Question

You decide to clone a gene using a plasmid vector featuring ampicillin resistance and the lacZ gene. However, when you try to grow the E. coli onto a plate containing X-gal and ampicillin, you see only blue colonies. Explain what most likely went wrong during the cloning process.

Answer 1

The bluw-white screening is a process where it allows the rapid and convenient detection of recombinant vector-based molecular cloning experiments. The vector is then inserted into a competent host cell viable for transformation, which are then grown in X-gal. Cells transformed with vectors containing recombinant DNA will produce white colonies and cells transformed with non-recombinant plasmids/vector grow into blue colonies.

This method is based on alpha-complementation of beta-galactosidase gene. When a recombinant gene is carried when transformed , lacZalpha sequence is carried in the vector /plasmid by which it disrupts the gene that produces alpha-peptide. so no beta-galactosidase may be formed. The prescence of active beta-galactosidase can be detected by X-gal, a colorless analog of lactoose that maybe cleaved by beta-galactosidase to form 5-bromo-4-chloro-indoxyl, which then spontaneously dimerizes and oxidizes to form a blue insoluble pigment.This results in a charactersticblue color in cells containing a functional beta-galactosidase.

Blue colonies therefore show that they may contain vector with an uninterrupted lacZalpha (therfore no insert) while white colonies where X-gal is hydrolyzed, indicate the presence of an insert lacZalpha which disrupts the formation of an active beta-galactosidase.

So, most likely we see only blue colonies because the vector/plasmid might not have the insert and X-gal isnt hydrolyzed.