A plasmid is a small antibiotic resistance), and can frequently be transmitted from one bacterium to another (even of another species) via horizontal gene transfer. While the chromosomes are big and contain all the essential information for living (an adequate analogy is the hard-drive of a computer), plasmids usually are very small and contain additional information (in this analogy, plasmids are the USB flash drives). Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms.
The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952. Plasmid sizes vary from 1 to over 1,000 kbp. The number of identical plasmids in a single cell can range anywhere from one to thousands under some circumstances. Plasmids can be found in all three major domains: Archaea, Bacteria, and Eukarya.
Plasmids are considered replicons, capable of replicating autonomously within a suitable host. However, plasmids, like viruses, are not considered by some to be a form of life. Plasmids can be transferred between bacterial hosts through a process known as bacterial conjugation. Because conjugation is a mechanism of horizontal gene transfer, plasmids can be considered part of the mobilome. Unlike viruses (which encase their genetic material in a protective protein coat called a capsid), plasmids are "naked" DNA and do not encode genes necessary to encase the genetic material for transfer to a new host. However, some classes of plasmids encode the conjugative "sex" pilus necessary for their own transfer.
Plasmid host-to-host transfer can also require changes in incipient host gene expression allowing the intentional uptake of the genetic element by
- International Society for Plasmid Biology and other Mobile Genetic Elements
- History of Plasmids with timeline
- Bacterial Transformation and Plasmid Recovery
- Piechaczek C, Fetzer C, Baiker A, Bode J, Lipps HJ (1999). "A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells". Nucleic Acids Res 27 (2): 426–428.
- Bode J , Fetzer CP, Nehlsen K, Scinteie M, Hinrichsen B-H, Baiker A, Piechazcek C, Benham C, Lipps HJ (2001). "The Hitchhiking principle: Optimizing episomal vectors for the use in gene therapy and biotechnology". Gene Ther Mol Biol 6: 33–46.
- Nehlsen K, Broll S, Bode J (2006). "Replicating minicircles: Generation of nonviral episomes for the efficient modification of dividing cells". Gene Ther Mol Biol 10: 233–244.
- Ehrhardt A, Haase R, Schepers A, Deutsch MJ, Lipps HJ, Baiker A. (2008). "Episomal vectors for gene therapy". Curr Gene Therapy 8 (3): 147–161.
- Argyros O, Wong SP, Niceta M, Waddington SN, Howe SJ, Coutelle C, Miller AD, Harbottle RP (2008). "Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector". Gene Therapy 15 (24): 1593–1605.
- Wong SP, Argyros O, Coutelle C, Harbottle RP (2009). "Strategies for the episomal modification of cells". Current Opinion in Molecular Therapeutics 11 (4): 433–441.
- Haase R, Argyros O, Wong SP, Harbottle RP, Lipps HJ, Ogris M, Magnusson T, Vizoso Pinto MG, Haas J, Baiker A. (2010). "pEPito: a significantly improved non-viral episomal expression vector for mammalian cells". BMC Biotechnol 10: 433–441.
- Klein, Donald W.; Prescott, Lansing M.; Harley, John (1999). Microbiology. Boston: WCB/McGraw-Hill.
- Smith, Christopher U. M. Elements of Molecular Neurobiology. Wiley. pp. 101, 111.
- Albert G. Moat, John W. Foster, Michael P. Spector (2002). Microbial Physiology. Wiley-Liss.
- Lederberg J (1952). "Cell genetics and hereditary symbiosis". Physiol. Rev. 32 (4): 403–430.
- Thomas, Christopher M; Summers, David (2008). "Bacterial Plasmids".
- Lipps G (editor). (2008). Plasmids: Current Research and Future Trends. Caister Academic Press.
- Sinkovics, J; Harvath J; Horak A. (1998). "The Origin and evolution of viruses (a review)". Acta microbiologica et immunologica Hungarica (St. Joseph's Hospital, Department of Medicine, University of South Florida College of Medicine, Tampa, FL, USA.: Akademiai Kiado) 45 (3–4): 349–90.
- Russell, David W.; Sambrook, Joseph (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory.
- Kandavelou K; Chandrasegaran S (2008). "Plasmids for Gene Therapy". Plasmids: Current Research and Future Trends. Caister Academic Press.
- 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): 3116–20.
- Kroll J, Klinter S, Schneider C, Voß I, Steinbüchel A (2010). "Plasmid addiction systems: perspectives and applications in biotechnology". Microb Biotechnol. 3 (6): 634–657.
- Gunge, N; Murata, K; Sakaguchi, K (July 1982). "Transformation of Saccharomyces cerevisiae with linear DNA killer plasmids from Kluyveromyces lactis". Journal of bacteriology 151 (1): 462–4.
- Cormier, Catherine. "PSI: Biology in the Spotlight". Retrieved 5 November 2012.
- Bacterial artificial chromosome
- Circular DNA
- Triparental mating
The use of plasmids as a technique in molecular biology is supported by bioinformatics software. These programs 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 LabGenius, pDraw32, Geneious, Lasergene, MacVector, GeneConstructionKit, ApE, Clone Manager, VectorFriends, and Vector NTI. These software help conduct entire experiments in silico before doing wet experiments .
Simulation of plasmids
Because of its tight conformation, supercoiled DNA migrates faster through a gel than linear or open-circular DNA..
At a specified, low voltage, the migration rate of small linear DNA fragments is a function of their length. Large linear fragments (over 20 kb 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.
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 continuously increasing yet different rates. Thus, the resolution of a gel decreases with increased voltage.
- 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). This can be modeled 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. This can be modeled 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 an integral twist, resulting in a compact form. This can be modeled 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.
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:
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).
In the latter, much larger volumes of bacterial suspension are grown from which a maxi-prep can be performed. In essence, this is a scaled-up miniprep followed by additional purification. This results in relatively large amounts (several micrograms) of very pure plasmid DNA.
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.
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.
Plasmid DNA extraction
- 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.
Other types of plasmids are often related to yeast cloning vectors that include:
Yeast are organisms that naturally harbour plasmids. Notable plasmids are 2 µm plasmids - small circular plasmids often used for genetic engineering of yeast, and linear pGKL plasmids from kluyveromyces lactis, that are responsible for killer phenotypes.
In contrast, virtually all biotechnologically used plasmids (such as pUC18, pBR322 and derived vectors) do not contain toxin-antitoxin addiction systems and thus need to be kept under antibiotic pressure to avoid plasmid loss.
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. 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 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.
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 that attempt to actively distribute a copy to both daughter cells. These systems, which include the parABS system and parMRC system, are often referred to as the partition system or partition function of a plasmid.
Plasmids can belong to more than one of these functional groups.
- Fertility F-plasmids, which contain tra genes. They are capable of conjugation and result in the expression of sex pilli.
- Resistance (R)plasmids, which contain genes that provide 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 bacteriocins, proteins that can kill other bacteria.
- Degradative plasmids, which enable the digestion of unusual substances, e.g. toluene and salicylic acid.
- Virulence plasmids, which turn the bacterium into a pathogen.
Another way to classify plasmids is by function. There are five main classes:
It is possible for plasmids of different types to coexist in a single cell. Several different plasmids have been found in E. coli. However, 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. Thus, plasmids can be assigned into incompatibility groups.
One way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative plasmids contain 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 be transferred only with the assistance of conjugative plasmids. 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.
In general, in eukaryotes, episomes are closed circular DNA molecules that are replicated in the nucleus. Viruses are the most common examples of this, such as herpesviruses, adenoviruses, and polyomaviruses. Other examples include aberrant chromosomal fragments, such as double minute chromosomes, that can arise during artificial gene amplifications or in pathologic processes (e.g., cancer cell transformation). Episomes in eukaryotes behave similarly to plasmids in prokaryotes in that the DNA is stably maintained and replicated with the host cell. Cytoplasmic viral episomes (as in poxvirus infections) can also occur. Some episomes, such as herpesviruses, replicate in a rolling circle mechanism, similar to bacterial phage viruses. Others replicate through a bidirectional replication mechanism (Theta type plasmids). In either case, episomes remain physically separate from host cell chromosomes. Several cancer viruses, including Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, are maintained as latent, chromosomally distinct episomes in cancer cells, where the viruses express oncogenes that promote cancer cell proliferation. In cancers, these episomes passively replicate together with host chromosomes when the cell divides. When these viral episomes initiate lytic replication to generate multiple virus particles, they in general activate cellular innate immunity defense mechanisms that kill the host cell.
Some strategies of gene therapy require the insertion of therapeutic genes at pre-selected chromosomal target sites within the human genome. 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. Plasmids encoding ZFN could help deliver a therapeutic gene to a specific site so that cell damage, cancer-causing mutations, or an immune response is avoided.
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. However, developments in Adeno-associated virus recombination techniques, and Zinc finger nucleases, have enabled the creation of a new generation of isogenic human disease models.
A plasmid can contain inserts of up to 30-40 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes, or yeast artificial chromosomes are used.
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 bacterium 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.
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 normally inserted into a plasmid that typically contains a number of features. These include a gene that makes the bacterial cells resistant to particular antibiotics (normally Kanamycin or Ampicillin), an origin of replication to allow the bacterial cells to replicate the plasmid DNA, and a multiple cloning site (MCS, or polylinker). A multiple cloning site is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. After the gene of interest is inserted, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria that 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 either breaks down the antibiotics or prevents it from inhibiting certain bacterial pathways. In this way, the antibiotics act as a filter to select only the bacteria containing the plasmid DNA. Now these bacteria can be grown in large amounts, harvested, and lysed (often using the alkaline lysis method) to isolate the plasmid of interest.
- Vectors 1
- Disease models 2.1
- Gene therapy 2.2
- Episomes 3
- Types 4
- Plasmid maintenance 5
- Yeast plasmids 6
- Plasmid DNA extraction 7
- Conformations 8
- Simulation of plasmids 9
- See also 10
- References 11
Further reading 12
- Episomes 12.1
- External links 13