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20-September-2008 09:29:09 - Plasmid Figure 1: Illustration of a bacterium with plasmids enclosed showing chromosomal DNA and plasmids. Figure 1: Illustration of a bacterium with plasmids enclosed showing chromosomal DNA and plasmids. A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA.1 In many cases, it is circular and double-stranded. 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 200 kilobase pairs kbp.2 The number of identical plasmids within a single cell can be zero, one, or 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 1952.3 Plasmids can be considered to be independent life-forms similar to viruses, since both are capable of autonomous replication in suitable host environments. However the plasmid-host relationship tends to be more symbiotic than parasitic although this can also occur for viruses, for example with endoviruses since plasmids can endow their hosts with useful packages of DNA to assist mutual survival in times of severe stress. For example, plasmids can convey antibiotic resistance to host bacteria, who may then survive along with their life-saving guests who are carried along into future host generations. Contents 1 Vectors 2 Gene Therapy 3 Types 4 Plasmid DNA extraction 5 Conformations 6 Simulation of plasmids 7 See also 8 References 9 External links 9.1 Episomes Vectors There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as in the top instance; whereas episomes, the lower example, integrate into the host chromosome. There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as in the top instance; whereas episomes, the lower example, integrate into the host chromosome. Further information: Vector molecular biology 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.2 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 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. Gene Therapy The success 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 or an immune response. Plasmid vectors 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 viable option in human cells. Plasmids encoding ZFN could be used to deliver a therapeutic gene to a pre-selected chromosomal site. This approach to gene therapy could be less problematic to the alternative viral-based delivery of therapeutic genes.4 Types Figure 4: Overview of Bacterial conjugation Figure 4: Overview of Bacterial conjugation 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. 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-Rplasmids, 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.5 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. 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.2 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. 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. 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. 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 Lasergene, GeneConstructionKit, and Vector NTI. See also Bacterial artificial chromosome Triparental mating Transposon Bacteriophage Provirus Segrosome References ^ Lipps G or. 2008. Plasmids: Current Research and Future Trends. Caister Academic Press. ISBN 978-1-904455-35-6. ^ a b c Russell, David W.; Sambrook, Joseph 2001. Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory. ^ LEDERBERG J 1952. Cell genetics and herary symbiosis. Physiol. Rev. 32 4: 403-30. ^ Kandavelou K; Chandrasegaran S 2008. Plasmids for Gene Therapy, Plasmids: Current Research and Future Trends. Caister Academic Press. ISBN 978-1-904455-35-6. ^ 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: 3116. doi:10.1073/pnas.83.10.3116. Klein, Donald W.; Prescott, Lansing M.; Harley, John 1999. Microbiology. Boston: WCB/McGraw-Hill. Smith, Christopher U. M.. Elements of Molecular Nerobiology. Wiley, 101,111. External links International Society for Plasmid Biology and other Mobile Genetic Elements History of Plasmids with timeline Episomes Overview: viral and non-viral episomes Replicating minicircles: novel non-viral episomes Retrieved from http://en..org/wiki/Plasmid Categories: Molecular biology | Mobile genetic elements | Molecular biology techniques | Microbiology Views Article Discussion this page History Personal tools Log in / create account Navigation Main page Contents Featured content Current events Random article Search Go Search Interaction Community portal Recent changes Contact Donate to Help Toolbox What links here Related changes Upload file Special pages Printable version Permanent link Cite this page Languages العربية Asturianu БългарÑ?ки Català Česky Dansk Deutsch Español Esperanto Euskara Ù?ارسی Français Galego Hrvatski Italiano עברית МакедонÑ?ки Bahasa Melayu Nederlands 日本語 ‪Norsk bokmÃ¥l‬ Occitan Polski Português Română РуÑ?Ñ?кий SlovenÅ¡Ä?ina СрпÑ?ки / Srpski Suomi Svenska Tiếng Việt Türkçe УкраїнÑ?ька West-Vlams ä¸æ–‡ This page was last modified on 7 August 2008, at 12:5
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