Gene Cloning with Plasmid Vector pBR322

Plasmid Vectors

Plasmid is an extrachromosomal, small, circular double-stranded DNA that is distinct from a cell’s chromosomal DNA. The plasmid behaves as a cloning vector, providing the replicative ability that enables the cloned gene to be propagated inside the host cell. Plasmids can replicate efficiently in bacterial hosts because each plasmid contains an origin of replication (ori) which is recognized by the DNA polymerases and other proteins that normally replicate the bacterium’s chromosomes. The host cell’s replicative machinery therefore propagates the plasmid along with any new genes that have been inserted into it.

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pBR322

• pBR322 plasmid is small contains just 4363 bp

• Has origin of replication

• Carries genes that code enzymes which enable the host bacterium to survive in the presence of two antibiotics: ampicillin (amp) and tetracycline (tet) resistant.

Mechanism of gene cloning with plasmid vector pBR322:

• An animal gene has been obtained from an animal DNA and a small E.coli plasmid has been purified and treated with the same RE, that cuts the plasmid in a single position.
• Then these two DNA molecules are added together with DNA ligase. So the injection of animal gene into the plasmid is completed.
• The recombinant plasmid is now reintroduced into E. coli, and the inserted gene does not disrupt its replicative ability. The plasmid is recognized by the DNA polymerases and other proteins that normally replicate the bacterium’s chromosomes.
• Then the plasmid as well as the inserted gene will be replicated and after the cell division, the daughter cells will possess the recombinant plasmid.
• More rounds of plasmid replication and cell division will cause in a colony of recombinant E. coli bacteria, each of which will contain multiple copies of the animal gene.

Screening of recombinant DNA using pBR322 vector:

After transformation, cells are plated onto ampicillin agar. All cells that contain a pBR322 plasmid, either recombinant or not, will divide and produce a colony. The colonies are then transferred onto tetracycline agar medium by replica plating, which results in the colonies on the second plate retaining the relative positions that they had on the first plate. Some colonies do not grow on the tetracycline agar because their cells contain recombinant pBR322 molecules that have a disrupted tetracycline-resistance gene. These are the colonies that we are looking for because they contain the cloned gene, so we go back to the ampicillin plate, from which samples of the cells can be recovered.