Plasmid

A plasmid is an extrachromosomal DNA molecule separate from the chromosomal DNA and capable of autonomous replication. In many cases, It is typically circular and double-stranded. It usually occurs naturally in bacteria, and is sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae).

The size of plasmids varies from 1 to over 400 kilobase pairs (kbp). There may be one copy, for large plasmids, to hundreds of copies of the same plasmid in a single cell, or even thousands of copies, for certain artificial plasmids selected for high copy number (such as the pUC series of plasmids). Plasmids can be part of the mobilome, since they are often associated with conjugation, a mechanism of horizontal gene transfer.

The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952.^[1]

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Antibiotic resistance

Plasmids often contain genes or gene cassettes that confer a selective advantage to the bacterium harboring them, such as the ability to make the bacterium antibiotic resistant.

Every plasmid contains at least one DNA sequence that serves as an origin of replication, or ori (a starting point for DNA replication), which enables the plasmid DNA to be duplicated independently from the chromosomal DNA (Figure 2). The plasmids of most bacteria are circular, like the plasmid depicted in Figure 2, but linear plasmids are also known, which superficially resemble the chromosomes of most eukaryotes.

Episomes

An episome is a plasmid that can be added, without integration, to the chromosomal DNA of the host organism (Fig. 3). In this situation, it can stay intact for a long time, be either diluted out or be duplicated with every cell division of the host, and become a basic part of its genetic makeup. The term is no longer commonly used for plasmids, since it is now clear that a region of homology with the chromosome such as a transposon will make a plasmid into an episome. In mammalian systems, the term episome refers to a circular DNA (such as a viral genome) that is maintained by noncovalent tethering to the host cell chromosome. In detail, we distinguish:

• Minichromosomes (MCs) that contain all elements for replication and segregation (telomeres, centromeres); MCs are usually obtained by chromosome engineering (e.g. telomere-seeding, the use of site-specific recombinases and/or the recombinase-mediated cassette exchange concept)
• Plasmids and Minicircles that stay extra-chromosomal (episomal) in non- or barely-dividing tissues (note: ´minicircles´ are plasmids devoid of prokaryotic sequence parts and thereby poor in inactivating CpG tracts)
• Replicating minicircles, i.e. plasmid derivatives that can recruit all components necessary for autonomous replication from the host cell. They replicate once per cell cycle and do not undergo epigenetic inactivation. In contrast to viral circular episomes (SV40, BPV, EBV, HSV) no viral components (proteins) are required for autonomous relpication.

Vectors

Plasmids used in genetic engineering are called vectors. They are used to transfer genes from one organism to another and typically contain a genetic marker conferring a phenotype that can be selected for or against. Most also contain a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location.Generally the polycloning site is present within the one of the antibiotic marker gene soppose tetracycline resistance gene in case of the pBR vector. So the insertion of the target DNA inactivates the marker gene.This is the basis of the selection of the recombinant and this process is known as the insertional inactivation.. See Applications below.^{[citation needed]}

Types

  One way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative plasmids contain so-called tra-genes, which perform the complex process of conjugation, the transfer of plasmids to another bacterium (Fig. 4). Non-conjugative plasmids are incapable of initiating conjugation, hence they can only be transferred with the assistance of conjugative plasmids, by 'accident'. An intermediate class of plasmids are mobilizable, and carry only a subset of the genes required for transfer. They can 'parasitise' a conjugative plasmid, transferring at high frequency only in its presence. Plasmids are now being used to manipulate DNA and may possibly be a tool for curing many diseases.

It is possible for plasmids of different types to coexist in a single cell. Seven different plasmids have been found in E. coli. But related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions. Therefore, plasmids can be assigned into compatibility groups.

Another way to classify plasmids is by function. There are five main classes:

• Fertility-F-plasmids, which contain tra-genes. They are capable of conjugation.
• Resistance-(R)plasmids, which contain genes that can build a resistance against antibiotics or poisons. Historically known as R-factors, before the nature of plasmids was understood.
• Col-plasmids, which contain genes that code for (determine the production of) bacteriocins, proteins that can kill other bacteria.
• Degradative plasmids, which enable the digestion of unusual substances, e.g., toluene or salicylic acid.
• Virulence plasmids, which turn the bacterium into a pathogen.

Plasmids can belong to more than one of these functional groups.

Plasmids that exist only as one or a few copies in each bacterium are, upon cell division, in danger of being lost in one of the segregating bacteria. Such single-copy plasmids have systems which attempt to actively distribute a copy to both daughter cells.

Some plasmids include an addiction system or "postsegregational killing system (PSK)", such as the hok/sok (host killing/suppressor of killing) system of plasmid R1 in Escherichia coli.^[2] They produce both a long-lived poison and a short-lived antidote. Daughter cells that retain a copy of the plasmid survive, while a daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate because of the lingering poison from the parent cell.

Applications

Plasmids serve as important tools in genetics and biochemistry labs, where they are commonly used to multiply (make many copies of) or express particular genes.^[3] Many plasmids are commercially available for such uses.

The gene to be replicated is inserted into copies of a plasmid which contains genes that make cells resistant to particular antibiotics. Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria which take up copies of the plasmid survive the antibiotic, since the plasmid makes them resistant. In particular, the protecting genes are expressed (used to make a protein) and the expressed protein breaks down the antibiotics. In this way the antibiotics act as a filter to select only the modified bacteria. Now these bacteria can be grown in large amounts, harvested and lysed (often using the alkaline lysis method) to isolate the plasmid of interest.

Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacteria produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for, for example, insulin or even antibiotics.

However, a plasmid can only contain inserts of about 1-10 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes or yeast artificial chromosomes could be used.

Plasmid DNA extraction

As alluded to above, plasmids are often used to purify a specific sequence, since they can easily be purified away from the rest of the genome. For their use as vectors, and for molecular cloning, plasmids often need to be isolated.

There are several methods to isolate plasmid DNA from bacteria, the archetypes of which are the miniprep and the maxiprep/bulkprep.^[3] The former can be used to quickly find out whether the plasmid is correct in any of several bacterial clones. The yield is a small amount of impure plasmid DNA, which is sufficient for analysis by restriction digest and for some cloning techniques.

In the latter, much larger volumes of bacterial suspension are grown from which a maxi-prep can be performed. Essentially this is a scaled-up miniprep followed by additional purification. This results in relatively large amounts (several micrograms) of very pure plasmid DNA.

In recent times many commercial kits have been created to perform plasmid extraction at various scales, purity and levels of automation. Commercial services can prepare plasmid DNA at quoted prices below $300/mg in milligram quantities and $15/mg in gram quantities (early 2007).

Conformations

Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during electrophoresis. The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest:

• "Nicked Open-Circular" DNA has one strand cut.
• "Linear" DNA has free ends, either because both strands have been cut, or because the DNA was linear in vivo. You can model this with an electrical extension cord that is not plugged into itself.
• "Relaxed Circular" DNA is fully intact with both strands uncut, but has been enzymatically "relaxed" (supercoils removed). You can model this by letting a twisted extension cord unwind and relax and then plugging it into itself.
• "Supercoiled" (or "Covalently Closed-Circular") DNA is fully intact with both strands uncut, and with a twist built in, resulting in a compact form. You can model this by twisting an extension cord and then plugging it into itself.
• "Supercoiled Denatured" DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation. You can model this by twisting a badly frayed extension cord and then plugging it into itself.

The rate of migration for small linear fragments is directly proportional to the voltage applied at low voltages. At higher voltages, larger fragments migrate at continually increasing yet different rates. Therefore the resolution of a gel decreases with increased voltage.

At a specified, low voltage, the migration rate of small linear DNA fragments is a function of their length. Large linear fragments (over 20kb or so) migrate at a certain fixed rate regardless of length. This is because the molecules 'reptate', with the bulk of the molecule following the leading end through the gel matrix. Restriction digests are frequently used to analyse purified plasmids. These enzymes specifically break the DNA at certain short sequences. The resulting linear fragments form 'bands' after gel electrophoresis. It is possible to purify certain fragments by cutting the bands out of the gel and dissolving the gel to release the DNA fragments.

Because of its tight conformation, supercoiled DNA migrates faster through a gel than linear or open-circular DNA.

See also

• Bacterial artificial chromosome
• Triparental mating
• Transposon
• Bacteriophage
• Provirus
• Segrosome

References

• Klein, Donald W.; Prescott, Lansing M.; Harley, John (1999). Microbiology. Boston: WCB/McGraw-Hill. 

1. ^ LEDERBERG J (1952). "Cell genetics and hereditary symbiosis". Physiol. Rev. 32 (4): 403-30.
2. ^ Gerdes K, Rasmussen PB, Molin S (1986). "Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells". Proc. Natl. Acad. Sci. U.S.A. 83 (10).
3. ^ ^{a b} Russell, David W.; Sambrook, Joseph (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory.