Plasmids are extrachromosomal genetic elements found in a variety of bacterial species. They are double-stranded, closed circular DNA molecules that range in size from 1 kb to greater than 200 kb. Often, plasmids contain genes coding for enzymes that, under certain circumstances, are advantageous to the bacterial host. Among the phenotypes conferred by different plasmids are:
resistance to antibiotics
production of antibiotics
degradation of complex organic compounds
production of colicins
production of enterotoxins
production of restriction and modification enzymes
Under natural conditions, many plasmids are transmitted to new hosts by a process similar to bacterial conjugation. In the laboratory, however, plasmids can be transferred to bacteria by an artificial process, known as transformation , in which they are introduced into bacteria that have been treated in ways that make some of the cells temporarily permeable to small DNA molecules (see section transformation).
For the most part, replication of plasmid DNA is carried out by the same set of enzymes used to duplicate the bacterial chromosome. Some plasmids are under stringent control, which means that their replication is coupled to that of the host. Plasmids under relaxed control, on the other hand, have copy numbers of 10-200. More importantly, the copy number can be increased to several thousands per cell if host protein synthesis is stopped (e.g., by treatment with chloramphenicol ) [108]. In the absence of protein synthesis, replication of relaxed plasmids continues, whereas replication of chromosomal DNA ceases.
A plasmid employed as a cloning vector should possess several properties. It should be relatively small and replicate in a relaxed fashion (like the used pUC vector). In addition, it should carry one or more selection markers to enable identification of transformants and to maintain the plasmid in the bacterial population. Finally, it should contain a single recognition site for one or more restriction enzymes in regions not essential for plasmid replication. Preferably, these restriction sites, into which foreign DNA can be inserted, should be located within the genes coding for selectable markers so that insertion of a foreign DNA fragment inactivates the gene.
The most commonly cited plasmid vector is
pBR322. An alternative to pBR322 cloning
of DNA is to use pUC plasmids.
These are modified pBR322
vectors with the ampicillin resistance gene
and an added polylinker site, similar to that in the M13mp
vectors . Also included in pUC plasmids is the 5'
end of the lac Z gene
( 
-galactosidase ), which is
disrupted by insertion of cloned fragments into the polylinker site, allowing a
colour selection of insert-bearing fragments.
The amino-terminal portion of the -galactosidase protein produced in the
infected cells is able to complement (" 
complementation") a defective
-galactosidase gene present on the F episome in the host cell. This
complementation produces active -galactosidase
(gal 
), which gives rise to a
blue colour when the phage and cells are grown in the presence of the inducer
isopropyl-thiogalactoside (IPTG , see figure fig:iptg) and the
chromogenic
substrate Xgal (see figure 3.1).
Insert-bearing plasmids will fail to complement the gal 
genotype of an -complementing
E. coli host.
The plasmid
transformant remains gal , and cannot metabolize the chromogenic
substrate Xgal to a blue
pigment. Resulting colonies, grown on LB/ampicillin plates supplemented with
IPTG and Xgal will remain white[109].
Figure: 5-Bromo-4-chloro-3-indolyl- -D-galactoside (Xgal)
Figure: Isopropyl- -D-thiogalactopyranoside (IPTG)