Biotechnology: Principles and Processes

Biology · Class 12

Simulation · Biology · Class 12

pBR322: finding recombinants by replica plating

From the lesson Cloning vectors in Biotechnology: Principles and Processes. Change the values and watch what happens.

pBR322: finding recombinants by replica platingBiology · Class 12

The idea behind it

NCERT §9.2.2 (i)-(iii)

  • 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.