Plasmids as Vectors, Autonomous Elements, Extrachromosomal Units, DNA Molecules, Bacterial Cell, Eukaryotic Cells

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Plasmids as vectors

Plasmids are defined as autonomous elements, whose genomes exist in the. cell as extrachromosomal units. They are self replicating circular (only rarely linear) duplex DNA molecules, which are maintained in a characteristic number of copies in a bacterial cell, yeast cell or even in organelles found in eukaryotic cells.

These plasmids can be single copy plasmids that are maintained as one plasmid DNA per cell or multicopy plasmids, which are maintained as 10-20 genomes per cell. There are also plasmids, which are under relaxed replication control, thus permitting their accumulation in very large numbers (upto 1000 copies per cell). These are the plasmids which are used as cloning vectors, due to their increased yield potential.

The Concept of Cloning a Foreign DNA Segment in Plasmid Vector

1. Plasmid pBR322	4. amp^R	7. organism for gene cloning
2. tet^R	5. plasmid confers resistance to both ampicillin and tetracycline	8. foreign DNA fragments
3. restriction endonuclease	6. restriction cut	9. tet^Rgene intect
10. foreign DNA insertion	11. amp^R gene ineffective

Circular plasmid DNA which is used as a vector, can be cleaved at one site with the help of a restriction enzyme to give a linear DNA molecule. A foreign DNA segment can now be inserted, by joining the ends of broken circular DNA to the two ends of foreign DNA, thus regenerating a bigger circular DNA molecule that can now be separated be gel electrophoresis on the basis of its size. Selection of bacterial colonies carrying chimeric plasmid DNA is also facilitated by the resistance genes, which the plasmid may carry against one or more antibiotics.

If a plasmid has two such genes conferring resistance against two antibiotics and if the foreign DNA insertion site lies within one of these two genes, then the chimeric vector loses resistance against one antibiotic, the gene for which has foreign DNA inserted within its structure.

In such a situation, the parent vector in bacterial cells can be selected by resistance against two antibiotics and the chimeric DNA can be selected by retention of resistanct1 against only one of the two antibiotics.

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