Plasmid

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Illustration of a bacterium showing chromosomal DNA and plasmids (Not to scale) Plasmid (english).svg
Illustration of a bacterium showing chromosomal DNA and plasmids (Not to scale)

A plasmid is a small, extrachromosomal DNA 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; however, plasmids are sometimes present in archaea and eukaryotic organisms. [1] [2] Plasmids often carry useful genes, such as antibiotic resistance and virulence. [3] [4] [5] While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain additional genes for special circumstances.

Contents

Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via transformation. Synthetic plasmids are available for procurement over the internet by various vendors using submitted sequences typically designed with software, if a design does not work the vendor may make additional edits from the submission. [6] [7] [8]

Plasmids are considered replicons , units of DNA capable of replicating autonomously within a suitable host. However, plasmids, like viruses, are not generally classified as life. [9] Plasmids are transmitted from one bacterium to another (even of another species) mostly through conjugation. [3] This host-to-host transfer of genetic material is one mechanism of horizontal gene transfer, and plasmids are considered part of the mobilome. Unlike viruses, which encase their genetic material in a protective protein coat called a capsid, plasmids are "naked" DNA and do not encode genes necessary to encase the genetic material for transfer to a new host; however, some classes of plasmids encode the conjugative "sex" pilus necessary for their own transfer. Plasmids vary in size from 1 to over 400 kbp, [10] and the number of identical plasmids in a single cell can range from one up to thousands.

History

The term plasmid was coined in 1952 by the American molecular biologist Joshua Lederberg to refer to "any extrachromosomal hereditary determinant." [11] [12] 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 refer to genetic elements that reproduce autonomously. [13] Later in 1968, it was decided that the term plasmid should be adopted as the term for extrachromosomal genetic element, [14] and to distinguish it from viruses, the definition was narrowed to genetic elements that exist exclusively or predominantly outside of the chromosome, can replicate autonomously, and contribute to transferring mobile elements between unrelated bacteria. [3] [4] [13]

Properties and characteristics

There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as with the top instance, whereas episomes, the lower example, can integrate into the host chromosome. Plasmid replication (english).svg
There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as with the top instance, whereas episomes, the lower example, can integrate into the host chromosome.

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 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 iterons, DnaA boxes, and an adjacent AT-rich region. [13] 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 episomes in prokaryotes. [15]

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 factors 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. [16] Plasmids can also provide bacteria with the ability to fix nitrogen. Some plasmids, called cryptic plasmids, play a crucial role in horizontal genes transfer, since they carry antibiotic-resistance genes. Thus they are important factors in spreading resistance, which can result in antibiotic treatment failures. [17]

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. Plasmids are generally circular, but examples of linear plasmids are also known. These linear plasmids require specialized mechanisms to replicate their ends. [13]

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, and is determined by how the replication initiation is regulated and the size of the molecule. Larger plasmids tend to have lower copy numbers. [15] Low-copy-number plasmids that exist only as one or a few copies in each bacterium are, upon cell division, in danger of being lost in one of the segregating bacteria. Such single-copy plasmids have systems that attempt to actively distribute a copy to both daughter cells. These systems, which include the parABS system and parMRC system, are often referred to as the partition system or partition function of a plasmid. [18]

Plasmids of linear form are unknown among phytopathogens with one exception, Rhodococcus fascians . [19]

Classifications and types

Overview of bacterial conjugation Conjugation.svg
Overview of bacterial conjugation
Electron micrograph of a DNA fiber bundle, presumably of a single bacterial chromosome loop DNA Under electron microscope Image 3576B-PH.jpg
Electron micrograph of a DNA fiber bundle, presumably of a single bacterial chromosome loop
Electron micrograph of a bacterial DNA plasmid (chromosome fragment) Plasmid em-en.jpg
Electron micrograph of a bacterial DNA plasmid (chromosome fragment)

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 genes which promote sexual conjugation between different cells. [15] In the complex process of conjugation, plasmids may be transferred from one bacterium to another via sex pili encoded by some of the transfer genes (see figure). [20] 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. [21]

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. [22] [23]

Another way to classify plasmids is by function. There are five main classes:

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. 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. [30]

Chromids

Chromids are elements that exist at the boundary between a chromosome and a plasmid, found in about 10% of bacterial species sequenced by 2009. These elements carry core genes and have codon usage similar to the chromosome, yet use a plasmid-type replication mechanism such as the low copy number RepABC. As a result, they have been variously classified as minichromosomes or megaplasmids in the past. [31] In Vibrio , the bacterium synchronizes the replication of the chromosome and chromid by a conserved genome size ratio. [32]

Vectors

Artificially constructed plasmids may be used as vectors in genetic engineering. 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 particular genes. [33] 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 is most frequently used for bacterial strains), an origin of replication to allow the bacterial cells to replicate the plasmid DNA, and a suitable site for cloning (referred to as a multiple cloning site).

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 include direct, inverted, and tandem repeats, which are known to be conspicuous in a large number of commercially available cloning and expression vectors. [34] Insertion sequences can also severely impact plasmid function and yield, by leading to deletions and rearrangements, activation, down-regulation or inactivation of neighboring gene expression. [35] Therefore, the reduction or complete elimination of extraneous noncoding backbone sequences would pointedly reduce the propensity for such events to take place, and consequently, the overall recombinogenic potential of the plasmid. [36] [37]

A schematic representation of the pBR322 plasmid, one of the first plasmids to be used widely as a cloning vector. Shown on the plasmid diagram are the genes encoded (amp and tet for ampicillin and tetracycline resistance respectively), its origin of replication (ori), and various restriction sites (indicated in blue). PBR322.svg
A schematic representation of the pBR322 plasmid, one of the first plasmids to be used widely as a cloning vector. Shown on the plasmid diagram are the genes encoded (amp and tet for ampicillin and tetracycline resistance respectively), its origin of replication (ori), and various restriction sites (indicated in blue).

Cloning

Plasmids are the most-commonly used bacterial cloning vectors. [38] These cloning vectors contain a site that allows DNA fragments to be inserted, for example a multiple cloning site or polylinker which has several commonly used restriction sites 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. These plasmids contain a selectable marker, 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 genes or reporter genes 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 cloning vector is typically used to clone DNA fragments of up to 15 kbp. [39] To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes, or yeast artificial chromosomes are used.

Protein Production

Insulin Human insulin 100IU-ml vial white background.jpg
Insulin

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, for example, utilizing the rapid reproduction of E.coli with a plasmid containing the insulin gene leads to a large production of insulin. [40] [41] [42]

Gene therapy

Plasmids may also be used for gene transfer as a potential treatment in gene therapy so that it may express the protein that is lacking in the cells. Some forms of gene therapy require the insertion of therapeutic genes at pre-selected chromosomal target sites within the human genome. Plasmid vectors are one of many approaches that could be used for this purpose. Zinc finger nucleases (ZFNs) offer a way to cause a site-specific double-strand break to the DNA genome and cause homologous recombination. Plasmids encoding ZFN could help deliver a therapeutic gene to a specific site so that cell damage, cancer-causing mutations, or an immune response is avoided. [43]

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 recombination techniques, and zinc finger nucleases, have enabled the creation of a new generation of isogenic human disease models.

Biogenetic Gene Cluster (BGC)

Plasmids assist in transporting biogenetic gene clusters - a set of gene that contain all the necessary enzymes that lead to the production of special metabolites (formally known as secondary metabolite). [44] A benefit of using plasmids to transfer BGC is demonstrated by using a suitable host that can mass produce specialized metabolites, some of these molecules are able to control microbial population. [45] Plasmids can contain and express several BGCs with a few plasmids known to be exclusive for transferring BGCs. [46] BGC's can also be transfers to the host organism's chromosome, utilizing a plasmid vector, which allows for studies in gene knockout experiments. [47] By using plasmids for the uptake of BGCs, microorganisms can gain an advantage as production is not limited to antibiotic resistant biosynthesis genes but the production of toxin s/antitoxins. [48]

Episomes

The term episome was introduced by François Jacob and Élie Wollman in 1958 to refer to extra-chromosomal genetic material that may replicate autonomously or become integrated into the chromosome. [49] [50] 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. [51] [52]

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. [53] 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. [54] [55] Viruses are the most common examples of this, such as herpesviruses, adenoviruses, and polyomaviruses, 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 infections) can also occur. Some episomes, such as herpesviruses, replicate in a rolling circle mechanism, similar to bacteriophages (bacterial phage viruses). Others replicate through a bidirectional replication mechanism (Theta type plasmids). In either case, episomes remain physically separate from host cell chromosomes. Several cancer viruses, including Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, are maintained as latent, chromosomally distinct episomes in cancer cells, where the viruses express oncogenes that promote cancer cell proliferation. In cancers, these episomes passively replicate together with host chromosomes when the cell divides. When these viral episomes initiate lytic replication to generate multiple virus particles, they generally activate cellular innate immunity 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 . [56] This variant produces both a long-lived poison and a short-lived antidote. Several types of plasmid addiction systems (toxin/ antitoxin, metabolism-based, ORT systems) were described in the literature [57] 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.[ clarification needed ]

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.

Plasmids in nature

Yeast plasmids

Yeasts naturally harbour various plasmids. Notable among them are 2 μm plasmids—small circular plasmids often used for genetic engineering of yeast—and linear pGKL plasmids from Kluyveromyces lactis , that are responsible for killer phenotypes. [58]

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

Plant mitochondrial plasmids

The mitochondria of many higher plants contain self-replicating, 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 two categories- circular and linear. [59] Circular plasmids have been isolated and found in many different plants, with those in Vicia faba and Chenopodium album 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 (as in C.album). [60] Linear plasmids have been identified in some plant species such as Beta vulgaris , Brassica napus, Zea mays , 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 from pathogenic fungi. [59] [61]

Study of plasmids

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, plasmids often need to be isolated.

There are several methods to isolate plasmid DNA from bacteria, ranging from the plasmid extraction kits (miniprep to the maxiprep or bulkprep), alkaline lysis, enzymatic lysis, and mechanical lysis . [33] The former can be used to quickly find out whether the plasmid is correct in any of several bacterial clones. The yield is a small amount of impure plasmid DNA, which is sufficient for analysis by restriction digest and for some cloning techniques.

In the latter, much larger volumes of bacterial suspension are grown from which a maxi-prep can be performed. 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. The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest:

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 digests are frequently used to analyse purified plasmids. These enzymes specifically break the DNA at certain short sequences. The resulting linear fragments form 'bands' after gel electrophoresis. It is possible to purify certain fragments by cutting the bands out of the gel and dissolving the gel to release the DNA fragments.

Because of its tight conformation, supercoiled DNA migrates faster through a gel than linear or open-circular DNA.

Software for bioinformatics and design

The use of plasmids as a technique in molecular biology is supported by bioinformatics software. These programs record the DNA sequence of plasmid vectors, help to predict cut sites of restriction enzymes, and to plan manipulations. Examples of software packages that handle plasmid maps are ApE, Clone Manager, GeneConstructionKit, Geneious, Genome Compiler, LabGenius, Lasergene, MacVector, pDraw32, Serial Cloner, UGENE, VectorFriends, Vector NTI, and WebDSV. These pieces of software help conduct entire experiments in silico before doing wet experiments. [62]

Plasmid collections

Many plasmids have been created over the years and researchers have given out plasmids to plasmid databases such as the non-profit organisations Addgene and BCCM/GeneCorner. One can find and request plasmids from those databases for research. Researchers also often upload plasmid sequences to the NCBI database, from which sequences of specific plasmids can be retrieved.

See also

Related Research Articles

<span class="mw-page-title-main">Bacterial conjugation</span> Method of bacterial gene transfer

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. This takes place through a pilus. It is a parasexual mode of reproduction in bacteria.

A bacterial artificial chromosome (BAC) 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 division. The bacterial artificial chromosome's usual insert size is 150–350 kbp. A similar cloning vector called a PAC has also been produced from the DNA of P1 bacteriophage.

<span class="mw-page-title-main">Cloning vector</span> Small piece of maintainable DNA

A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites. The vector and the foreign DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.

<span class="mw-page-title-main">Yeast artificial chromosome</span> Genetically engineered chromosome derived from the DNA of yeast

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 kb, the inserted sequences can be cloned and physically mapped using a process called chromosome walking. This is the process that was initially used for the Human Genome Project, however due to stability issues, YACs were abandoned for the use of bacterial artificial chromosome

<span class="mw-page-title-main">Prophage</span> Bacteriophage genome that is integrated into a bacterial cell

A prophage is a bacteriophage genome that is integrated into the circular bacterial chromosome or exists as an extrachromosomal plasmid within the bacterial cell. Integration of prophages into the bacterial host is the characteristic step of the lysogenic cycle of temperate phages. Prophages remain latent in the genome through multiple cell divisions until activation by an external factor, such as UV light, leading to production of new phage particles that will lyse the cell and spread. As ubiquitous mobile genetic elements, prophages play important roles in bacterial genetics and evolution, such as in the acquisition of virulence factors.

<span class="mw-page-title-main">Genetic transformation</span> Genetic alteration of a cell by uptake of genetic material from the environment

In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

<span class="mw-page-title-main">Transduction (genetics)</span> Transfer process in genetics

Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector. An example is the viral transfer of DNA from one bacterium to another and hence an example of horizontal gene transfer. Transduction does not require physical contact between the cell donating the DNA and the cell receiving the DNA, and it is DNase resistant. Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell's genome.

A cosmid is a type of hybrid plasmid that contains a Lambda phage cos sequence. Often used as cloning vectors in genetic engineering, cosmids can be used to build genomic libraries. They were first described by Collins and Hohn in 1978. Cosmids can contain 37 to 52 kb of DNA, limits based on the normal bacteriophage packaging size. They can replicate as plasmids if they have a suitable origin of replication (ori): for example SV40 ori in mammalian cells, ColE1 ori for double-stranded DNA replication, or f1 ori for single-stranded DNA replication in prokaryotes. They frequently also contain a gene for selection such as antibiotic resistance, so that the transformed cells can be identified by plating on a medium containing the antibiotic. Those cells which did not take up the cosmid would be unable to grow.

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 vector which allows the insert sequence to be replicated and/or expressed in the target cell. This gene can be cloned from a naturally occurring gene, or synthetically constructed. The vector can be delivered using physical, chemical or viral methods. Typically, the vectors used in DNA constructs contain an origin of replication, a multiple cloning site, and a selectable marker. Certain vectors can carry additional regulatory elements based on the expression system involved.

Extrachromosomal DNA 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 extrachromosomal DNA exist, and, while some of these serve important biological functions, they can also play a role in diseases such as cancer.

A genomic library is a collection of overlapping DNA fragments that together make up the total genomic DNA of a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA. In order to construct a genomic library, the organism's DNA is extracted from cells and then digested with a restriction enzyme to cut the DNA into fragments of a specific size. The fragments are then inserted into the vector using DNA ligase. Next, the vector DNA can be taken up by a host organism - commonly a population of Escherichia coli or yeast - with each cell containing only one vector molecule. Using a host cell to carry the vector allows for easy amplification and retrieval of specific clones from the library for analysis.

Fosmids are similar to cosmids but are based on the bacterial F-plasmid. The cloning vector is limited, as a host can only contain one fosmid molecule. Fosmids can hold DNA inserts of up to 40 kb in size; often the source of the insert is random genomic DNA. A fosmid library is prepared by extracting the genomic DNA from the target organism and cloning it into the fosmid vector. The ligation mix is then packaged into phage particles and the DNA is transfected into the bacterial host. Bacterial clones propagate the fosmid library. The low copy number offers higher stability than vectors with relatively higher copy numbers, including cosmids. Fosmids may be useful for constructing stable libraries from complex genomes. Fosmids have high structural stability and have been found to maintain human DNA effectively even after 100 generations of bacterial growth. Fosmid clones were used to help assess the accuracy of the Public Human Genome Sequence.

Plant transformation vectors are plasmids that have been specifically designed to facilitate the generation of transgenic plants. The most commonly used plant transformation vectors are T-DNA binary vectors and are often replicated in both E. coli, a common lab bacterium, and Agrobacterium tumefaciens, a plant-virulent bacterium used to insert the recombinant DNA into plants.

A P1-derived artificial chromosome, or PAC, is a DNA construct derived from the DNA of P1 bacteriophages and Bacterial artificial chromosome. It can carry large amounts of other sequences for a variety of bioengineering purposes in bacteria. It is one type of the efficient cloning vector used to clone DNA fragments in Escherichia coli cells.

In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

QMCF Technology is an episomal protein production system that uses genetically modified mammalian cells and specially designed plasmids. QMCF plasmids carry a combination of regulatory sequences from mouse polyomavirus (Py) DNA replication origin which in combination with Epstein-Barr virus (EBV) EBNA-1 protein binding site as nuclear retention elements ensure stable propagation of plasmids in mammalian cells. In addition the vectors carry the selection marker operational for selection of plasmid carrying bacteria and QMCF cells, bacterial ColE1 origin of replication, and cassette for expression of protein of interest. QMCF cell lines express Large-T antigen and EBNA-1 proteins which bind the viral sequences on the QMCF plasmid and hence support plasmid replication and maintenance in the cells. QMCF Technology has several important differences compared to commonly known transient expression and stable cell line expression systems. Unlike in transient expression system, QMCF Technology enables to maintain episomally replicating QMCF plasmids inside the cells for up to 50 days thus providing an option for production phase of 2–3 weeks. Therefore, the production levels of QMCF Technology are higher. Another difference is the option of establishing expression cell banks within one week, which is not feasible with transient system. Compared to usage of stable cell line, QMCF technology is a rapid method leaving out time-consuming clone selection step during cell line development.

<span class="mw-page-title-main">Plasmid-mediated resistance</span> Antibiotic resistance caused by a plasmid

Plasmid-mediated resistance is the transfer of antibiotic resistance genes which are carried on plasmids. Plasmids possess mechanisms that ensure their independent replication as well as those that regulate their replication number and guarantee stable inheritance during cell division. By the conjugation process, they can stimulate lateral transfer between bacteria from various genera and kingdoms. Numerous plasmids contain addiction-inducing systems that are typically based on toxin-antitoxin factors and capable of killing daughter cells that don't inherit the plasmid during cell division. Plasmids often carry multiple antibiotic resistance genes, contributing to the spread of multidrug-resistance (MDR). Antibiotic resistance mediated by MDR plasmids severely limits the treatment options for the infections caused by Gram-negative bacteria, especially family Enterobacteriaceae. The global spread of MDR plasmids has been enhanced by selective pressure from antimicrobial medications used in medical facilities and when raising animals for food.

<span class="mw-page-title-main">Molecular cloning</span> Set of methods in molecular biology

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 method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.

Bacterial recombination is a type of genetic recombination in bacteria characterized by DNA transfer from one organism called donor to another organism as recipient. This process occurs in three main ways:

Integrative and conjugative elements (ICEs) are mobile genetic elements present in both gram-positive and gram-negative bacteria. In a donor cell, ICEs are located primarily on the chromosome, but have the ability to excise themselves from the genome and transfer to recipient cells via bacterial conjugation.

References

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