Name: Genetic Engineering in Health
Uploaded: 2019-07-19
Duration: 5 min 10 s
Description: Genetic Engineering holds the key to the door of wide possibilities. Watch this video to learn how genes are being edited and stitched and how they impact humanity and life!

Question

For selection of recombinants, insertional inactivation of antibiotic marker has been superseded by insertional inactivation of a marker gene coding for a chromogenic substrate.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

Accepted Answer

The recombinant cell is formed by the insertion of a foreign gene into the cell by recombinant DNA technology. The recombinant cells are not easy to identify. These are identified by the presence of selectable markers in them. In the vector pBR322, two antibiotic-resistant genes (amp^Rand tet^R) are present that act as selectable markers and help in the identification of transformed cells.

For this, the cells are first plated on to an antibiotic plate containing ampicillin. These cells are then replica plated on to the plate containing second antibiotic i.e., tetracycline. The non-transformed cells will grow on both the plates while transformed cells will grow only on the ampicillin plate because the tetracycline has been inactivated by the insertion of recombinant DNA. This whole process, however, is very laborious and takes too much of time.

An alternative method of this is the insertional inactivation of a marker that codes for a chromogenic substrate. E.g. if the recombinant DNA is inserted within the coding sequence of the β-galactosidase gene, it results in the inactivation of this enzyme (called insertional inactivation). When the cells are treated with a chromogenic substance, the cells that produce blue colour are non transformed cells. While the cells which do not take up the stain represent the transformed cells in which the gene for β galactosidase has been deactivated. Thus, the cells which do not appear blue can be considered as the transformed cells. This process takes less time and energy thus, is more preferable.

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