pBR322 Plasmid Vector & Mode of Selection of Transformants - Practice Questions & MCQ

• This was the first artificial cloning vector constructed in 1977 by Bolivar and Rodriguez.
• In pBR322:
  • p denotes that it is a plasmid
  • BR stands for Bolivar and Rodriguez who constructed this plasmid
  • 322 is the number given to distinguish this plasmid from others developed in the same laboratory
• Vector pBR322 has restriction sites for HindIII, EcoR I, BamHI, Sal I, Pvu II, Pst I, Cla I.
• It has antibiotic resistance genes for ampicillin (ampR) and tetracycline (tetR).
• Rop codes for the proteins involved in the replication of the plasmid.

Mode of Selection Through Antibiotic Resistance:

• In pBR322, foreign DNA can be ligated at the BamHI site of the tetracycline resistance gene.
• The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA but can still be selected out from non-recombinant ones by plating the transformants on ampicillin containing medium. 
• The transformants growing on ampicillin containing medium are then transferred on a medium containing tetracycline. 
• The recombinants will grow in ampicillin containing medium but not on that containing tetracycline. But, nonrecombinants will grow on the medium containing both the antibiotics.
• In this case, one antibiotic resistance gene helps in selecting the transformants, whereas the other antibiotic resistance gene gets ‘inactivated due to insertion’ of alien DNA, and helps in the selection of recombinants.

Mode of Selection Through Insertional Inactivation:

• Selection of recombinants due to inactivation of antibiotics is a cumbersome procedure because it requires simultaneous plating on two plates having different antibiotics. 
• Therefore, alternative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate (X-gal in this case). 
• In this, a recombinant DNA is inserted within the coding sequence of an enzyme, â-galactosidase. 
• This results in inactivation of the enzyme, which is referred to as insertional inactivation. 
• The presence of a chromogenic 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 â-galactosidase and the colonies do not produce any colour, these are identified as recombinant colonies.