopyranoside. This is a reagent of molecular biology which is used as a mimic of allolactose. This is a lactose metabolite which triggers the transcription of lac operon.

IPTG binds to the lac repressor. This binding of the IPTG causes the release of tetrameric repressor from the lac operator in an allosteric manner. This allows the transcription of the genes in the lac operon such as the gene encoding for β galactosidase, which hydrolyses β galactosides into monosaccharides.

Since IPTG cannot be metabolised by E.coli, the expression levels of lac promoter/operator genes do not vary.

Now, X-gal is a substance that can be metabolised by β galactosidase to form a blue product. Thus, cells expressing beta-galactosidase grown in the presence of IPTG and X-Gal will turn blue. Whereas, in the cells where the DNA fragment has been inserted into lac Z(one of the genes for beta-galactosidase), there will be no action upon X-Gal and the cells will not turn blue. Thus we can identify the cells with recombinant plasmid rather than non recombinant plasmid. So, the DNA component namely the recombinant plasmid will produce such phenotype.