Question

For selection of recombinants, insertional inactivation of antibiotic marker has been superceded by insertional inactivation of a marker gene coding for a chromogenic substrate. Give reasons.

Answer 1

Selection of recombinants due to the inactivation of antibiotics is a laborious process as it requires:

1. A vector with two antibiotic resistance marker
2. Preparation of two kinds of media plate, with one antibiotic each.

Transformed cells are first plated on that antibiotic plate which has not been intentionally inactivated (ampicillin) and incubated overnight for the growth of transformants. For the selection of recombinants, these transformants are Replica plated on a second antibiotic (tetracycline) plate (which got inactivated due to insertion of the gene). Non-Recombinants grow on both plates (one carrying ampicillin and the other carrying tetracycline ) while recombinants will grow only on the ampicillin plate.

This entire exercise is laborious and takes more time (two overnight incubation). However, if we choose the second option (insertional inactivation of a marker that produces colour in the presence of a chromogenic compound), we can distinguish between the recombinants and non-recombinants on a single medium plate (containing one antibiotic and the chromogenic compound) after overnight growth.

Hence I would choose a marker which produces a coloured compound but gets inactivated due to the insertion of foreign DNA.