Cloning Vectors: Plasmids and Bacteriophages

Cutting out a gene of interest is only useful if that gene can then be carried into a host cell and copied there indefinitely. This is the job of a cloning vector -- a DNA molecule, usually a bacterial plasmid or a bacteriophage genome, that is capable of independent replication inside a host cell and can be engineered to carry a foreign DNA insert along with it.

Not every plasmid or phage genome makes a good cloning vector straight out of nature; a useful vector needs to be deliberately engineered to have a small set of essential features. First, it needs an origin of replication (ori), a specific DNA sequence at which replication of the plasmid begins; this is what allows the vector -- and any foreign DNA joined to it -- to be copied by the host cell's replication machinery and passed on to daughter cells. Second, it needs at least one selectable marker, typically a gene conferring resistance to a specific antibiotic (such as ampicillin or tetracycline resistance); this allows a researcher to distinguish host cells that have successfully taken up the vector (which will survive and grow on a medium containing that antibiotic) from the vast majority of host cells that have not taken up any vector at all (which will die on that medium). Third, it needs one or more unique restriction sites -- recognition sequences for common restriction enzymes that occur only once in the whole vector -- positioned where a foreign DNA fragment can be inserted without disrupting the vector's own essential genes; this region is often called the cloning site.

pBR322, one of the earliest and most widely used cloning vectors, illustrates all three features clearly. It is a small circular plasmid carrying its own origin of replication (ori), two different antibiotic-resistance genes -- ampR (conferring ampicillin resistance) and tetR (conferring tetracycline resistance) -- and several unique restriction sites, including sites for PstI (located within the ampR gene) and for BamHI and SalI (located within the tetR gene). It also carries a gene called rop, which is involved in regulating the copy number of the plasmid inside the host cell. …