Plasmids - pBR 322 Vectors

• pBR322 is an artificial plasmid vector used in gene cloning and is regarded as the “workhorse” of the cloning laboratory.
• It is derived from the E. coli plasmid ColE1 and contains 4,362 base pairs of DNA.
• The name 'pBR322' comes from Bolivar and Rodriguez, who developed this vector.
• Another reconstructed plasmid vector mentioned along with it is pUC, named after the University of California.

• pBR322 is small in size, so it can be easily taken up by the host cell.
• Its small size also makes it comparatively more stable inside the host.
• It contains genes for resistance to two antibiotics: ampicillin and tetracycline.
• These antibiotic resistance genes act as selectable markers and help identify transformed host cells.

• ampR gene: gives resistance to ampicillin.
• tetR gene: gives resistance to tetracycline.
• ori (origin of replication): the site from where replication of the plasmid begins.
• rop gene: codes for proteins involved in the replication of the plasmid.

Plasmid pBR322 showing restriction sites (Hind III, EcoR I, BamH I, Sal I, Pvu II, Pst I, Cla I), ori and antibiotic resistance genes (amp^R and tef^R). rop codes for the proteins involved in the replication of the plasmid. 

• pBR322 contains several unique restriction sites where foreign DNA can be inserted.
• The restriction sites mentioned in the source are BamHI, HindIII, SalI, PvuII, PstI, EcoRI, and ClaI.
• These sites are named after the restriction enzymes that recognise and cut them.

• The BamHI restriction site is located within the tetracycline resistance gene (tetR).
• When foreign DNA is inserted at the BamHI site, the tetR gene becomes non-functional.
• As a result, the recombinant plasmid loses resistance to tetracycline.
• This property helps in identifying recombinant plasmids from non-recombinant ones.

• Recombinant plasmids contain foreign DNA inserted into the BamHI site of tetR, so they lose tetracycline resistance.
• Such recombinants can grow in a medium containing only one antibiotic, such as ampicillin, but they do not grow in a medium containing both antibiotics.
• Non-recombinant plasmids do not carry the insert, so both ampR and tetR remain functional.
• Therefore, non-recombinant plasmids can grow in a medium containing both antibiotics.
• In this way, the presence or absence of functional antibiotic resistance genes helps in selecting E. coli colonies carrying the desired foreign DNA.