Biotech Basics: A Plasmid Primer

What are plasmids?

 In 1952, Nobel Prize Winner Joshua Lederberg introduced the term ‘plasmid’, a double-stranded, circular DNA molecule that naturally exists in bacterial cells and some eukaryotes (Fig. 1). Plasmids have been used by molecular biologists to study, analyze, and clone genes. They are capable of self-replicating and are distinct from the cells’ chromosomal DNA. Although not typically essential for the cell’s survival, an important aspect of plasmids is the genetic advantage they provide to the bacterial cell by expressing antibiotic resistance genes. This characteristic primarily impacts the treatment of humans and animals with disease.

Figure 1:  A bacterial cell containing a plasmid.
Figure 1: A bacterial cell containing a plasmid. ©Edvotek 2014

What do they contain?

Figure 2: Plasmid Features.
Figure 2: Plasmid Features.

The three main components of a plasmid (Fig. 2) are origin of replication, selectable marker gene (antibiotic resistance gene), and a multiple cloning site (MCS). The origin of replication is a 50-100 base pair sequence in the plasmid where replication is initiated.  Antibiotic resistance is a selectable marker that protects the plasmid from an antibiotic that would normally kill it (e.g. tetracycline, ampicillin).  The MCS is a sequence of DNA which can be cleaved by restriction enzymes for cloning purposes.

What are the different conformations?

 For plasmids to be used for research and other scientific purposes, plasmid DNA has to be isolated from its bacterial host (E. coli). Plasmid isolation techniques include the mini-prep and maxi-prep methods. The mini prep is plasmid preparation done on a small scale and maxi-prep is plasmid preparation done on a large scale.

Figure 3: Plasmid Confirmations. ©Edvotek 2014
Figure 3: Plasmid Confirmations. ©Edvotek 2014

Once the plasmid DNA has been isolated, different conformations can be observed on an agarose gel. The majority of ‘uncut’ plasmid DNA exists in the supercoiled conformation. This is the native conformation found in vivo and migrates the fastest (and farthest) in an agarose gel.

Although most of the uncut DNA is supercoiled, other conformations are also present due to mechanical manipulation during plasmid purification. This results in a loss of the supercoiling and gives conformations like, ‘nicked’ and ‘linear’. Upon digestion with a restriction enzyme or after the DNA has been ‘cut’, the conformation is linear and one or more bands are observed depending on the number of restriction enzymes used (Fig. 3.)

What are they used for?

 The multiple cloning site in the plasmid DNA allows it to be used as a vector into which a gene of interest can be inserted after utilizing the appropriate enzymes to cut the plasmid. This technique is commonly known as recombinant DNA technology or molecular cloning. DNA recombinant technology has allowed scientists to create sequences that are not normally found in biological systems and can be used for biotechnology, medicine, and research.

Figure 4:  Molecular Cloning Strategies
Figure 4: Molecular Cloning Strategies

For more information about transformation, or the process through which bacteria can acquire exogenous DNA from the surrounding environment, check out our Quick Guide on the subject!

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