Finding the recombinants

Only a few cells take up a plasmid, and only some of those plasmids carry the insert. So we must select the right cells. Recombinant cells are made to differ in some visible trait: antibiotic resistance, a protein such as β-galactosidase or the glowing GFP, or the need for a nutrient such as leucine.

Direct selection. If the plasmid carries an antibiotic-resistance gene, grow the cells on that antibiotic: cells without the plasmid die, and those with it grow.

Insertional inactivation. Here, the insert is placed inside one of two marker genes, which switches it off. Take a plasmid with ampR and tetR, and put the insert into ampR:

• On a tetracycline plate (the master plate), all cells carrying the plasmid grow, with or without the insert.
• The colonies are copied onto an ampicillin plate (the replica plate) by pressing a membrane onto the master and then onto the new plate.
• Recombinant cells, whose ampR gene was broken, cannot grow on ampicillin.

So the colonies that grow on the master but not on the replica are the recombinants.

Blue-white selection is quicker. The insert goes into the lacZ gene, which makes β-galactosidase. The cells are grown on ampicillin with two chemicals, X-gal and IPTG. Where lacZ is intact, β-galactosidase turns X-gal into a blue pigment. Where the insert has broken lacZ, there is no enzyme, and the colonies stay white. So white colonies are recombinant.

GFP (green fluorescent protein, from a jellyfish) can be used the same way: with the insert inside the GFP gene, recombinant bacteria do not glow under UV light.