Plasmids as Cloning Vehicles

Basic Properlies of Plasmids
Plasmids arc widely used as cloning vehicles but before discussing their use in this context it is appropriate to review some of their basic properties. Plasmids are replicons which are stably inherited in an extra chromosomal slate. It should be emphasized that extrachromosomal nucleic acid molec ules are not necessarily plasmids. for the defimiton given above impliesgenetic homogeneity, constant monomeric unit size and the ability to replicate independently of the chromosome. Thus the heterogeneous circular DNA molecules which are found in Bacillus mezaterium (Carltoa & Helinski 1969) are not necessarily plasmids. The definition given above. however, does Include the prophagcs of those temperate phages. e.g. P1. which are maintained in an extrachromosomal state, as opposed to those Such as X (see next chapter) which are maintained by integration into the host chromosome. Also included arc the replicative forms of the filamcnt ous coliphages which specify the continued production and release of  phage particles without concomitant cell lysis. Plasmids are widely distributed throughout the prokaryotes. vary in size from less than I x 10’ daltons to greater than 200 x 10’. and are generally dispcnsabk. Some of the plenetypes which these plasmids confer on their host cells are listed in Table 3.1. Plasmids to which phenotypic traits have not yet been ascribed are caHed cryptic plasmids.

Pbmids can be caIegoized into one of the two majol types—conjugathe or non-conjugativc—dcpcnding on whether or no they carry a set of genes. called the Ira genes, that promotes bacictial conjugation. Plasmids can also be categoriied on the basis of their being maintained as multiple copies per cdl (rekaxed pbsnids) or a limited number of copies per ccli (stnngeni plasmids). The replication of stringent plasmids is of necessity coupled to chromosome replication, hence their low copy number. Generally. conjugative plasmids arc of a relatively high mol. wt. and arc present as1-3 copies per chromosome whcrcas non-conjugative plasmids arc of low mol. wL. and present as multiple copies per cell (Table 3.2). An exception is the conjugative plasmid R6K which has a mol. wi. of 25 x 10’ daltons and is maintained as a relaxed plasmid.

Plasmid incornpciibiliiy is the inability of two diffcrcrn plasmids to coexist in the same host cell an the abscncc of selection pressure. The turn incompatibility can only be used swhcn it is certain that entry of the second plasmid has taken place and that DNA reirktion is not insolscd. Goups of pLamids which arc mutually incompatible arc considered to belong to the same incompatibility class. Currently. over 25 incompatibility groups have been defined among plasmids of E. ctW,s and 7 for plasinids of Staphylococcus aureus. Wkh one exception, incompatibility is of relatively little importance for gene manipulation. Plasmids belonging to incompali. bility cLass P. e.g. RP4. arc termed promiscio.s for they are capable of promoing their own transfer to a wide range of Giam•ncgativc bacteria and of being stably maintained in these diverse hosts. Such promiscuous plasmids thus offer the potcrnial of readily transferring cloned DNA  rnolcceles Into a wide range of gcnctI environments. An esiremcly useful article cxplainh.ag the tcrni olu used in pla-smid genetics is that of Novick ci eL (1976). A much fuller discussio. of the topics outhned above is provided by FalLow (1975).

• The Purification of Plasmid DNA