Q.i) What do you mean by ‘insertional inactivation’?

pBR322 carries the ampR (ampicillin-resistance) and tetR (tetracycline-resistance) genes as its two selectable markers.

pBR322 is one of the most widely used cloning vectors (plasmids). To let researchers pick out the bacterial cells that have taken it up, it carries two selectable marker genes, each giving resistance to an antibiotic:

1. ampR — confers resistance to the antibiotic ampicillin.
2. tetR — confers resistance to the antibiotic tetracycline. …

Since DNA is hydrophilic and cannot cross the (largely hydrophobic) cell membrane on its own, bacterial cells must first be chemically and thermally treated to temporarily increase membrane/wall permeability, allowing DNA uptake.

DNA is a hydrophilic (water-loving, charged) molecule, so it cannot pass directly through the hydrophobic cell membrane of a bacterial host cell. To allow recombinant DNA to enter, the host bacterium must first be made 'competent' to take it up:

1. Bacterial cells are treated with a specific concentration of a divalent cation, most commonly calcium chloride (CaCl2). This treatment increases the efficiency with which DNA can enter the bacterium, by increasing the porosity of the cell wall/membrane.
2. The recombinant DNA is then incubated together with these competent cells on ice. …

Selectable markers are genes carried on a cloning vector that let scientists distinguish transformed cells (which took up the vector/foreign DNA) from non-transformed cells.

When foreign DNA is introduced into a host cell (transformation), only a small fraction of the host cells actually take it up. To identify and select only these transformed cells, cloning vectors are engineered to carry a selectable marker gene alongside the gene of interest. Common selectable markers are genes conferring resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, which are otherwise lethal to the host bacterium (e.g. E. coli).

Role in genetic engineering:

• After transformation, cells are grown on a medium containing the relevant antibiotic.
• Only cells that have successfully taken up the vector (and hence the resistance gene) survive and grow; untransformed cells are killed. …