Usefulness of Nalural Plasmids is Cloning ‘ehides

Usefulness of Nalural Plasmids is Cloning Vehides : 
The term naiural’ i% used loosely in this contezt to dcsciibe plasmids which were not constructed an vitro for the sole purpose of cloning. Col El is a naturally occurring plasmid which specifics the producton of a
bacieriocin, colicin El. By necessity this plasmid aLso carries a gene which confers on host cells immunity to colicin El. RSF 2124 is a derwatise of Col El which carries a transposon specifying ampicillin resistance. The
cxact origm of pSC 101 is not clear but for the purposes of this discussaon we shall consider i to be a naiural’ plasmid. Details of these plasmids arc hnun rn T2hIi

To clone DNA in pSCIOI, the plasmid DNA and the DNA to be inserted are digested with Eco RI, mixed and treated with DNA ligase. The ligated molecules are then used to transform a suitable recipient to tetracycline- resistance. Unfortunately there is no easy genetical method of distinguishing chimaeras from reconstituted vector DNA unless the insert confers a new phcnotypc on the transformants. Two examples of the use of pSCIOI for cloning DNA are presented in the next section. Cloning with Col El as the
vector is a little simpler. Transformants are selected on the basis of immunity to colicin El and chimaeras recognized by their inability to produce colicin El. Unfortunately screening for immunity to colicin El is
not technically simple and plasmid RSF 2124 is more useful in this respect since transformants are selected by virtue of their ampicillin resistance. Col El and plasmids derived from it (see later) have a distinct advantage over pSClOl—thcy can be enriched with chloramphenicol. When chloramphenicol is added to a late log-phase culture of a Col El-containing strain of E. coil, chromosome replication ceases because of the need for corninued protein synthesis. However, the cessation of prolein synthesis has no effect on Cot El replication such that after 10-12 hours oser 30% of the DNA a the cells is plasmid DNA (Hcrshflckl ci al. 1974). Since there may be 1000-3000 copies of the plasmid in each cell it is easy io sec why chloramphcmcol enrichment is a useful step in plasmid isolation.