Plasmids and plasmid vectors

Plasmids are small circles of double-stranded DNA that live outside the main chromosome and replicate by themselves. They are found in bacteria, archaea and even yeast. They range from a few thousand base pairs to more than 100 kbp, and when the cell divides, each daughter cell gets at least one copy. Plasmids that can also join the chromosome are called episomes.

Natural plasmids come in several types:

• R-plasmids carry genes for resistance to drugs (ampicillin, tetracycline, chloramphenicol). They are the most used for cloning, because their resistance genes make ready-made markers.
• Col plasmids make colicins, proteins that kill other bacteria.
• F-plasmids carry genes for transfer: they build a pilus, a tube through which DNA passes to another cell.

Plasmids also differ in copy number, how many copies a cell keeps:

• High-copy plasmids are not controlled by the chromosome's replication, so they make 10-30 copies per cell: relaxed replication. They give higher yields of the cloned gene.
• Low-copy plasmids follow the chromosome's control, with only one or a few copies: stringent replication.

Scientists then trimmed natural plasmids into better tools. pBR313 had relaxed replication, the tetR and ampR genes, and many unique restriction sites, but it was 9 kb long, and more than half of it was not needed. Cutting out the unneeded DNA gave pBR322, only 4,361 bp, which kept the ori, ampR, tetR and unique sites. Small vectors enter host cells more easily, and pBR322 became the most widely used cloning vector.

The next improvement was the multiple cloning site (MCS), or polylinker: a short, synthetic stretch of DNA packed with many unique restriction sites in a row, so that almost any enzyme can be used to insert a gene. In the vector pUC19 (2.686 kb), the MCS sits inside the lacZ gene. An insert breaks lacZ, which allows blue/white selection of recombinants: colonies with the insert stay white.