![plasmid (english)](https://upload.wikimedia.org/wikipedia/commons/c/cf/Plasmid_%28english%29.svg)
A plasmid is a small,
extrachromosomal DNA
Extrachromosomal DNA (abbreviated ecDNA) is any DNA that is found off the chromosomes, either inside or outside the nucleus of a cell. Most DNA in an individual genome is found in chromosomes contained in the nucleus. Multiple forms of extrachromo ...
molecule within a cell that is physically separated from
chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in
bacteria
Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...
; however, plasmids are sometimes present in
archaea
Archaea ( ; singular archaeon ) is a domain of single-celled organisms. These microorganisms lack cell nuclei and are therefore prokaryotes. Archaea were initially classified as bacteria, receiving the name archaebacteria (in the Archaebac ...
and
eukaryotic organisms. In nature, plasmids often carry genes that benefit the survival of the organism and confer selective advantage such as
antibiotic resistance
Antimicrobial resistance (AMR) occurs when microbes evolve mechanisms that protect them from the effects of antimicrobials. All classes of microbes can evolve resistance. Fungi evolve antifungal resistance. Viruses evolve antiviral resistance. ...
. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain only additional genes that may be useful in certain situations or conditions.
Artificial plasmids A DNA construct is an artificially-designed segment of DNA borne on a vector that can be used to incorporate genetic material into a target tissue or cell. A DNA construct contains a DNA insert, called a transgene, delivered via a transformation v ...
are widely used as
vectors in
molecular cloning
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word ''cloning'' refers to the fact that the metho ...
, serving to drive the replication of
recombinant DNA
Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be foun ...
sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via
transformation
Transformation may refer to:
Science and mathematics
In biology and medicine
* Metamorphosis, the biological process of changing physical form after birth or hatching
* Malignant transformation, the process of cells becoming cancerous
* Trans ...
. Synthetic plasmids are available for procurement over the internet.
Plasmids are considered ''
replicons'', units of DNA capable of replicating autonomously within a suitable host. However, plasmids, like
virus
A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea.
Since Dmitri Ivanovsky's 1 ...
es, are not generally classified as
life
Life is a quality that distinguishes matter that has biological processes, such as signaling and self-sustaining processes, from that which does not, and is defined by the capacity for growth, reaction to stimuli, metabolism, energ ...
. Plasmids are transmitted from one bacterium to another (even of another species) mostly through
conjugation
Conjugation or conjugate may refer to:
Linguistics
* Grammatical conjugation, the modification of a verb from its basic form
* Emotive conjugation or Russell's conjugation, the use of loaded language
Mathematics
* Complex conjugation, the chang ...
. This host-to-host transfer of genetic material is one mechanism of
horizontal gene transfer
Horizontal gene transfer (HGT) or lateral gene transfer (LGT) is the movement of genetic material between Unicellular organism, unicellular and/or multicellular organisms other than by the ("vertical") transmission of DNA from parent to offsprin ...
, and plasmids are considered part of the
mobilome
The mobilome is the entire set of mobile genetic elements in a genome. Mobilomes are found in eukaryotes, prokaryotes, and viruses. The compositions of mobilomes differ among lineages of life, with transposable elements being the major mobile elem ...
. Unlike viruses, which encase their genetic material in a protective protein coat called a
capsid
A capsid is the protein shell of a virus, enclosing its genetic material. It consists of several oligomeric (repeating) structural subunits made of protein called protomers. The observable 3-dimensional morphological subunits, which may or may ...
, 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. Plasmids vary in size from 1 to over 400 k
bp,
and the number of identical plasmids in a single
cell
Cell most often refers to:
* Cell (biology), the functional basic unit of life
Cell may also refer to:
Locations
* Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery w ...
can range anywhere from one to thousands under some circumstances.
History
The term ''plasmid'' was introduced in 1952 by the American
molecular biologist
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
Joshua Lederberg
Joshua () or Yehoshua ( ''Yəhōšuaʿ'', Tiberian: ''Yŏhōšuaʿ,'' lit. 'Yahweh is salvation') ''Yēšūaʿ''; syr, ܝܫܘܥ ܒܪ ܢܘܢ ''Yəšūʿ bar Nōn''; el, Ἰησοῦς, ar , يُوشَعُ ٱبْنُ نُونٍ '' Yūšaʿ ...
to refer to "any extrachromosomal hereditary determinant." The term's early usage included any bacterial genetic material that exists extrachromosomally for at least part of its replication cycle, but because that description includes bacterial viruses, the notion of plasmid was refined over time to comprise genetic elements that reproduce autonomously.
Later in 1968, it was decided that the term plasmid should be adopted as the term for extrachromosomal genetic element, and to distinguish it from viruses, the definition was narrowed to genetic elements that exist exclusively or predominantly outside of the chromosome and can replicate autonomously.
Properties and characteristics
![Plasmid replication (english)](https://upload.wikimedia.org/wikipedia/commons/4/42/Plasmid_replication_%28english%29.svg)
In order for plasmids to replicate independently within a cell, they must possess a stretch of DNA that can act as an
origin of replication
The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by semi ...
. The self-replicating unit, in this case, the plasmid, is called a
replicon. A typical bacterial replicon may consist of a number of elements, such as the gene for plasmid-specific replication initiation protein (Rep), repeating units called
iteron
Iterons are directly repeated DNA sequences which play an important role in regulation of plasmid copy number in bacterial cells. It is one among the three negative regulatory elements found in plasmids which control its copy number. The others in ...
s,
DnaA
Introduction
Based on the Replicon Model, a positively active initiator molecule contacts with a particular spot on a circular chromosome called the replicator to start DNA replication. DnaA is a protein that activates initiation of DNA replica ...
boxes, and an adjacent AT-rich region.
[ Smaller plasmids make use of the host replicative enzymes to make copies of themselves, while larger plasmids may carry genes specific for the replication of those plasmids. A few types of plasmids can also insert into the host chromosome, and these integrative plasmids are sometimes referred to as ]episome
An episome is a special type of plasmid, which remains as a part of the eukaryotic genome without integration. Episomes manage this by replicating together with the rest of the genome and subsequently associating with metaphase chromosomes during m ...
s in prokaryote
A prokaryote () is a single-celled organism that lacks a nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Greek πρό (, 'before') and κάρυον (, 'nut' or 'kernel').Campbell, N. "Biology:Concepts & Connec ...
s.
Plasmids almost always carry at least one gene. Many of the genes carried by a plasmid are beneficial for the host cells, for example: enabling the host cell to survive in an environment that would otherwise be lethal or restrictive for growth. Some of these genes encode traits for antibiotic resistance or resistance to heavy metal, while others may produce virulence factor
Virulence factors (preferably known as pathogenicity factors or effectors in plant science) are cellular structures, molecules and regulatory systems that enable microbial pathogens (bacteria, viruses, fungi, and protozoa) to achieve the following ...
s that enable a bacterium to colonize a host and overcome its defences or have specific metabolic functions that allow the bacterium to utilize a particular nutrient, including the ability to degrade recalcitrant or toxic organic compounds.[ Plasmids can also provide bacteria with the ability to fix nitrogen. Some plasmids, however, have no observable effect on the phenotype of the host cell or its benefit to the host cells cannot be determined, and these plasmids are called cryptic plasmids.
Naturally occurring plasmids vary greatly in their physical properties. Their size can range from very small mini-plasmids of less than 1-kilobase pairs (kbp) to very large megaplasmids of several megabase pairs (Mbp). At the upper end, little differs between a megaplasmid and a ]minichromosome
A minichromosome is a small chromatin-like structure resembling a chromosome and consisting of centromeres, telomeres and replication origins but little additional genetic material. They replicate autonomously in the cell during cellular division. ...
. Plasmids are generally circular, but examples of linear plasmids are also known. These linear plasmids require specialized mechanisms to replicate their ends.
Plasmids may be present in an individual cell in varying number, ranging from one to several hundreds. The normal number of copies of plasmid that may be found in a single cell is called the plasmid copy number
In cellular biology, the plasmid copy number is the number of copies of a given plasmid in a cell. To ensure survival and thus the continued propagation of the plasmid, they must regulate their copy number. If a plasmid has too high of a copy numb ...
, and is determined by how the replication initiation is regulated and the size of the molecule. Larger plasmids tend to have lower copy numbers. Low-copy-number plasmids that exist only as one or a few copies in each bacterium are, upon cell division
Cell division is the process by which a parent cell (biology), cell divides into two daughter cells. Cell division usually occurs as part of a larger cell cycle in which the cell grows and replicates its chromosome(s) before dividing. In eukar ...
, 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
The parABS system is a broadly conserved molecular mechanism for plasmid partitioning and chromosome segregation in bacteria. Originally identified as a genetic element required for faithful partitioning of low-copy-number plasmids, it consists of ...
and parMRC system
The ParMRC system is a mechanism for sorting DNA plasmids to opposite ends of a bacterial cell during cell division. It has three components: ParM, an actin-like protein that forms a long filament to push two plasmids apart, ParR, which binds the ...
, are often referred to as the partition system or partition function of a plasmid.
Classifications and types
Plasmids may be classified in a number of ways. Plasmids can be broadly classified into conjugative plasmids and non-conjugative plasmids. Conjugative plasmids contain a set of transfer gene
Transfer genes or tra genes (also transfer operons or tra operons), are some genes necessary for non-sexual Bacterial conjugation, transfer of genetic material in both gram-positive and gram-negative bacteria. The tra locus includes the pilin gene ...
s which promote sexual conjugation between different cells. In the complex process of conjugation
Conjugation or conjugate may refer to:
Linguistics
* Grammatical conjugation, the modification of a verb from its basic form
* Emotive conjugation or Russell's conjugation, the use of loaded language
Mathematics
* Complex conjugation, the chang ...
, plasmids may be transferred from one bacterium to another via sex pili encoded by some of the transfer genes (see figure). 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 can also be classified into incompatibility groups. A microbe can harbour different types of plasmids, but different plasmids can only exist in a single bacterial cell if they are compatible. If two plasmids are not compatible, one or the other will be rapidly lost from the cell. Different plasmids may therefore be assigned to different incompatibility groups depending on whether they can coexist together. Incompatible plasmids (belonging to the same incompatibility group) normally share the same replication or partition mechanisms and can thus not be kept together in a single cell.
Another way to classify plasmids is by function. There are five main classes:
* Fertility F-plasmid
The fertility factor (first named F by one of its discoverers Esther Lederberg; also called the sex factor in '' E. coli'' or the F sex factor; also called F-plasmid) allows genes to be transferred from one bacterium carrying the factor to another ...
s, which contain ''tra'' genes. They are capable of conjugation
Conjugation or conjugate may refer to:
Linguistics
* Grammatical conjugation, the modification of a verb from its basic form
* Emotive conjugation or Russell's conjugation, the use of loaded language
Mathematics
* Complex conjugation, the chang ...
and result in the expression of sex pili.
* Resistance (R) plasmids, which contain genes that provide resistance against antibiotic
An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and prevention of ...
s or poison
Poison is a chemical substance that has a detrimental effect to life. The term is used in a wide range of scientific fields and industries, where it is often specifically defined. It may also be applied colloquially or figuratively, with a broa ...
s. Historically known as R-factors, before the nature of plasmids was understood.
* Col plasmids, which contain genes that code for bacteriocin
Bacteriocins are proteinaceous or peptidic toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). They are similar to yeast and paramecium killing factors, and are structurally, functionally, and ec ...
s, protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
s that can kill other bacteria.
* Degradative plasmids, which enable the digestion of unusual substances, e.g. toluene
Toluene (), also known as toluol (), is a substituted aromatic hydrocarbon. It is a colorless, water-insoluble liquid with the smell associated with paint thinners. It is a mono-substituted benzene derivative, consisting of a methyl group (CH3) at ...
and salicylic acid
Salicylic acid is an organic compound with the formula HOC6H4CO2H. A colorless, bitter-tasting solid, it is a precursor to and a metabolite of aspirin (acetylsalicylic acid). It is a plant hormone, and has been listed by the EPA Toxic Substance ...
.
* Virulence plasmids, which turn the bacterium into a pathogen
In biology, a pathogen ( el, πάθος, "suffering", "passion" and , "producer of") in the oldest and broadest sense, is any organism or agent that can produce disease. A pathogen may also be referred to as an infectious agent, or simply a germ ...
. e.g. Ti plasmid
A tumour inducing (Ti) plasmid is a plasmid found in pathogenic species of ''Agrobacterium'', including ''A. tumefaciens, ''A. rhizogenes'', ''A. rubi'' and ''A. vitis''.
Evolutionarily, the Ti plasmid is part of a family of plasmids carried b ...
in ''Agrobacterium tumefaciens
''Agrobacterium radiobacter'' (more commonly known as ''Agrobacterium tumefaciens'') is the causal agent of crown gall disease (the formation of tumours) in over 140 species of eudicots. It is a rod-shaped, Gram-negative soil bacterium. Sympto ...
''
Plasmids can belong to more than one of these functional groups.
RNA plasmids
Although most plasmids are double-stranded DNA molecules, some consist of single-stranded DNA, or predominantly double-stranded RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydr ...
. RNA plasmids are non-infectious extrachromosomal linear RNA replicons, both encapsidated and unencapsidated, which have been found in fungi and various plants, from algae to land plants. In many cases, however, it may be difficult or impossible to clearly distinguish RNA plasmids from RNA viruses and other infectious RNAs.
Vectors
Artificially constructed plasmids may be used as vectors in genetic engineering
Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including t ...
. These plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to clone and amplify (make many copies of) or express
Express or EXPRESS may refer to:
Arts, entertainment, and media Films
* '' Express: Aisle to Glory'', a 1998 comedy short film featuring Kal Penn
* '' The Express: The Ernie Davis Story'', a 2008 film starring Dennis Quaid
Music
* ''Express'' ...
particular genes. A wide variety of 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 for their use. These include a gene that confers resistance to particular antibiotics (ampicillin
Ampicillin is an antibiotic used to prevent and treat a number of bacterial infections, such as respiratory tract infections, urinary tract infections, meningitis, salmonellosis, and endocarditis. It may also be used to prevent group B strepto ...
is most frequently used for bacterial strains), an origin of replication
The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by semi ...
to allow the bacterial cells to replicate the plasmid DNA, and a suitable site for cloning (referred to as a multiple cloning site A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only o ...
).
DNA structural instability can be defined as a series of spontaneous events that culminate in an unforeseen rearrangement, loss, or gain of genetic material. Such events are frequently triggered by the transposition of mobile elements or by the presence of unstable elements such as non-canonical (non-B) structures. Accessory regions pertaining to the bacterial backbone may engage in a wide range of structural instability phenomena. Well-known catalysts of genetic instability
Genome instability (also genetic instability or genomic instability) refers to a high frequency of mutations within the genome of a cellular lineage. These mutations can include changes in nucleic acid sequences, chromosomal rearrangements or aneu ...
include direct, inverted, and tandem repeats, which are known to be conspicuous in a large number of commercially available cloning and expression vectors. Insertion sequences can also severely impact plasmid function and yield, by leading to deletion
Deletion or delete may refer to:
Computing
* File deletion, a way of removing a file from a computer's file system
* Code cleanup, a way of removing unnecessary variables, data structures, cookies, and temporary files in a programming language
* ...
s and rearrangements, activation, down-regulation
In the biological context of organisms' production of gene products, downregulation is the process by which a cell decreases the quantity of a cellular component, such as RNA or protein, in response to an external stimulus. The complementary proce ...
or inactivation of neighboring gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. The ...
. Therefore, the reduction or complete elimination of extraneous noncoding
Non-coding DNA (ncDNA) sequences are components of an organism's DNA that do not encode protein sequences. Some non-coding DNA is transcribed into functional non-coding RNA molecules (e.g. transfer RNA, microRNA, piRNA, ribosomal RNA, and regula ...
backbone sequences would pointedly reduce the propensity for such events to take place, and consequently, the overall recombinogenic potential of the plasmid.
Cloning
Plasmids are the most-commonly used bacterial cloning vectors. These cloning vectors contain a site that allows DNA fragments to be inserted, for example a multiple cloning site A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only o ...
or polylinker which has several commonly used restriction sites Restriction sites, or restriction recognition sites, are located on a DNA molecule containing specific (4-8 base pairs in length) sequences of nucleotides, which are recognized by restriction enzymes. These are generally palindromic sequences (beca ...
to which DNA fragments may be ligated. After the gene of interest is inserted, the plasmids are introduced into bacteria by a process called transformation
Transformation may refer to:
Science and mathematics
In biology and medicine
* Metamorphosis, the biological process of changing physical form after birth or hatching
* Malignant transformation, the process of cells becoming cancerous
* Trans ...
. These plasmids contain a selectable marker A selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, and gen ...
, usually an antibiotic resistance gene, which confers on the bacteria an ability to survive and proliferate in a selective growth medium containing the particular antibiotics. The cells after transformation are exposed to the selective media, and only cells containing the plasmid may survive. In this way, the antibiotics act as a filter to select only the bacteria containing the plasmid DNA. The vector may also contain other marker gene In biology, a marker gene may have several meanings. In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA. In particular, there are tw ...
s or reporter gene
In molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Such genes are called reporters because the charac ...
s to facilitate selection of plasmids with cloned inserts. Bacteria containing the plasmid can then be grown in large amounts, harvested, and the plasmid of interest may then be isolated using various methods of plasmid preparation
A plasmid preparation is a method of DNA extraction and purification for plasmid DNA. Many methods have been developed to purify plasmid DNA from bacteria that directly exist in the environment or bacteria that grown at laboratory level. These m ...
.
A plasmid cloning vector is typically used to clone DNA fragments of up to 15 kbp. To clone longer lengths of DNA, lambda phage
''Enterobacteria phage λ'' (lambda phage, coliphage λ, officially ''Escherichia virus Lambda'') is a bacterial virus, or bacteriophage, that infects the bacterial species ''Escherichia coli'' (''E. coli''). It was discovered by Esther Lederb ...
with lysogeny genes deleted, cosmid
A cosmid is a type of hybrid plasmid that contains a Lambda phage ''cos'' sequence. They are often used as a cloning vector in genetic engineering. Cosmids can be used to build genomic libraries. They were first described by Collins and Hohn in ...
s, bacterial artificial chromosome A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually '' E. coli''. F-plasmids play a crucial role because they contain partition ...
s, or yeast artificial chromosome
Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, ''Saccharomyces cerevisiae'', which is then ligated into a bacterial plasmid. By inserting large fragments of DNA, from 100–1000 ...
s are used.
Protein production
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 the protein the gene codes for, for example, insulin
Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the ''INS'' gene. It is considered to be the main anabolic hormone of the body. It regulates the metabolism o ...
.
Gene therapy
Plasmids may also be used for gene transfer as a potential treatment in gene therapy
Gene therapy is a medical field which focuses on the genetic modification of cells to produce a therapeutic effect or the treatment of disease by repairing or reconstructing defective genetic material. The first attempt at modifying human DN ...
so that it may express the protein that is lacking in the cells. Some forms of gene therapy
Gene therapy is a medical field which focuses on the genetic modification of cells to produce a therapeutic effect or the treatment of disease by repairing or reconstructing defective genetic material. The first attempt at modifying human DN ...
require the insertion of therapeutic gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
s at pre-selected chromosomal
A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
target sites within the human genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...
. Plasmid vectors are one of many approaches that could be used for this purpose. Zinc finger nuclease
Zinc-finger nucleases (ZFNs) 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 specific desired DNA sequences and this enables zinc ...
s (ZFNs) offer a way to cause a site-specific double-strand break
DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA dam ...
to the DNA genome and cause homologous recombination
Homologous recombination is a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids (usually DNA as in cellular organisms but may ...
. Plasmids encoding ZFN could help deliver a therapeutic gene to a specific site so that cell damage
Cell damage (also known as cell injury) is a variety of changes of stress that a cell suffers due to external as well as internal environmental changes. Amongst other causes, this can be due to physical, chemical, infectious, biological, nutritiona ...
, cancer-causing mutations, or an immune response
An immune response is a reaction which occurs within an organism for the purpose of defending against foreign invaders. These invaders include a wide variety of different microorganisms including viruses, bacteria, parasites, and fungi which could ...
is avoided.
Disease models
Plasmids were historically used to genetically engineer the embryonic stem cells of rats 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 viruses (AAV) are small viruses that infect humans and some other primate species. They belong to the genus ''Dependoparvovirus'', which in turn belongs to the family ''Parvoviridae''. They are small (approximately 26 nm in di ...
recombination techniques, and zinc finger nucleases
Zinc-finger nucleases (ZFNs) 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 specific desired DNA sequences and this enables zinc ...
, have enabled the creation of a new generation of 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 s ...
.
Episomes
The term ''episome'' was introduced by François Jacob
François Jacob (17 June 1920 – 19 April 2013) was a French biologist who, together with Jacques Monod, originated the idea that control of enzyme levels in all cells occurs through regulation of transcription. He shared the 1965 Nobel Prize i ...
and Élie Wollman
Élie Léo Wollman (July 4, 1917 – June 1, 2008) was a French microbial geneticist who first described plasmids (what he termed "episomes"), and served as vice director of research for the Pasteur Institute for twenty years. He was awarded t ...
in 1958 to refer to extra-chromosomal genetic material that may replicate autonomously or become integrated into the chromosome. Since the term was introduced, however, its use has changed, as ''plasmid'' has become the preferred term for autonomously replicating extrachromosomal DNA. At a 1968 symposium in London some participants suggested that the term ''episome'' be abandoned, although others continued to use the term with a shift in meaning.
Today, some authors use ''episome'' in the context of prokaryotes to refer to a plasmid that is capable of integrating into the chromosome. The integrative plasmids may be replicated and stably maintained in a cell through multiple generations, but at some stage, they will exist as an independent plasmid molecule. In the context of eukaryotes, the term ''episome'' is used to mean a non-integrated extrachromosomal closed circular DNA molecule that may be replicated in the nucleus. Viruses are the most common examples of this, such as herpesviruses, adenoviruses
Adenoviruses (members of the family ''Adenoviridae'') are medium-sized (90–100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. Their name derives from thei ...
, and polyomavirus
''Polyomaviridae'' is a family of viruses whose natural hosts are primarily mammals and birds. As of 2020, there are six recognized genera and 117 species, five of which are unassigned to a genus. 14 species are known to infect humans, while othe ...
es, but some are plasmids. 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
''Poxviridae'' is a family of double-stranded DNA viruses. Vertebrates and arthropods serve as natural hosts. There are currently 83 species in this family, divided among 22 genera, which are divided into two subfamilies. Diseases associated wit ...
infections) can also occur. Some episomes, such as herpesviruses, replicate in a rolling circle
Rolling circle replication (RCR) is a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genom ...
mechanism, similar to bacteriophage
A bacteriophage (), also known informally as a ''phage'' (), is a duplodnaviria virus that infects and replicates within bacteria and archaea. The term was derived from "bacteria" and the Greek φαγεῖν ('), meaning "to devour". Bacteri ...
s (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
Kaposi's sarcoma-associated herpesvirus (KSHV) is the ninth known human herpesvirus; its formal name according to the International Committee on Taxonomy of Viruses (ICTV) is ''Human gammaherpesvirus 8'', or HHV-8 in short. Like other herpesvirus ...
, are maintained as latent, chromosomally distinct episomes in cancer cells, where the viruses express oncogenes
An oncogene is a gene that has the potential to cause cancer. In tumor cells, these genes are often mutated, or expressed at high levels. 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 generally activate cellular innate immunity
The innate, or nonspecific, immune system is one of the two main immunity strategies (the other being the adaptive immune system) in vertebrates. The innate immune system is an older evolutionary defense strategy, relatively speaking, and is the ...
defense mechanisms that kill the host cell.
Plasmid maintenance
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
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
''. This variant produces both a long-lived poison
Poison is a chemical substance that has a detrimental effect to life. The term is used in a wide range of scientific fields and industries, where it is often specifically defined. It may also be applied colloquially or figuratively, with a broa ...
and a short-lived antidote
An antidote is a substance that can counteract a form of poisoning. The term ultimately derives from the Greek term φάρμακον ἀντίδοτον ''(pharmakon) antidoton'', "(medicine) given as a remedy". Antidotes for anticoagulants are s ...
. Several types of plasmid addiction systems (toxin/ antitoxin, metabolism-based, ORT systems) were described in the literature
Literature is any collection of written work, but it is also used more narrowly for writings specifically considered to be an art form, especially prose fiction, drama, and poetry. In recent centuries, the definition has expanded to include ...
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.
In contrast, plasmids used in biotechnology, such as pUC18, pBR322 and derived vectors, hardly ever contain toxin-antitoxin addiction systems, and therefore need to be kept under antibiotic pressure to avoid plasmid loss.
Yeast plasmids
Yeast
Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constitut ...
s naturally harbour various plasmids. Notable among them are 2 μm plasmids—small circular plasmids often used for genetic engineering
Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including t ...
of yeast—and linear pGKL plasmids from ''Kluyveromyces lactis
''Kluyveromyces lactis'' is a ''Kluyveromyces'' yeast commonly used for genetic studies and industrial applications. Its name comes from the ability to assimilate lactose and convert it into lactic acid.
''Kluyveromyces lactis'' (formerly ''Sa ...
'', that are responsible for killer phenotypes
A killer is someone or something that kills, such as a murderer or a serial killer.
Killer may also refer to:
Arts, entertainment, and media Fictional characters
* Killer (''Home and Away''), a character from ''Home and Away''
* Killer Kane, ...
.
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 be lost during budding.
Plant mitochondrial plasmids
The mitochondria of many higher plants contain self-replicating
Self-replication is any behavior of a dynamical system that yields construction of an identical or similar copy of itself. Biological cells, given suitable environments, reproduce by cell division. During cell division, DNA is replicated and ca ...
, extra-chromosomal linear or circular DNA molecules which have been considered to be plasmids. These can range from 0.7 kb to 20 kb in size. The plasmids have been generally classified into to two categories- circular and linear. Circular plasmids have been and found in many different plants, with those in ''Vicia faba
''Vicia faba'', commonly known as the broad bean, fava bean, or faba bean, is a species of vetch, a flowering plant in the pea and bean family Fabaceae. It is widely cultivated as a crop for human consumption, and also as a cover crop. Variet ...
and Chenopodium album
''Chenopodium album'' is a fast-growing weedy annual plant in the genus ''Chenopodium''. Though cultivated in some regions, the plant is elsewhere considered a weed. Common names include lamb's quarters, melde, goosefoot, wild spinach and fat-h ...
'' being the most studied and whose mechanism of replication is known. The circular plasmids can replicate using the θ model of replication (as in ''Vicia faba'') and through rolling circle replication
Rolling circle replication (RCR) is a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genom ...
(as in ''C.album''). Linear plasmids have been identified in some plant species such as ''Beta vulgaris
''Beta vulgaris'' (beet) is a species of flowering plant in the subfamily Betoideae of the family Amaranthaceae. Economically, it is the most important crop of the large order Caryophyllales. It has several cultivar groups: the sugar beet, of gre ...
'', ''Brassica napus
Rapeseed (''Brassica napus ''subsp.'' napus''), also known as rape, or oilseed rape, is a bright-yellow flowering member of the family Brassicaceae (mustard or cabbage family), cultivated mainly for its oil-rich seed, which naturally contains a ...
, Zea mays
Maize ( ; ''Zea mays'' subsp. ''mays'', from es, maíz after tnq, mahiz), also known as corn (North American and Australian English), is a cereal grain first domesticated by indigenous peoples in southern Mexico about 10,000 years ago. Th ...
'', etc. but are rarer than their circular counterparts.
The function and origin of these plasmids remains largely unknown. It has been suggested that the circular plasmids share a common ancestor, some genes in the mitochondrial plasmid have counterparts in the nuclear DNA suggesting inter-compartment exchange. Meanwhile, the linear plasmids share structural similarities such as invertrons with viral DNA and fungal plasmids, like fungal plasmids they also have low GC content, these observations have led to some hypothesizing that these linear plasmids have viral origins, or have ended up in plant mitochondria through horizontal gene transfer
Horizontal gene transfer (HGT) or lateral gene transfer (LGT) is the movement of genetic material between Unicellular organism, unicellular and/or multicellular organisms other than by the ("vertical") transmission of DNA from parent to offsprin ...
from pathogenic fungi.
Plasmid DNA extraction
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
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word ''cloning'' refers to the fact that the metho ...
, plasmids often need to be isolated.
There are several methods to isolate plasmid DNA from bacteria, ranging from the miniprep to the maxiprep or 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
A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed ''DNA fragmentation'' (this term is used for other procedures as well). Hartl and Jones describe it this way:
...
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 hundred micrograms) of very pure plasmid DNA.
Many commercial kits have been created to perform plasmid extraction at various scales, purity, and levels of automation.
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, from Ancient Greek ἤλεκτρον (ḗlektron, "amber") and φόρησις (phórēsis, "the act of bearing"), is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric fie ...
. 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). 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
DNA supercoiling refers to the amount of twist in a particular DNA strand, which determines the amount of strain on it. A given strand may be "positively supercoiled" or "negatively supercoiled" (more or less tightly wound). The amount of a st ...
'' (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
An extension cord (US), power extender, drop cord, or extension lead (UK) is a length of flexible electrical power cable (flex) with a AC power plugs and sockets, plug on one end and one or more sockets on the other end (usually of the same type ...
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 continuously increasing yet different rates. Thus, 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 'respirate', with the bulk of the molecule following the leading end through the gel matrix. 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'' (this term is used for other procedures as well). Hartl and Jones describe it this way:
...
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 is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...
. 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.
Software for bioinformatics and design
The use of plasmids as a technique in molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
is supported by bioinformatics
Bioinformatics () is an interdisciplinary field that develops methods and software tools for understanding biological data, in particular when the data sets are large and complex. As an interdisciplinary field of science, bioinformatics combi ...
software
Software is a set of computer programs and associated documentation and data. This is in contrast to hardware, from which the system is built and which actually performs the work.
At the lowest programming level, executable code consists ...
. These programs record the DNA sequence of plasmid vectors, help to predict cut sites of restriction enzymes
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
, and to plan manipulations. Examples of software packages that handle plasmid maps are ApE, Clone Manager
Clone Manager is a commercial bioinformatics software work suite of Sci-Ed, that supports molecular biologists with data management and allows them to perform certain ''in silico
In biology and other experimental sciences, an ''in silico'' exp ...
, GeneConstructionKit, Geneious, Genome Compiler
Twist Bioscience is a public biotechnology company based in South San Francisco that manufactures synthetic DNA and DNA products for customers in a wide range of industries. Twist was founded in 2013 by Emily Leproust, Bill Banyai, and Bill Pec ...
, LabGenius, Lasergene, MacVector
MacVector is a commercial sequence analysis application for Apple Macintosh computers running Mac OS X. It is intended to be used by Molecular biology, molecular biologists to help analyze, design, research and document their experiments in the la ...
, pDraw32, Serial Cloner, VectorFriends, Vector NTI
Vector NTI was a commercial bioinformatics software package used by many life scientists in the early 2000s to work, among other things, with nucleic acids and proteins '' in silico''. It allowed researchers to, for example, plan a DNA cloning ...
, and WebDSV. These pieces of software help conduct entire experiments in silico before doing wet experiments.
Plasmid collections
Many plasmids have been created over the years and researchers have given out plasmids to plasmid databases such as the non-profit organisation
Addgene
an
BCCM/LMBP
One can find and request plasmids from those databases for research.
Researchers also often upload plasmid sequences to th
NCBI database
from which sequences of specific plasmids can be retrieved.
See also
References
Further reading
General works
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Episomes
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External links
International Society for Plasmid Biology and other Mobile Genetic Elements
What is Biotechnology
{{Authority control
Gene delivery
Mobile genetic elements
Molecular biology
Molecular biology techniques