Plasmid

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Image:Plasmids.png Plasmids are (typically) circular double-stranded DNA molecules separate from the chromosomal DNA (Fig. 1) and capable of autonomous replication. They usually occur in bacteria, sometimes in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae). Their size varies from 1 to over 400 kilobase pairs (kbp). There are anywhere from one copy, for large plasmids, to hundreds of copies of the same plasmid present in a single cell.

1 Antibiotic resistance
2 Episomes
3 Vectors
4 Types of plasmid
5 Applications of plasmids
6 Plasmid DNA extraction
7 Conformations
8 See also
9 External links

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Antibiotic resistance

Image:Example plasmid.png

Plasmids often contain genes or gene-cassettes that confer a selective advantage to the bacterium harboring them, e.g., 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 (Fig. 2)

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Episomes

Episomes are plasmids that can integrate themselves into the chromosomal DNA of the host organism (Fig. 3). For this reason, they can stay intact for a long time, be duplicated with every cell division of the host, and become a basic part of its genetic makeup. This 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 makes a plasmid into an episome.

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Vectors

Image:Plasmid episome.png

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 polylinker or multiple cloning site (MCS), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. See also 'Applications of plasmids', below.

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Types of plasmid

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 sexual 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. These plasmids can 'parasitise' another plasmid, transferring at high frequency in the presence of a conjugative plasmid.

It is possible for several different types of plasmids to coexist in a single cell, e.g., seven different plasmids have been found in E. coli. On the other hand, related plasmids are often 'incompatible', resulting in the loss of one of them from the cell line. Therefore, plasmids can Image:Conjugative plasmids.png be assigned into incompatibility groups, depending on their ability to coexist in a single cell. These incompatibility groupings are due to the regulation of vital plasmid functions.

An obvious way of classifying 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) colicines, proteins that can kill other bacteria.
Degrative 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)". These plasmids 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. This is an example of plasmids as selfish DNA.

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Applications of plasmids

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. There are many plasmids that are commercially available for such uses. Initially, the gene to be replicated is inserted in a plasmid. These plasmids contain, in addition to the inserted gene, one or more genes capable of providing antibiotic resistance to the bacterium that harbors them. The plasmids are next inserted into bacteria by a process called transformation, which are then grown on specific antibiotic(s). Bacteria which took up one or more copies of the plasmid then express (make protein from) the gene that confers antibiotic resistance. This is typically a protein which can break down any antibiotics that would otherwise kill the cell. As a result, only the bacteria with antibiotic resistance can survive, the very same bacteria containing the genes to be replicated. The antibiotic(s) will, however, kill those bacteria that did not receive a plasmid, because they have no antibiotic resistance genes. In this way the antibiotic(s) acts as a filter selecting out only the modified bacteria. Now these bacteria can be grown in large amounts, harvested and lysed to isolate the plasmid of interest.

Another major use of plasmids is to make large amounts of proteins. In this case you grow the 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.

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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 archaetypes of which are the miniprep and the maxiprep. 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 ug) 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.

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Conformations

When performing DNA electrophoresis, plasmid DNA may appear in the following five conformations:

"Supercoiled" (or "Covalently Closed-Circular") DNA is fully intact with both strands uncut.
"Relaxed Circular" DNA is fully intact with both strands uncut, but has been enzymatically "relaxed" (supercoils removed).
"Supercoiled Denatured" DNA, is not a "natural" form present in vivo. It is a contaminent often produced in small quantities following excessive alkaline lysis; both strands are uncut but are not correctly paired, resulting in a compacted plasmid form.
"Nicked Open-Circular" DNA has one strand cut.
"Linearized" DNA has both strands cut site at only one site.

The relative electrophoretic mobility (speed) of these DNA conformations in a gel are as follows:

Nicked Open Circular (slowest)
Linear
Relaxed Circular
Supercoiled Denatured
Supercoiled (fastest)

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. Enzymes specifically break the DNA at certain short sequences. The resulting linear fragments form 'bands' after gel electrophoresis.

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