Plasmids are self – reproducing, double – stranded, rounded DNA molecules that are retained in bacteria as independent extra chromosomal entities. Plasmids can be transferred simply from generation to generation bacterium, this process is called transformation. Therefore, they are used frequently in recombinant DNA application.
In maximum cases both DNA strands are intact circles, the molecules are termed as CCC DNA (covalently closed circles DNA). If one strand is intact then the molecules are stated as open circle or OC DNA.
Properties of plasmids: The properties of a plasmid as cloning vectors include:
· Low MW(molecular weight) & possible small size.
· Ability to confer selectable phenotypic trait on host cells.
· Its replication is not linked to the replication of chromosomal DNA.
· Single sites for a large number of restriction end nuclease.
· Have origins of replication and selective markers that are recognized in these cells.
· Have remarkable properties useful in genetic manipulation: ---------
ü They can pass from one cell to another.
ü Induced, from one species of bacteria to another.
Why large DNA is not incorporated into plasmid?
The plasmid size is very significant property for their selection as cloning vectors. A plasmid vector should be as small size as far as possible, because: --
8 Small DNA is less prone to damage by shearing during isolation.
8 This increased stability will allow relatively violent methods of processing like deproteinization of plasmid to get pure DNA.
8 Small molecules are more efficiently reserved by bacteria during transformation process.
Thus large DNA molecules are not incorporated into plasmid.
EXPRESSION VECTOR
The protein production by rDNA technology is done through the use of expression vectors that are mainly an assembly of covalently linked segments of DNA each performing a specific function.
Desirable properties of an expression vector:
ü Expression vectors must have a strong promoter (its activity is controlled by either temperature or level of inducer or repressor).
ü Also have an initiation codon.
ü One restriction site within or a small distance downstream from the initiation codon.
ü The origin of replication
ü The marker gene (coding for antibiotic resistance).
ü Fusion of the eukaryotic gene to the gene of a large protein related with the plasmid.
