Explain how recombinants and non- recombinants are differentiated on the basis of colour production in the presence of a chremogenic substrate. Name that procedure. Describe the temperature treatment (-3 step-) that enhances the bacteria to take up the rDNA.
Answers
Answer:
Insertional inactivation” . In this recombinants & non recombinants are differentiated on the basis of the ability to produce colour in the presence of a chromosomic substrate – In this, a rDNA is inserted in an enzyme – a galactosidase – leads to inactivation of the enzyme which does not produce colour due to insertion.
(i) Host cells are incubated with rDNA on ice.
(ii) Followed by placing them briefly at 4 -10°C heat.
(iii) Then transforming them back on ice.
This enables the host cells (bacteria) to take up the rDNA
Alternative selectable markers have been developed which differentiate recombinants from the non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. In this, a recombinant DNA is inserted within the coding sequence of an enzyme, ( -galactosidase. This results into inactivation of the enzyme, which is referred i to as insertional inactivation. The presence of a chromogenic j substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the -galactosidase and the colonies do not produce any colour, these are identified as recombinant colonies.