Plasmid
Overview
 
In microbiology
Microbiology
Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell , cell clusters or no cell at all . This includes eukaryotes, such as fungi and protists, and prokaryotes...

 and genetics
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....

, a plasmid is a DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 molecule that is separate from, and can replicate independently of, the chromosomal DNA
GDNA
Genomic deoxyribonucleic acid is chromosomal DNA, in contrast to extrachromosomal DNAs like plasmids. It is often abbreviated as gDNA. The genome of an organism is the biological information of heredity which is passed from one generation of organism to the next...

. They are double-stranded and, in many cases, circular. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...

 (e.g., the 2-micrometre ring in Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...

).

Plasmid sizes vary from 1 to over 1,000 kbp
Base pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...

. The number of identical plasmids in a single cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....

 can range anywhere from one to even thousands under some circumstances.
Encyclopedia
In microbiology
Microbiology
Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell , cell clusters or no cell at all . This includes eukaryotes, such as fungi and protists, and prokaryotes...

 and genetics
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....

, a plasmid is a DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 molecule that is separate from, and can replicate independently of, the chromosomal DNA
GDNA
Genomic deoxyribonucleic acid is chromosomal DNA, in contrast to extrachromosomal DNAs like plasmids. It is often abbreviated as gDNA. The genome of an organism is the biological information of heredity which is passed from one generation of organism to the next...

. They are double-stranded and, in many cases, circular. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...

 (e.g., the 2-micrometre ring in Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...

).

Plasmid sizes vary from 1 to over 1,000 kbp
Base pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...

. The number of identical plasmids in a single cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....

 can range anywhere from one to even thousands under some circumstances. Plasmids can be considered part of the mobilome
Mobilome
The mobilome is the total of all mobile genetic elements in a genome. Elements that can move within the genome are the major constituents of the mobilome in eukaryotes. In prokaryotes, however, mobile genetic elements that can move between genomes, like prophages and plasmids, are also an...

 because they are often associated with conjugation
Bacterial conjugation
Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells...

, a mechanism of horizontal gene transfer
Horizontal gene transfer
Horizontal gene transfer , also lateral gene transfer , is any process in which an organism incorporates genetic material from another organism without being the offspring of that organism...

.

The term plasmid was first introduced by the American
United States
The United States of America is a federal constitutional republic comprising fifty states and a federal district...

 molecular biologist
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

 Joshua Lederberg
Joshua Lederberg
Joshua Lederberg ForMemRS was an American molecular biologist known for his work in microbial genetics, artificial intelligence, and the United States space program. He was just 33 years old when he won the 1958 Nobel Prize in Physiology or Medicine for discovering that bacteria can mate and...

 in 1952.

Plasmids are considered "replicon
Replicon (genetics)
A replicon is a DNA molecule or RNA molecule, or a region of DNA or RNA, that replicates from a single origin of replication.For most prokaryotic chromosomes, the replicon is the entire chromosome. One notable exception found comes from archaea, where two Sulfolobus species have been shown to...

s", capable of autonomous replication within a suitable host. Plasmids can be found in all three major domains
Domain (biology)
In biological taxonomy, a domain is the highest taxonomic rank of organisms, higher than a kingdom. According to the three-domain system of Carl Woese, introduced in 1990, the Tree of Life consists of three domains: Archaea, Bacteria and Eukarya...

: Archaea
Archaea
The Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon...

, Bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...

, and Eukarya. Similar to virus
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...

es, plasmids are not considered by some to be a form of "life". 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
Pilus
right|thumb|350px|Schematic drawing of bacterial conjugation. 1- Donor cell produces pilus. 2- Pilus attaches to recipient cell, brings the two cells together. 3- The mobile plasmid is nicked and a single strand of DNA is then transferred to the recipient cell...

 necessary for their own transfer. Plasmid host-to-host transfer requires direct, mechanical transfer by conjugation
Bacterial conjugation
Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells...

 or changes in host gene expression allowing the intentional uptake of the genetic element by transformation
Transformation (genetics)
In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...

. Microbial transformation with plasmid DNA is neither parasitic nor symbiotic in nature, because 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 the proteins produced may act as toxin
Toxin
A toxin is a poisonous substance produced within living cells or organisms; man-made substances created by artificial processes are thus excluded...

s under similar circumstances. Plasmids can also provide bacteria with the ability to fix elemental nitrogen
Nitrogen
Nitrogen is a chemical element that has the symbol N, atomic number of 7 and atomic mass 14.00674 u. Elemental nitrogen is a colorless, odorless, tasteless, and mostly inert diatomic gas at standard conditions, constituting 78.08% by volume of Earth's atmosphere...

 or to degrade recalcitrant organic compounds that provide an advantage when nutrients are scarce.

Vectors

Plasmids used in genetic engineering
Genetic engineering
Genetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...

 are called vectors
Vector (molecular biology)
In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes...

.
Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express
Protein expression
Protein expression is a subcomponent of gene expression. It consists of the stages after DNA has been translated into polypeptide chains, which are ultimately folded into proteins...

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
Multiple cloning site
A multiple cloning site , also called a polylinker, is a short segment of DNA which contains many restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. MCSs are commonly used during...

 (MCS, or polylinker), which is a short region containing several commonly used restriction sites
Restriction sites
Restriction sites, or restriction recognition sites, are locations on a DNA molecule containing specific sequences of nucleotides, which are recognized by restriction enzymes...

 allowing the easy insertion of DNA fragments at this location. Next, the plasmids are inserted into bacteria by a process called transformation
Transformation (genetics)
In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...

. 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 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
Lysis
Lysis refers to the breaking down of a cell, often by viral, enzymic, or osmotic mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a "lysate"....

 (often using the alkaline lysis
Alkaline lysis
Alkaline lysis is a method used in molecular biology to break cells open to isolate plasmid DNA or other cell components such as proteins. Bacteria containing the plasmid of interest are first grown, then lysed with a strong alkaline buffer consisting of a detergent sodium dodecyl sulfate and a...

 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 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
Insulin
Insulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....

 or even antibiotic
Antibiotic
An antibacterial is a compound or substance that kills or slows down the growth of bacteria.The term is often used synonymously with the term antibiotic; today, however, with increased knowledge of the causative agents of various infectious diseases, antibiotic has come to denote a broader range of...

s.

However, a plasmid can contain inserts of only about 1–10 kbp
Base pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...

. To clone longer lengths of DNA, lambda phage
Lambda phage
Enterobacteria phage λ is a temperate bacteriophage that infects Escherichia coli.Lambda phage is a virus particle consisting of a head, containing double-stranded linear DNA as its genetic material, and a tail that can have tail fibers. The phage particle recognizes and binds to its host, E...

 with lysogeny genes deleted, cosmid
Cosmid
A cosmid, first described by Collins and Hohn in 1978, is a type of hybrid plasmid that contains cos sequences, DNA sequences originally from the Lambda phage. Cosmids can be used to build genomic libraries....

s, bacterial artificial chromosome
Bacterial artificial chromosome
A bacterial artificial chromosome is a DNA construct, based on a functional fertility plasmid , used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell...

s, or yeast artificial chromosome
Yeast artificial chromosome
A yeast artificial chromosome is a vector used to clone DNA fragments larger than 100 kb and up to 3000 kb. YACs are useful for the physical mapping of complex genomes and for the cloning of large genes...

s are used.

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. However, developments in Adeno-associated virus
Adeno-associated virus
Adeno-associated virus is a small virus which infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of...

 recombination techniques, and Zinc finger nucleases, have enabled the creation of a new generation of isogenic human disease models
Isogenic human disease models
Isogenic human disease models are a family of cells that are selected or engineered to accurately model the genetics of a specific patient population, in vitro . They are provided with a genetically matched ‘normal cell’ to provide an isogenic system to research disease biology and novel...

.

Gene therapy

The success of some strategies of gene therapy
Gene therapy
Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development...

 depends on the efficient insertion of therapeutic gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...

s at the appropriate chromosomal
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...

 target sites within the human genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....

, 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 nuclease
Zinc finger nuclease
Zinc-finger nucleases are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes...

s (ZFNs) offer a way to cause a site-specific double-strand break to the DNA genome and cause homologous recombination
Homologous recombination
Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks...

. 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.

Episomes

Episomes are the eukaryotic equivalent of bacterial plasmids. 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
Polyomavirus
Polyomavirus is the sole genus of viruses within the family Polyomaviridæ. Murine polyomavirus was the first polyomavirus discovered by Ludwik Gross in 1953. Subsequently, many polyomaviruses have been found to infect birds and mammals...

. Other examples include aberrant chromosomal fragments, such as double minute chromosomes
Double minute
Double minutes are small fragments of extrachromosomal DNA, which have been observed in a large number of human tumors including breast, lung, ovary, colon, and most notably, neuroblastoma. They are a manifestation of gene amplification during the development of tumors, which give the cells...

, 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. In either case, episomes remain physically separate from host cell chromosomes. Several cancer viruses, including Epstein-Barr virus
Epstein-Barr virus
The Epstein–Barr virus , also called human herpesvirus 4 , is a virus of the herpes family and is one of the most common viruses in humans. It is best known as the cause of infectious mononucleosis...

 and Kaposi's sarcoma-associated herpesvirus
Kaposi's sarcoma-associated herpesvirus
Kaposi's sarcoma-associated herpesvirus is one of seven currently known human cancer viruses, or oncoviruses. It is also the eighth human herpesvirus; its formal name according to the International Committee on Taxonomy of Viruses is HHV-8. Like other herpesviruses, its informal name is used...

, 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
Lytic cycle
The lytic cycle is one of the two cycles of viral reproduction, the other being the lysogenic cycle. The lytic cycle is typically considered the main method of viral replication, since it results in the destruction of the infected cell...

 to generate multiple virus particles, they in general activate cellular innate immunity defense mechanisms that kill the host cell.

Types

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
Bacterial conjugation
Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells...

, 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.

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. 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
    Bacterial conjugation
    Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells...

    .
  • Resistance (R)plasmids, which contain genes that can build a resistance against antibiotic
    Antibiotic
    An antibacterial is a compound or substance that kills or slows down the growth of bacteria.The term is often used synonymously with the term antibiotic; today, however, with increased knowledge of the causative agents of various infectious diseases, antibiotic has come to denote a broader range of...

    s or poison
    Poison
    In the context of biology, poisons are substances that can cause disturbances to organisms, usually by chemical reaction or other activity on the molecular scale, when a sufficient quantity is absorbed by an organism....

    s and help bacteria produce pili
    Pilus
    right|thumb|350px|Schematic drawing of bacterial conjugation. 1- Donor cell produces pilus. 2- Pilus attaches to recipient cell, brings the two cells together. 3- The mobile plasmid is nicked and a single strand of DNA is then transferred to the recipient cell...

    . Historically known as R-factors, before the nature of plasmids was understood.
  • Col plasmids, which contain genes that code for bacteriocin
    Bacteriocin
    Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain. They are typically considered to be narrow spectrum antibiotics, though this has been debated...

    s, protein
    Protein
    Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

    s that can kill other bacteria.
  • Degradative plasmids, which enable the digestion of unusual substances, e.g. toluene
    Toluene
    Toluene, formerly known as toluol, is a clear, water-insoluble liquid with the typical smell of paint thinners. It is a mono-substituted benzene derivative, i.e., one in which a single hydrogen atom from the benzene molecule has been replaced by a univalent group, in this case CH3.It is an aromatic...

     and salicylic acid
    Salicylic acid
    Salicylic acid is a monohydroxybenzoic acid, a type of phenolic acid and a beta hydroxy acid. This colorless crystalline organic acid is widely used in organic synthesis and functions as a plant hormone. It is derived from the metabolism of salicin...

    .
  • Virulence plasmids, which turn the bacterium into a pathogen
    Pathogen
    A pathogen gignomai "I give birth to") or infectious agent — colloquially, a germ — is a microbe or microorganism such as a virus, bacterium, prion, or fungus that causes disease in its animal or plant host...

    .


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
Cell division
Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...

, 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 are often referred to as the partition system or partition function of a plasmid.

Some plasmids or microbial hosts include an addiction system or postsegregational killing system (PSK), such as the hok/sok
Hok/sok system
The hok/sok system is a postsegregational killing mechanism employed by the R1 plasmid in Escherichia coli. It was the first type I toxin-antitoxin pair to be identified through characterisation of a plasmid-stabilising locus...

 (host killing/suppressor of killing) system of plasmid R1 in Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...

. This variant produces both a long-lived poison
Poison
In the context of biology, poisons are substances that can cause disturbances to organisms, usually by chemical reaction or other activity on the molecular scale, when a sufficient quantity is absorbed by an organism....

 and a short-lived antidote
Antidote
An antidote is a substance which can counteract a form of poisoning. The term ultimately derives from the Greek αντιδιδοναι antididonai, "given against"....

. Several types of plasmid addiction systems (toxin/ antitoxin, metabolism-based, ORT systems) were described in the literature
Literature
Literature is the art of written works, and is not bound to published sources...

 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 are often related to yeast cloning vectors that 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
Plasmid preparation
A plasmid preparation is a method used to extract and purify plasmid DNA. Many methods have been developed to purify plasmid DNA from bacteria...

 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
Restriction digest
A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed DNA fragmentation...

 and for some cloning techniques.

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.

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 .

Conformations

Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during electrophoresis
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...

. 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
    DNA supercoil
    DNA supercoiling refers to the over- or under-winding of a DNA strand, and is an expression of the strain on the polymer. Supercoiling is important in a number of biological processes, such as compacting DNA. Additionally, certain enzymes such as topoisomerases are able to change DNA topology to...

    " (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
    Extension cord
    An extension cord, power extender, or extension lead is a length of flexible electrical power cable with a plug on one end and one or more sockets on the other end . The term usually refers to mains extensions but is also used to refer to extensions for other types of cabling...

     and then plugging it into itself.
  • "Supercoiled Denatured
    Denaturation (biochemistry)
    Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...

    " 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 continuously 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 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 digest
Restriction digest
A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed DNA fragmentation...

s 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
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...

. 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
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

 is supported by bioinformatics
Bioinformatics
Bioinformatics is the application of computer science and information technology to the field of biology and medicine. Bioinformatics deals with algorithms, databases and information systems, web technologies, artificial intelligence and soft computing, information and computation theory, software...

 software. These programmes record the DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 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
Geneious
Geneious is suite of cross-platform bioinformatics software applications developed by Biomatters Ltd.- Features :Geneious comes in a Basic version that is free for academic use, and a commercial Pro version with added features. Geneious bundles various bioinformatics tools under one hood with an...

, Lasergene, GeneConstructionKit, ApE, Clone Manager, and Vector NTI
Vector NTI
Vector NTI is a bioinformatics software package. The current versions are v11.5.1 for Windows/PCs and v7.1 for Macs, but only supporting Mac OS X v10.3 .- Features :* create, annotate, analyse, and share DNA/protein sequences...

.

See also

  • Bacterial artificial chromosome
    Bacterial artificial chromosome
    A bacterial artificial chromosome is a DNA construct, based on a functional fertility plasmid , used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell...

  • Triparental mating
    Triparental mating
    Triparental mating is a form of Bacterial conjugation where a conjugative plasmid present in one bacterial strain assists the transfer of a mobilizable plasmid present in a second bacterial strain into a third bacterial strain. Plasmids are introduced into bacteria for such purposes as...

  • Transposon
    Transposon
    Transposable elements are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's...

  • Bacteriophage
    Bacteriophage
    A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid...

  • Provirus
    Provirus
    A provirus is a virus genome that is integrated into the DNA of a host cell.This state can be a stage of virus replication, or a state that persists over longer periods of time as either inactive viral infections or an endogenous retrovirus. In inactive viral infections the virus will not replicate...

  • Segrosome
    Segrosome
    Segrosomes are protein complexes that ensure accurate segregation of plasmids or chromosomes during bacterial cell division.Just as higher forms of life have evolved a complex mitotic apparatus to partition duplicated DNA during cell division, bacteria require a specialized apparatus to partition...


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