Draw a labelled diagram of the E. coli cloning vector pBR322. Explain how recombinants are selected using it.
How to lay out the answer. Follow the frame, then write it in your own words.
1 · Define a cloning vector. A cloning vector is a DNA molecule, such as a plasmid, that carries foreign DNA into a host and replicates there.
2 · Draw pBR322. [Diagram: circle with ori, rop, ampR, tetR; sites Hind III, EcoR I, Cla I, BamH I, Sal I, Pvu II, Pst I, Pvu I]
3 · List its features.
ori: replication and copy number.
ampR and tetR: selectable markers.
Single sites for several restriction enzymes.
rop: replication proteins.
4 · Insert the foreign DNA. Ligate it into the BamH I site inside tetR, which inactivates tetR.
5 · Select transformants. Plate on ampicillin: only cells carrying a plasmid grow.
6 · Select recombinants. Transfer to tetracycline: recombinants do not grow, non-recombinants do.
7 · Give the easier method. Insert inside the β-galactosidase gene: with a chromogenic substrate, recombinants give colourless colonies and non-recombinants blue.
8 · Close in one line. So insertional inactivation of a marker gene separates recombinants from non-recombinants.
Scoring tip: Put every restriction site on the right gene: BamH I and Sal I in tetR, Pst I and Pvu I in ampR. Examiners check this.
