pBR322: finding recombinants by replica plating.

PBR322 insertional inactivation: insert at BamH I (tetR) or Pst I/Pvu I (ampR), transform, replica-plate on ampicillin and tetracycline.

Pick out the recombinant colonies: those that grow on ampicillin but are missing from the tetracycline replica (BamH I insert)

Expecting recombinants with an insert at BamH I to grow on tetracycline.

• 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.

Teaching this idea? Put the simulation on your own page or LMS with this code.