Question

pBR322 is a plasmid. It has two antibiotic resistance genes a the amp gene encoding the ampicillin resistance protein and the gene yet encoding the tetracycline resistance protein. In the plasmid, there is only one EcoRV restriction enzyme recognition sequence within the Tet gene. One EcoRI restriction enzyme recognition sequence between Tet gene and amp gene. Is amp gene a regulated gene or nonregulated gene. justify

Answer 1

pBR322 is the first artificial cloning vector constructed by boliver and Rodriguez. It contains ori, which allows to produce multiple copies per cell, it has two selectable markers ,( tetracycline and amplicillin - antibiotic resistance genes), and cloning sites for 12 endonucleases. of which two unique sites ( Pst I and Pvu I) are located in the ampicillin gene. Bam HI and Sal I within tetracyclin gene.

The presence of these unique restriction sites withi these antibiotic resistance genes permits us to easily select the transformants with pBR322. By inserting DNA fragments with enzyme Pst I or Pvu I , the DNA is get inserted within the ampicillin resistance gene, making this antibiotic resistance gene nonfunctional. Those bacterial cell which contains recombinant pBR322 cannot grow in the presence of ampicillin but can can grow in presence of tetracycline. If we insert DNA fragments with enzyme BamHI or Sal I, making the antibiotic resistance gene nonfunctional. then bacteria can grow only on ampicillin but can't on tetracycline. Hence ampicillin gene is a regulated gene.