Cloning vectors

Plasmids and bacteriophages replicate inside bacterial cells independently of the chromosomal DNA. Phages have very high copy numbers; some plasmids have one or two copies per cell and others 15-100, sometimes more.

DNA linked to a plasmid or phage is multiplied to that vector's copy number. Modern vectors are engineered to make linking foreign DNA and selecting recombinants easy.

Origin of replication (ori): the sequence where replication starts. Any DNA linked to it can replicate in the host, and ori also controls the copy number, so a high-copy ori is used to recover many copies.

Selectable marker: it identifies and eliminates non-transformants and lets transformants grow. Genes for resistance to ampicillin, chloramphenicol, tetracycline or kanamycin are useful markers for E. coli, since normal E. coli resists none of these.

Cloning sites: the vector should have very few, preferably single, recognition sites for the common restriction enzymes. Several sites would cut it into many fragments and complicate cloning.

pBR322, an E. coli vector, carries ori, ampR and tetR, rop (which codes for proteins involved in plasmid replication), and sites for Hind III, EcoR I, BamH I, Sal I, Pvu II, Pst I and Cla I.

Foreign DNA ligated at the BamH I site inside tetR makes the recombinant lose tetracycline resistance. On ampicillin all transformants grow. Moved to tetracycline, recombinants fail while non-recombinants grow on both antibiotics.

One resistance gene thus selects transformants, while the other, inactivated by the insert, picks out recombinants. Needing two plates makes this cumbersome.

Easier: insert the DNA into the coding sequence of the enzyme β-galactosidase (insertional inactivation). With a chromogenic substrate, colonies without an insert turn blue; recombinant colonies stay colourless.