Plasmid DNA 101

A plasmid is extra chromosomal DNA that is not incorporated in the main helix of genomic DNA. Plasmids occur in nature and can be found everywhere – in plants, insects, and bacteria. Scientists have discovered various applications for plasmids including gene therapy and DNA vaccines. These applications deliver the plasmid to a specific target in an organism to obtain a desired response. Plasmids can be delivered as a naked molecule or encapsulated in a nanoparticle. Other delivery methods are being developed to improve plasmid delivery efficiency and increase serum survival lengths (see Minivector DNAs).

Most plasmids are designed to contain an origin of replication, a gene of interest and a gene for antibiotic resistance. In a process called cloning, a gene is inserted into an empty plasmid backbone (vector) using genetic engineering. Plasmids are specifically designed to replicate in bacteria without gene expression; yet, in their intended target, they express the gene without replication.                  

Once a plasmid is assembled with the proper components, it is transformed into a competent cell. In this process, electrical current or chemistry opens the bacterial pores to accept the plasmid, and the competent cells end up with several copies of the plasmid. To isolate only the plasmid-containing bacteria, the cells are cultured with the antibiotic corresponding to their antibiotic resistant gene.

 Since the plasmid includes a resistance marker to the antibiotic, a cell that accepts the plasmid will be resistant to the antibiotic. Cells that reject the plasmid will not be resistant and will be instantly killed. The resistant cells are frozen as the master cell bank (MCB), and the MCB is used to make a working cell bank (WCB). Upon completion of cell banking, the WCB is then fermented to replicate enough plasmid for use in animal studies and clinical trials.

-Henry H.

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