3.6 Plasmid Cloning Vectors for E. coli

There are hundreds of different plasmids that today may be used for cloning experiments with E. coli. We will concentrate on describing the plasmid known as pBR322 which was developed by Bolivar and Boyer.

The plasmid known as pBR322 was developed by Bolivar and Boyer in 1977. This plasmid has 4,362 base pairs that includes a special sequence of DNA that is needed as an origin (the ori site) for DNA replication and that allows for efficient replication. Usually 10--20 copies of the circular DNA are produced in each bacterial cell that harbors the plasmid.

Figure 20. The pBR322 plasmid

Two genes of pBR322 confer resistance to antibiotics to any cell that contains the plasmid. AmpR confers resistance to amplicillin and tetR confers resistance to tetracycline to cells containing the plasmids. AmpR is a gene that codes for the periplasmic enzyme beta-lactamase that cleaves the ring structure found in amphicillin, which is a penicillin antibiotic. TetR is a gene that codes for a protein that modifies the bacterial cell wall and prevents tetracycline from entering the cell.

Multiple restriction endonuclease sites are present where foreign DNA fragments may be inserted.

"Relaxed" plasmid DNA replication continues in the presence of chloramphenicol. An interesting feature of this plasmid is that "relaxed" plasmid DNA replication continues even in the presence of an inhibitor of protein synthesis such as chloramphenicol. This feature allows increased yields of plasmid/cell of up to 100-fold.