Types

One way off grouping plasmids is by to their ability to transfer to other bacteria. Conjugative plasmids contain so-called tra-constrained , which perform the complex process off conjugation , the sexual transfer off plasmids to another bacterium (fig. 4). Non-conjugative plasmids unable are off initiating conjugation, hence they edge only Be transferred with the assistance off conjugative plasmids, by “accident”. Year intermediate class off plasmids are mobilizable , and curry only has subset off the obstructed required for transfer. They edge “parasitize” has conjugative plasmid, transferring At high frequency only in its presence.

Possible It is for plasmids off different types to coexist in has individual concealment. Different Seven plasmids cuts been found in E. coli . Goal related plasmids are often incompatible, in the judicious that only one off them survive in the concealment line, due to the regulation off vital plasmid functions. Therefore, plasmids edge Be assigned into compatibility groups .

Another way to classify plasmids is by function. Five There are hand classes:

• Fertility- F-plasmid S , which contain tra-constrained. They able are off conjugation.
• Resistance- (R) plasmids , which contain constrained that edge build has resistance against Antibiotic S gold Poison S. Historically known ace R-factors, before the natural off plasmids was understood.
• Collar-plasmids , which contain constrained that code for (given the production off) Colicine S, Protein S that edge kill other bacteria.
• Degrative plasmids , which enable the digestion off unusual substances, e.g., Toluene gold Salicylic acid.
• Virulence plasmids , which turn the bacterium into has Pathogen.

Plasmids edge belong to more than one off thesis functional groups.

Plasmids that exist only ace one gold has few copies in each bacterium are, upon Cell division, in danger off being lost in one off the segregating bacteria. Such individual-Copy plasmids cuts systems which attempt to actively distribute has Copy to both daughter concealments.

Summon plasmids include year addiction system gold " postsegregational killing system (PSK) ". They produce both has length-lived Poison and has shorts-lived Antidote. Daughter concealments that retain has Copy off the plasmid survives, while has daughter concealment that fails to inherit the plasmid dies gold suffers has reduced growth-spleen because off the lingering poison from the relative concealment.

Applications

Plasmids serf important ace tools in genetics and biochemistry labs, where they are commonly used to multiply (make many copies off) constrained gold express train particular. Many plasmids are commercially available for such use.

The obstructs off to Be replicated is inserted into copies has plasmid which contains obstructed that make concealments resisting to particular antibiotics. Next, the plasmids are inserted into bacteria by has process called transformation . Then, the bacteria are exposed to the particular antibiotics. Only bacteria which take up copies off the plasmid survives the antibiotic, since the plasmid makes them resistant. In particular, the protecting constrained are expressed (used to make has protein) and the expressed protein station-wagons down the antibiotics. In this way the antibiotics act ace has filter to select only the modified bacteria. Now thesis bacteria edge Be grown in broad amounts, harvested and Lysis D to isolate the plasmid off interest.

Another major uses plasmids is to make off broad amounts off proteins. In this box you grow bacteria containing plasmid harboring the embarrassment off interest has. Just ace the bacteria produces proteins to confer its antibiotic resistance, it broad edge also Be induced to produce amounts off proteins from the inserted embarrassment. This has cheap and easy way off farmhouse-producing has embarrassment gold the protein it then codes for, for example, Insulin gold even Antibiotic S.

Plasmid DNA extraction

Ace alluded to above, plasmids are often used to purify has specific sequence, since they edge easily Be purified away from the rest off the genome. For to their uses ace vectors, and for molecular cloning, plasmids often need to Be isolated.

There are several methods to isolate plasmid DNA from bacteria, the archaetypes off which are the miniprep and the maxiprep . To form edge correct Be used to quickly find out whether the plasmid is in any off several bacterial clones. The yield has off small amount impure plasmid DNA, which is sufficient for analysis by Restriction digest and for nap cloning technical.

In to lath, much larger volumes off bacterial suspension are grown from which has maximum-Prep edge Be performed. Essentially this has scaled-up miniprep followed by additional purification. Broad This results in relatively amounts (several ug) off very pure plasmid DNA.

In recent times many commercial kits cuts been created to perform plasmid extraction At various scales, purity and levels off automation. Commercial services edge prepares 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 off five conformations, which (for has given size) run different At speeds in has freezing during Electrophoresis. The conformations are listed below in order off electrophoretic mobility (speed for has given applied voltage) from slowest to fastest:

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

The misses off migration for small linear fragments is directly proportional to the voltage applied At low voltages. At higher voltages, larger fragments migrate different At continually increasing yet spleens. Therefore the resolution off has freezing decreases with increased voltage.

At has specified, low voltage, the migration misses off small linear DNA fragments has function off to their length. Broad linear fragments (over 20kb gold so) migrate At has some off fixed spleen regardless length. This is because the molecules “reptate”, with the bulk off the molecule following the leading end through the freezing matrix. Restriction digest S are frequently used to analyzes purified plasmids. Thesis enzymes specifically station-wagon the DNA At certain shorts sequences. The resulting linear fragments form “bands” after Freezing electrophoresis. Possible It is to purify certain fragments by cutting the bands out off the freezing and dissolving the freezing to release the DNA fragments.

See also

• Bacterial artificial chromosome

External links

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A plasmide indicates in microbiology or molecular biology a Molécule of DNA disctincte of the chromosomal DNA, capable of autonomous replication. The term plasmide was introduced by the American molecular biologist Joshua Lederberg in 1952.

The plasmides are generally circular. They are quasi-exclusivement in the Bactérie S, except notable for the plasmide 2Mu that one finds lodged by the micro-organism eucaryote Saccharomyces cerevisiae (yeast of the baker).

A bacterial cell can contain a copy of it, for large the plasmides, or of the hundreds for artificial plasmides (built by genetic engineering at ends of gene cloning). The Bactérie S generally comprise 5 to 30 copies of them, the Levure S between 50 and 100 specimens per cell.

Several plasmides different can coexist in the same cell under condition of their mutual compatibility. Some plasmides are able to be integrated into the Chromosome S; one calls these plasmides épisome S.

The plasmides take part in the horizontal transfers of genes between the bacterial populations, and thus in the dissemination of genes conferring of the selective advantages (for example resistances to antibiotics or factors of virulence). The mobility of the plasmides (by conjugation) within the bacterial populations increases the spectrum of host of genes implied in virulence. These genes n the other hand offer a selective advantage for the plasmide and the bacteria hosts. One thus conceives nature quasi-ubiquitaire and persistent plasmides in the pathogenic bacteria.

Réplication and transmission

Each plasmide contains at least a sequence of DNA which is used as Origine of replication, or ori (a starting point of Réplication of the DNA), allowing the DNA plasmidic to be duplicated independently of the bacterial chromosome or saccharomycian (Figure 2), by using the “machinery” of the host cell. The plasmides can be circular, or sometimes linear, presenting a surface resemblance to the chromosomes eucaryotes.

As the plasmides present in the bacteria usually do not carry of Gène S essential with the cell Procaryote, their perenniality in a line of Bactérie S thus depends on various means of Stabilization of the plasmides, which results from various selection processes and environmental conditions.

The plasmides can be transmitted of a bacterium mother to a bacterium girl thanks to the bacterial conjugation via pili sexual. During the cellular division, the plasmides are distributed in a completely random way thus (contrary to the chromosomes), even if the probability remains weak, it may be that one of the two cells girls does not have any plasmide. The probability increases with the reduction in the number of plasmides present in the cell mother.

See also: Stabilization of the plasmides

Épisomes

A épisome is a plasmide which can be integrated in the chromosomal DNA of the cell-host (fig. 3). So there can remain intact for long periods, be duplicated with each cellular Division of the host, and become integral part of his genetic inheritance. The term is not any more of use for the plasmides, since it was established that an area of homology with the chromosome, like a Transposon, was made of a plasmide a épisome. In the mammalian systems, the term épisome refers to a circular DNA (like a viral genome) attached to the chromosome of the cell-host in a not-covalent way.

Vectors

Genetic marker conferring has Phenotype that edge Be selected for gold against. Most also contain has Polylinker gold Multiple cloning site (MCS), which has off shorts area containing several commonly used Restriction sites allowing the easy insertion DNA fragments At this hiring. See Applications below. -->Existant in a natural state, the plasmides in addition are very much used in the laboratories like vector of Clonage . This technology is usually used in molecular biology.

Properties coded by the plasmides

The plasmides conjugatifs

See also: Conjugation (genetic)

The plasmides conjugatifs are the first plasmides which were discovered in the bacterium Escherichia coli in the Années 1950. They are called also factors of fertility or plasmides F . These plasmides confers on the bacterium host the capacity of synthesis of pili known as sexual. Via these pili, the carrying bacterium (donneuse) can transfer a copy from the plasmide F by process of bacterial conjugation. The plasmides F at least have an origin of replication and all genes necessary to the synthesis of the pili and transfer of the plasmide. Some plasmides F are épisomes, i.e. they can be integrated in the chromosomal Génome.

Plasmides of resistance

The plasmides of resistance, also called plasmides or factors R , code resistances to the antibiotics and the heavy metals.

In 1959, with the Japan, one found among patients reached of bacterial Dysenterie an insensitivity to any treatment Antibiotique. In fact, the responsible bacterium, Shigella dysentariae , carried genes of resistance to several antibiotics never yet met. Thereafter, one found of it in other bacteria (like E. coli ) and one called these plasmides R .

These plasmides can protect the cell by various means: The synthesis of a protein of resistance to toxic substance: it will neutralize (by it denaturing, hydrolizing, etc) the activity Toxique of the substance. The plasmides can also modify the properties of envelope of the cell and return it Imperméable to toxic substance (as it is the case for heavy metals).

Metabolic plasmides

The metabolic plasmides carry genes allowing the use of some Nutriment S. At E. coli , the genes carried by these plasmides are for example: the use of the Citrate like source of Carbon, production of Sulfur, the hydrolysis of the Urea. At the Salmonelle S one observed the degradation of the Lactose what is completely unusual at this bacterial kind. The majority of these plasmides code the synthesis of one or several enzymes.

Plasmides of virulence

It proves that the pathogenic bacteria very often lodge plasmides conjugatifs which take part in the pathogenicity. The plasmides of virulence carry genes coding of the factors of virulence, having a role in the capacity Pathogène of the bacteria. For example the Escherichia coli enterotoxigenic (ETEC) responsible for the Diarrhée for the traveller (or tourista ) lodge at least two plasmides, one carrying genes coding a factor of colonization, the other coding of toxins.

In the same way, the determinants of the invasive capacity of the Shigella are carried by a plasmide (pInv). In other pathogenic bacteria (for example Salmonella ), these plasmides codes a proteinic complex located on the wall of the bacterium: it is the complex pili-adhésine which makes it possible the bacterium to adhere on hydrocarbon receivers located at the surface of certain cells eucaryotes in particular the Entérocyte S.

Plasmides of bactériocines

These plasmides codes the synthesis of an extracellular protein whose biosynthesis is lethal for the producing bacterium like for the other surrounding not-producing bacteria. However, these plasmides codes also a second intracellular protein of resistance to this first toxin. The Bactériocine S act on vital functions of the bacterium.

At E. coli , one will find various categories of bactériocines (colicines coded by the plasmides collar ) and for example the gene colE1 code a Endonucléase and the gene colE3 , a Ribonucléase which inactive the Ribosome S.

See also: Structure and operation of a opéron poison-antidote

Manufacture of plasmides recombining

To supplement

Application in genetic engineering

Application for the production of molecule

One wishes to cause production of by bacteria some Protéine (protein of interest). It is via plasmides that one will introduce into cultures of Bactérie S a gene coding our protein of interest and a gene of resistance to a Antibiotique X. One will select the bacteria having integrated the plasmides while making push the bacterial colonies on a medium containing antibiotic X; the bacteria not having integrated the plasmide will not develop.

Application for the sequencing

This application is particularly useful in genetic Engineering for the Séquençage of DNA: DNA of an unspecified cell is difficult with séquencer because it very often exceeds 40.000 KB (1 Kilo bases = 1.000 bases) and is in small quantity. Thus, to make the sequencing easier, one cuts out the DNA to be analyzed and the DNA plasmidic with a Enzyme of restriction, under specific conditions; the DNA with séquencer will be integrated in the DNA plasmidic and the plasmide will be transferred in the bacterial Hyaloplasme. Once put in culture, the bacterium will retort the DNA plasmidic (and thus the fragment with séquencer) in great quantity. After having extracted the DNA with séquencer, and having eliminated the remainder from DNA plasmidic by enzymes of restriction, one recovers the fragments of DNA reproduced to several thousands of specimens, which one can now séquencer easily .

External bonds

• Integration and Excision off has off Plasmid Flash Animation
• International Society for Plasmid Biology and other Mobile Genetic Elements
• History Plasmids with timeline

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