In: Biology
3. Describe a method to isolate E. coli mutants that do not transcribe lacZ?
The blue white colony screening method can be used for isolating lac Z mutants of E.coli. This screening method is extensively used in cloning experiments. Lac Z gene encodes enzyme beta galactosidase. Beta galactosidase breaks down lactose as well as X gal.
We use X gal (indole derivative and analog of lactose) to detect beta galactosidase activity. We supplement X gal in our nutrient agar plates before spreading the E.coli culture and incubate the plate overnight at 37 degree celsius.
If lac Z is active then functional beta galactosidase will be synthesized. When X gal is broken by functional beta galactosidase, it gives rise to a chemical named 5-bromo-4 chloro-indoxyl, which dimerizes and gives blue colour. Hence E. coli with functional lac Z will appear blue.
If functional beta galactosidase is not present then X gal will not be cleaved and it will remain colourless and hence white E. coli colony will be found on the culture plate.
E coli -blue colony-functional beta galactosidase- functional lac z
E.coli-White colony- non functional beta galactosidase- mutated lac Z.
So, white E coli colonies will be unable to transcribe lac Z.