VPB 321: Animal Biotechnology (2+1)

PLASMIDS

• Plasmids are small, circular, extrachromosomal DNA molecules found in bacteria, which can replicate on their own, outside of a host cell.
• They have a cloning limit of 100 to 10,000 base pairs or 0.1-10 kilobases (kb).
• A plasmid vector is made from natural plasmids by removing unnecessary segments and adding essential sequences.
• Plasmids make excellent cloning vectors for various laboratory techniques, including recombinant DNA.

Features of pBR322

• pBR322 is a plasmid and the most commonly used E. coli cloning vectors.
• pBR322 was the first artificial plasmid , created in 1977.
• It was named after its Mexican creators, p standing for plasmid, and BR for Bolivar and Rodriguez.
• pBR322 is 4361 base pairs  in length and contains a replicon region (source plasmid pMB1), the ampR gene, encoding the ampicillin resistance protein (source plasmid [RSF2124]) and the tetR gene, encoding the tetracycline resistance protein (source plasmid pSC101).
• The plasmid has unique restriction sites for more than forty restriction enzymes.
• 11 of these 40 sites lie within the tetR gene.
• There are 2 sites for restriction enzymes HindIII and ClaI within the promoter of the tetR gene.
• There are 6 key restriction sites inside the ampR gene.
• The origin of replication or ori site in this plasmid is pMB1 (a close relative of ColE1).
• The ori encodes two RNAs (RNAI and RNAII) and one protein (called Rom or Rop).
• The circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the count increases through the tet genes.
• The ampicillin resistance gene is a penicillin beta-lactamase.
• Promoters P1 and P3 are for the beta-lactamase gene.