plasmid questions -2

2. Replication of plasmid ColE1 is catalyzed by the host encoded DNA polymerase I protein (Pol I). In wild-type cells, Pol I is present in large excess over that needed for plasmid replication. Initiation of plasmid replication is down regulated by the plasmid encoded Rop protein which is present in limiting amounts. If the protein synthesis inhibitor chloramphenicol was added to sensitive cells that carry a ColE1 plasmid, what would happen to the plasmid copy number and why?

ANSWER: Addition of chloramphenicol will prevent synthesis of new proteins, including both Pol I and Rop. Pol I is present in excess, so even in the absence of protein synthesis there is sufficient Pol I in the cells for many rounds of plasmid replication. In contrast, Rop is limiting, so in the absence of new protein synthesis there is insufficient Rop to inhibit plasmid replication. This result in multiple rounds of plasmid replication and the accumulation of plasmids to a copy number much greater than normally found (up to about 1000 copies of the plasmid per cell), a process called plasmid amplification. This can be used as a trick to facilitate purification of plasmid DNA from cells.