Host and vector: the two partners

Gene cloning has two main steps. First, the gene we want, called the insert, is isolated. Then it is joined to a carrier DNA, the vector. The vector with its insert is recombinant DNA, and it is put into a host cell, which copies it and can make its product.

Think of the vector as a vehicle: the gene cannot reach the host and multiply on its own, so it travels inside the vector. But the vehicle needs a driver. The vector brings the DNA sequences needed for its replication (such as its origin of replication); the host brings the machinery: the enzymes and proteins that actually copy the DNA. So cloning is a two-part system.

A good host:

• lets the rDNA in easily;
• does not destroy it as foreign DNA;
• supplies all the enzymes needed to copy and express it.

The most used prokaryotic host is E. coli (strain K12): a rod-shaped, Gram-negative bacterium that doubles about every 20 minutes, so it multiplies very fast. Bacillus subtilis is used when the protein should be secreted out of the cell. Among eukaryotes, yeast is the most used host.

A good vector:

• is small, yet can carry a large insert;
• has an origin of replication (ori), so it can copy itself inside the host;
• has unique restriction sites: each enzyme should cut it at only one place, to open it for the insert. With several sites, it would be chopped into pieces;
• has at least one selectable marker, such as the tetR or ampR gene for resistance to tetracycline or ampicillin, so that cells that took up the vector (transformants) can be picked out: only they survive on the antibiotic.

Each kind of host has its own vectors: E. coli uses plasmids (such as pBR322) and the virus phage lambda; yeast uses YACs (yeast artificial chromosomes); plants use the Ti plasmid (pTi); animal cells use viruses such as vaccinia.