Plasmid

A plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double stranded and, in many cases, circular. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae).

Plasmid size varies from 1 to over 1,000 kilobase pairs (kbp).The number of identical plasmids within a single cell can range anywhere from one to even thousands under some circumstances. Plasmids can be considered to 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.
Plasmids are considered transferable genetic elements, or "replicons", capable of autonomous replication within a suitable host. Plasmids can be found in all three major domains, Archea, Bacteria and Eukarya. Similar to viruses, plasmids are not considered a form of "life" as it is currently defined. Unlike viruses, plasmids are "naked" DNA and do not encode genes necessary to encase the genetic material for transfer to a new host, though some classes of plasmids encode the sex pilus necessary for their own transfer. Plasmid host-to-host transfer requires direct, mechanical transfer by conjugation or changes in host gene expression allowing the intentional uptake of the genetic element by transformation. Microbial transformation with plasmid DNA is neither parasitic nor symbiotic in nature, since each implies the presence of an independent species living in a commensal or detrimental state with the host organism. Rather, plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. Plasmids also can provide bacteria with an ability to fix elemental nitrogen or to degrade recalcitrant organic compounds which provide an advantage under conditions of nutrient deprivation.

Vectors:

There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as with the top instance; whereas episomes, the lower example, integrate into the host chromosome.

Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and 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. 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, 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.

 Applications:

 Disease Models:

Plasmids were historically used to genetically engineer the embryonic stem cells of rats in order to create rat genetic disease models. The limited efficiency of plasmid based techniques precluded their use in the creation of more accurate human cell models. Fortunately, developments in Adeno-associated virus recombination techniques, and Zinc finger nucleases, have enabled the creation of a new generation of isogenic human disease models.

 Gene therapy:

The success of some strategies of gene therapy depends on the efficient insertion of therapeutic genes at the appropriate chromosomal target sites within the human genome, without causing cell injury, oncogenic mutations (cancer) or an immune response. Plasmid vectors are one of many approaches that could be used for this purpose. Zinc finger nucleases (ZFNs) offer a way to cause a site-specific double strand break to the DNA genome and cause homologous recombination. This makes targeted gene correction a possibility in human cells. Plasmids encoding ZFN could be used to deliver a therapeutic gene to a pre-selected chromosomal site with a frequency higher than that of random integration. Although the practicality of this approach to gene therapy has yet to be proven, some aspects of it could be less problematic than the alternative viral-based delivery of therapeutic genes.
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 'parasitize' 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. Several 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 (transfer of genetic material between bacteria which are touching).
• Resistance-(R)plasmids, which contain genes that can build a resistance against antibiotics or poisons and help bacteria produce pili. 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 (one that causes disease).

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 or microbial hosts 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.^[8] This variant produces both a long-lived poison and a short-lived antidote. Several types of plasmid addiction systems (toxin/ antitoxin, metabolism-based, ORT systems) were described in the literature^[9] and used in biotechnical (fermentation) or biomedical (vaccine therapy) applications. 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. Finally, the overall productivity could be enhanced.

Yeast Plasmids:

Other types of plasmids, often related to yeast cloning vectors include:

• Yeast integrative plasmid (YIp), yeast vectors that rely on integration into the host chromosome for survival and replication, and are usually used when studying the functionality of a solo gene or when the gene is toxic. Also connected with the gene URA3, that codes an enzyme related to the biosynthesis of pyrimidine nucleotides (T, C);
• Yeast Replicative Plasmid (YRp), which transport a sequence of chromosomal DNA that includes an origin of replication. These plasmids are less stable, as they can "get lost" during the budding.

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. 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^[update]).

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.
• "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.
• "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.
• "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.

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 'resperate', 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.

Simulation of plasmids:

The use of plasmids as a technique in molecular biology is supported by bioinformatics software. These programmes record the DNA sequence of plasmid vectors, help to predict cut sites of restriction enzymes, and to plan manipulations. Examples of software packages that handle plasmid maps are Geneious, Lasergene, GeneConstructionKit, ApE, and Vector NTI.

Ti plasmid:

The structure of the Ti plasmid

Ti plasmid is a circular plasmid that often, but not always, is a part of the genetic equipment that Agrobacterium tumefaciens and Agrobacterium rhizogenes use to transduce its genetic material to plants. The Ti plasmid is lost when Agrobacterium is grown above 28°C. Such cured bacteria do not induce crown galls, i.e. they become avirulent. pTi and pRi share little sequence homology but are functionally rather similar. The Ti plasmids are classified into different types based on the type of opine produced by their genes. The different opines specified by pTi are octopine, nopaline, succinamopine and leucinopine.
The plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206,479 nucleotides long, the GC content is 56% and 81% of the material is coding genes. There are no pseudogenes.

The modification of this plasmid is very important in the creation of transgenic plants, but only in dicotyledon plants.

Virulence Region:

Genes in the virulence region are grouped into the operons virABCDEFG, which code for the enzymes responsible for mediating transduction of T-DNA to plant cells.
 virA codes for a receptor which reacts to the presence of phenolic compounds such as acetosyringone,, syringealdehyde or acetovanillone which leak out of damaged plant tissues.

• virB encodes proteins which produce a pore/pilus-like structure.
• virC binds the overdrive sequence.
• virD1 and virD2 produce endonucleases which target the direct repeat borders of the T-DNA segment, beginning with the right border
• virG activates vir-gene expression after binding to a consensus sequence, once it has been phosphorylated by virA.

R1 plasmid:

The R1 Plasmid is a plasmid that was first isolated from Salmonella paratyphi bacteria in 1963.
The R1 plasmid imparts multi-drug antibiotic resistance to its host bacteria.
It’s known as a “low copy” plasmid, meaning that it exists in relatively few copies in any given bacteria. Because of its low copy nature, R1 must rely on “type II” segregation system to ensure that at least one copy is contained in each daughter cell after mitosis.
Some genes on the R1 plasmid are:

• ParM is a prokaryotic actin homologue which provides the force to drive copies of the R1 plasmid to opposite ends of rod shaped bacteria before mitosis.
• The Hok/sok system a postsegregational killing system of the plasmid.
• CopA-like RNA, an antisense RNA involved in replication control of the plasmid.

Plasmid preparation:

Plasmid miniprep. 0.8% agarose gel ethidium bromide-stained.

A plasmid preparation is a method used to extract and purify plasmid DNA. Many methods have been developed to purify plasmid DNA from bacteria. These methods invariably involve three steps:

• Growth of the bacterial culture
• Harvesting and lysis of the bacteria
• Purification of plasmid DNA

Growth of the bacterial culture:

Plasmids are almost always purified from liquid bacteria cultures, usually E. coli, which have been transformed and isolated. Virtually all plasmid vectors in common use encode one or more antibiotic resistance genes as a selectable marker (Ex :kanamycin,Ampicillin), which allows bacteria that have been successfully transformed to multiply uninhibited.

Harvesting and lysis of the bacteria:

When bacteria are lysed under alkaline conditions both DNA and proteins are precipitated. Some scientists reduce the concentration of NaOH used to 0.1M in order to reduce the occurrence of ssDNA. After the addition of acetate-containing neutralization buffer the large and less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids can renature and stay in solution.

Preparations by size:

Kits are available from varying manufacturers to purify plasmid DNA, which are named by size of bacterial culture and corresponding plasmid yield. In increasing order, these are the miniprep, midiprep, maxiprep, megaprep, and gigaprep. The plasmid DNA yield will vary depending on the plasmid copy number, type and size, the bacterial strain, the growth conditions, and the kit.

Minipreparation:

Minipreparation of plasmid DNA is a rapid, small-scale isolation of plasmid DNA from bacteria. It is based on the alkaline lysis method invented by the researchers Birnboim and Doly in 1979. The extracted plasmid DNA resulting from performing a miniprep is itself often called a "miniprep". Minipreps are used in the process of molecular cloning to analyze bacterial clones. A typical plasmid DNA yield of a miniprep is 20 to 30 µg depending on the cell strain.

Miniprep Protocols:

http://www.protocol-online.org/prot/Molecular_Biology/Plasmid/Miniprep/

Midipreparation;

The starting E. coli culture volume is 15-25 ml of LB broth and the expected DNA yield is 100-350 µg.

Maxipreparation:

The starting E. coli culture volume is 100-200 ml of LB broth and the expected DNA yield is 500-850 µg.

Megapreparation:

The starting E. coli culture volume is 500 ml – 2.5 L of LB broth and the expected DNA yield is 1.5-2.5 mg.

Gigapreparation:

The starting E. coli culture volume is 2.5-5 L of LB broth and the expected DNA yield is 7.5–10 mg.

Purification of plasmid DNA:

Addition of phenol/chloroform can dissolve and denature proteins, like DNase. This is especially important if the plasmids are to be used for enzyme digestion. Otherwise, smearing may occur in enzyme restricted form of plasmid DNA.