Hubbry Logo
PlasmidPlasmidMain
Open search
Plasmid
Community hub
Plasmid
logo
8 pages, 0 posts
0 subscribers
Be the first to start a discussion here.
Be the first to start a discussion here.
Plasmid
Plasmid
Plasmid
View on Wikipedia
from Wikipedia

Diagram 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[1] and archaea;[2] however plasmids are sometimes present in eukaryotic organisms as well.[3][page needed][4] Plasmids often carry useful genes, such as those involved in antibiotic resistance, virulence,[5][6][7] secondary metabolism[8] and bioremediation.[9][10] 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.

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.[11][12][13]

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.[14] Plasmids are transmitted from one bacterium to another (even of another species) mostly through conjugation.[5] 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,[15] and the number of identical plasmids in a single cell can range from one up to thousands.

History

[edit]

The term plasmid was coined in 1952 by the American molecular biologist Joshua Lederberg to refer to "any extrachromosomal hereditary determinant."[16][17] 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.[18] Later in 1968, it was decided that the term plasmid should be adopted as the term for extrachromosomal genetic element,[19] 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.[5][6][18]

Properties and characteristics

[edit]
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.[18] 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.[20]

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.[21] Plasmids can also provide bacteria with the ability to fix nitrogen. Some plasmids, called cryptic plasmids, don't appear to provide any clear advantage to its host, yet still persist in bacterial populations.[22] However, recent studies show that they may play a role in antibiotic resistance by contributing to heteroresistance within bacterial populations.[23]

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.[18]

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.[20] 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.[24]

Further information: Regulatory region of repBA gene

Plasmids of linear form are unknown among phytopathogens with one exception, Rhodococcus fascians.[25]

Classifications and types

[edit]
Overview of bacterial conjugation
Electron micrograph of a DNA fiber bundle, presumably of a single bacterial chromosome loop
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.[20] 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).[26] 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.[27]

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.[28][29] Incompatibility typing (or Inc typing) was traditionally achieved by genetic phenotyping methods, testing whether cells stably transmit plasmid pairs to their progeny.[30] This has largely been superseded by genetic methods such as PCR, and more recently by whole-genome sequencing methods with bioinformatic tools such as PlasmidFinder.[31]

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 and result in the expression of sex pili.[17][32] F-plasmids are categorized as either (+) or (-) and contribute to the difference of being a donor or recipient during conjugation.[17][32][33]
  • Resistance (R) plasmids, which contain genes that provide resistance against antibiotics or antibacterial agents was first discovered in 1959.[34] R-factors where seen as the contributing factor for the spread of multidrug resistance in bacteria, some R-plasmids assist in transmissibility of other specifically non- self transmissible R-factors.[35][36] Historically known as R-factors, before the nature of plasmids was understood.
  • Col plasmids, which contain genes that code for bacteriocins, proteins that can kill other bacteria.
  • Degradative plasmids, which enable the digestion of unusual substances, e.g. toluene and salicylic acid.
  • Virulence plasmids, which turn the bacterium into a pathogen. e.g. Ti plasmid in Agrobacterium tumefaciens. Bacteria under selective pressure will keep plasmids containing virulence factors as it is a cost - benefit for survival, removal of the selective pressure can lead to the loss of a plasmid due to the expenditure of energy needed to keep it is no longer justified.[7][37]

Plasmids can belong to more than one of these functional groups.

Sequence-based plasmid typing

[edit]

With the wider availability of whole genome sequencing which is able to capture the genetic sequence of plasmids, methods have been developed to cluster or type plasmids based on their sequence content. Plasmid multi-locus sequence typing (pMLST) is based on chromosomal Multilocus sequence typing by matching the sequence of replication machinery genes to databases of previously classified sequences. If the sequence allele matches the database, this is used as the plasmid classification, and therefore has higher sensitivity than a simple presence or absence test of these genes.[31]

A related method is to use average nucleotide identity between plasmids to find close genetic neighbours. Tools which use this approach include COPLA[38] and MOB-cluster.[39]

Creating typing classifications using unsupervised learning, that is without a pre-existing database or 'reference-free', has been shown to be useful in grouping plasmids in new datasets without biasing or being limited to representations in a pre-built database—tools to do this include mge-cluster.[40] As plasmid frequently change their gene content and order, modelling genetic distances between them using methods designed for point mutations can lead to poor estimates of the true evolutionary distance between plasmids. Tools such as pling find homologous sequence regions between plasmids, and more accurately reconstruct the number of evolutionary events (structural variants) between each pair, then use unsupervised clustering approaches to group plasmids.[41]

RNA plasmids

[edit]

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.[42]

Chromids

[edit]
Main article: Chromid

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.[43] In Vibrio, the bacterium synchronizes the replication of the chromosome and chromid by a conserved genome size ratio.[44]

Vectors

[edit]
Further information: Vector (molecular biology)

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.[45] 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.[46] 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.[47] 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.[48][49]

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

[edit]
Main article: Cloning vector

Plasmids are the most-commonly used bacterial cloning vectors.[50] 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.[51] To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes, or yeast artificial chromosomes are used.

Suicide Vectors (plasmids)

[edit]

Suicide vectors are plasmids that are unable to replicate in the host cell and therefore have to integrate in the chromosome or disappear.[52] One example of these vectors are pMQ30 plasmid. This plasmid has SacB gene from Bacillus subtilis which can be induced by sucrose and will be lethal when expressed in Gram-negative bacteria.[53] The benefit of this system( two-step success monitoring ) shows when the experiment design needs a target gene to be integrated into the chromosome of the bacterial host. In the first step after transforming the host cells with the plasmid, a media with specific antibiotic could be used to select for bacteria that contain the plasmid. The second step makes sure that only the bacteria with integrated plasmid would survive. Since the plasmid contain the SacB gene that will induce toxicity in presence of sucrose, only the bacteria would survive and grow that has the plasmid integrated in their chromosome.

Protein Production

[edit]
Main article: Expression vector
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.[54][55][56]

Gene therapy

[edit]
Main article: Vectors in 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.[57]

Disease models

[edit]

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.

Biosynthetic Gene Cluster (BGC)

[edit]

Plasmids assist in transporting biosynthetic 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).[58] 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.[59][60] Plasmids can contain and express several BGCs with a few plasmids known to be exclusive for transferring BGCs.[60] BGC's can also be transfers to the host organism's chromosome, utilizing a plasmid vector, which allows for studies in gene knockout experiments.[61] 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 toxins/antitoxins.[62]

Episomes

[edit]
Main article: Episome

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.[63][64] 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.[65][66]

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.[67]

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.[68][69] 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

[edit]
Main article: Addiction module

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.[70] 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[71] 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 eukaryotes

[edit]

Plasmids replicated and expressed by the eukaryotic cell's machinery may reside in the cytoplasm or the cell nucleus, with different plasmids having different preferences. In addition, eukaryotes have two endosymbiotic organelles with their own genome and genetic machinery – the mitochondrion and the chloroplast – and each of these can carry plasmids in addition to the main organellar genome molecule.

Eukaryotic cytoplasmic plasmids

[edit]

Yeast cytoplasmic plasmids

[edit]

The natural linear pGKL plasmids from Kluyveromyces lactis are responsible for killer phenotypes. They can be put into the common baker's yeast S. cerevisiae.[72]

Eukaryotic nuclear plasmids

[edit]

Plasmids enter the nucleus during cell division, being rolled up in the nuclear envelope as it is reformed. As a result, nuclear plasmids do not work in non-dividing cells.[73]

Yeast nuclear plasmids

[edit]

The natural 2 micron plasmid (named after the fact that it is about 2 μm in size) is found in the common baker's yeast S. cerevisiae. It resides in the nucleus in 30–40 copies per cell. It is often modified for genetic engineering of yeast.[74]

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. They are usually modified into cloning vectors when studying the functionality of a solo gene or when the gene is toxic. Also connected with the gene URA3 which 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.

Mammalian nuclear plasmids

[edit]

Artificial plasmids are put into mammalian cells to make them produce a certain gene product. They are also used to identify and quantitatively study regulatory elements, with the assumption that these elements on a plasmid would act similarly to a copy on the chromosome.[75]

Interaction with chromatin

[edit]

Being DNA sequences in the nucleus, the plasmid molecule also interactes with histone proteins.

  • In yeast, pBR327 produces well-formed chromatin thanks to the alignment of nucleosomes by Histone H5. A specific 800bp region is found to induce this alignment, without which it does not occur.[76]
  • Mammals: the HEK 293 cell forms nucleosomes around both intact and damaged pEGFP-N1, but the structure is anomalous compared to proper chromatin from chromosomes.[77] In 2025, it was discovered that formation of chromatin is guided by the sequences of the plasmid (like it is on the chromosome), but only some sequences produce the "native" structure as found on the chromosome.[75]

Eukaryotic organellar plasmids

[edit]

Natural plant mitochondrial plasmids

[edit]

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.[78] 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).[79] 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.[78][80]

Study of plasmids

[edit]

Plasmid DNA extraction

[edit]

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 .[45] 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

[edit]

Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during electrophoresis. The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest:

  • Nicked open-circular DNA has one strand cut.
  • Relaxed circular DNA is fully intact with both strands uncut but has been enzymatically relaxed (supercoils removed). This can be modeled by letting a twisted extension cord unwind and relax and then plugging it into itself.
  • Linear DNA has free ends, either because both strands have been cut or because the DNA was linear in vivo. This can be modeled with an electrical extension cord that is not plugged into itself.
  • Supercoiled (or covalently closed-circular) DNA is fully intact with both strands uncut, and with an integral twist, resulting in a compact form. This can be modeled by twisting an extension cord and then plugging it into itself.
  • Supercoiled denatured DNA is similar to 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 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

[edit]
Main article: List of genetic engineering software

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.[81]

Plasmid collections

[edit]

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. There have been multiple efforts to create curated and quality controlled databases from these uploaded sequences; an early example is by Orlek et al,[82] which limited itself to Enterobacteriaceae plasmids, while COMPASS also encompassed plasmids from other bacteria. More recently, PLSDB[83] was made as a more up to date curated database of NCBI plasmids, and as of 2024 contains over 72,000 entries.[84] A similar database is pATLAS, which additionally includes visual analytics tools to show relationships between plasmids.[85] The largest plasmid database made from publicly available data is IMG/PR, which not only contains full plasmid sequences retrieved from NCBI, but novel plasmid genomes found from metagenomes and metatranscriptomes.[86]

Other datasets have been created by sequencing and computing plasmid genomes from pre-existing bacterial collections, e.g. the NORM collection[87][88] and the Murray Collection.[89][90]

mRNA vaccines

[edit]

In 2025, researchers from Canada and the US revealed that Covid-19 mRNA vaccines contain high amounts of residual DNA (371-1,548 ng/dose for Pfizer and 1,130-6,280 for Moderna), far exceeding the maximum level (10 ng/dose) that was recommended from the WHO and the FDA. SV40 sequences were observed in Pfizer products. They also analyzed data from VERS to examine an association between serious adverse events (SAEs) and mRNA products, suggesting a positive correlation between SAEs and residual DNA levels. The study concluded that transparent information about manufacturing processes should be provided by regulators and industry, and stricter guidelines should be set for safety.[91]

See also

[edit]

References

[edit]

Further reading

[edit]
[edit]
Revisions and contributorsEdit on WikipediaRead on Wikipedia

Plasmid

View on Grokipedia
from Grokipedia
A plasmid is an extrachromosomal, usually circular, double-stranded molecule that is self-replicating and capable of autonomous replication independent of the host cell's chromosomal , most commonly found in and . These molecules typically range in size from a few thousand to hundreds of thousands of base pairs and can exist in multiple copies within a single cell, influencing bacterial and through . The term "plasmid" was coined by in 1952 to describe any extrachromosomal genetic element, with early studies in the late focusing on their role in antibiotic resistance. Plasmids play a critical role in microbial adaptation by carrying accessory genes that confer traits such as antibiotic resistance, virulence factors, metabolic capabilities, or toxin production, which can be rapidly disseminated between cells via conjugation, transformation, or transduction. They are classified by various criteria, including replication mechanism (e.g., theta-type, rolling-circle, or strand displacement), (predominantly circular but occasionally linear), mobility (conjugative, mobilizable, or nonmobilizable), and incompatibility groups that determine coexistence within the same host. In natural environments, plasmids contribute to bacterial diversity and ecosystem dynamics, such as or heavy metal tolerance, while also posing challenges in clinical settings through the spread of multidrug resistance. Beyond their ecological significance, plasmids have revolutionized as essential tools for , serving as vectors to introduce, express, and propagate foreign genes in host organisms like . Landmark developments, including the 1973 construction of recombinant plasmids using restriction enzymes like , enabled the production of insulin and other therapeutics, marking the birth of modern technology. Today, engineered plasmids incorporate features like selectable markers, promoters, and origins of replication for tunable copy numbers, supporting applications in , vaccine development, and across kingdoms of life.

History

Discovery and Early Observations

The discovery of plasmids began with foundational experiments on bacterial genetics in the mid-20th century. In 1946, Joshua Lederberg and Edward L. Tatum demonstrated genetic recombination in Escherichia coli through conjugation, a process where genetic material is transferred between bacterial cells, revealing the existence of non-chromosomal hereditary elements responsible for this inheritance. Their work, using auxotrophic mutants, showed that traits could be exchanged independently of the main chromosome, laying the groundwork for understanding extrachromosomal DNA. Building on these findings, Lederberg coined the term "plasmid" in 1952 to describe any extrachromosomal genetic particle capable of and transmission, distinguishing it from viral or cytoplasmic factors. Concurrently, in the early 1950s, studies on the factor (F-factor) in E. coli highlighted its in promoting conjugation, suggesting a distinct genetic entity. By 1958, François Jacob and Élie L. Wollman refined this concept, introducing the term "" for autonomously replicating elements that could integrate into or excise from the bacterial , based on their analysis of the . A pivotal technique developed by and Wollman further elucidated these elements. Their interrupted experiments, conducted in the late 1950s and detailed in 1958 publications, involved mechanically disrupting conjugating bacterial pairs at timed intervals using a blender, allowing mapping of gene transfer and confirmation that the F-factor was an extrachromosomal entity initiating mobilization. This method provided of plasmid-mediated transfer, shifting the view from chromosomal recombination alone to involvement of independent DNA loops. Early links to practical implications emerged in 1959 when Riichi Ochiai and colleagues observed the transfer of multiple resistance (e.g., to , , , and ) between Shigella strains and E. coli , attributing it to a transferable factor later identified as an R-plasmid. These observations, among the first to connect plasmids to resistance, underscored their role in bacterial adaptability and set the stage for broader microbiological investigations.

Key Milestones in Research and Applications

In 1969, Donald B. Clewell and Donald R. Helinski isolated the first plasmid, , from as a supercoiled circular DNA-protein complex, marking a pivotal advancement that enabled detailed studies of plasmid structure and function. The development of technology in 1972–1973 by , Herbert W. Boyer, and Stanley N. revolutionized plasmid applications, with Berg demonstrating the joining of DNA from different sources using and lambda phage, and and Boyer creating the first plasmid-based gene cloning system in bacteria by inserting foreign DNA into E. coli plasmids via restriction enzymes. This breakthrough facilitated the controlled propagation of recombinant genes and laid the foundation for genetic engineering. Berg received the 1980 for his contributions to methodology, sharing it with and for related advancements in nucleic acid biochemistry. During the 1970s, the discovery and characterization of type II restriction endonucleases by , Hamilton O. Smith, and —enzymes that precisely cleave DNA at specific sequences—combined with DNA ligases such as T4 ligase, enabled efficient plasmid manipulation and vector construction. These tools were instrumental in the creation of the first synthetic gene cloned into a plasmid in 1977, when Boyer and colleagues inserted a chemically synthesized gene into E. coli, demonstrating the feasibility of producing eukaryotic proteins in bacterial hosts. In recent years, plasmids have integrated with CRISPR-Cas9 systems for advanced , beginning with the 2012 demonstration by , , and colleagues of Cas9-mediated cleavage of plasmid DNA and using , which expanded plasmids' role in programmable . In 2020, Charpentier and Doudna were awarded the for the development of CRISPR-Cas9, a method utilizing plasmid vectors for precise . Additionally, has advanced with the design of minimal plasmids, such as pJL1 reported in 2018 by Michael Jewett and team, which strips non-essential elements to optimize cell-free protein expression and reduce metabolic burden in host cells.

Properties and Characteristics

Molecular Structure

Plasmids are small, extrachromosomal, circular, double-stranded DNA molecules that exist independently of the bacterial chromosome. These molecules typically range in size from 1 to 200 kilobase pairs (kb), though natural plasmids exhibit significant variability, with small plasmids often under 10 kb and large megaplasmids exceeding 1 megabase pair (Mb). In their native state within cells, plasmids adopt a supercoiled topology, where is twisted upon itself to form a compact structure that facilitates cellular processes and packaging. Linear forms are exceedingly rare among natural plasmids, which are predominantly covalently closed circular. At the molecular level, plasmids contain essential core components that enable their autonomous existence, including an (ori) sequence that serves as the starting point for , as well as genes for partitioning to ensure equitable distribution during . Selectable markers, such as resistance genes, are common accessory elements that confer advantages like survival under selective pressures, while modular genetic elements including promoters and terminators regulate within the plasmid. The genetic content of plasmids is divided into housekeeping genes, which maintain the plasmid's replication and stability, and accessory genes that provide adaptive traits to the host, such as those involved in factors, metabolic pathways, or production. This modular organization allows plasmids to integrate diverse functional modules while preserving the core elements necessary for propagation.

Replication Mechanisms

Plasmids replicate autonomously within host cells, primarily using two distinct mechanisms: replication for most circular forms and rolling-circle replication for smaller, often single-stranded or linear variants. These processes rely on a combination of plasmid-encoded and host-derived enzymes to ensure faithful duplication of the genetic material. replication, the predominant mode for circular bacterial plasmids, initiates at a specific origin region known as oriV, where a plasmid-encoded initiator protein, typically called Rep, binds to repeated sequences called iterons to unwind the DNA and recruit the host replication machinery. This leads to the formation of a bidirectional replication in many cases, such as in the R1 plasmid, where two forks proceed outward from oriV, creating a -shaped intermediate observable under electron microscopy; however, unidirectional replication occurs in plasmids like , with a single traversing the entire molecule. The process involves host enzymes including III for nucleotide addition, DnaB for unwinding , DnaG for synthesizing primers on the lagging strand, and topoisomerases I and IV to relieve torsional stress ahead of the advancing forks. Some plasmids, such as those in enterobacteria, depend on the host initiator protein to facilitate open complex formation at oriV, mirroring chromosomal initiation at oriC. In contrast, rolling-circle replication, employed by certain small plasmids like pT181 in staphylococci, begins with the Rep initiator protein introducing a site-specific nick at the double-stranded origin (dso), exposing a 5' end that serves as a primer for leading-strand synthesis by host DNA polymerase. The displaced single strand is coated by host single-strand binding proteins, and replication proceeds unidirectionally, generating a linear single-stranded intermediate that is later converted to double-stranded form through synthesis of the complementary strand using host and polymerase. This mechanism avoids the bidirectional complexity of theta replication and is suited to compact genomes, with Rep also possessing ligase activity in some cases to seal nicks during termination. Unlike theta modes, rolling-circle replication does not typically involve DnaA but heavily relies on host elongation factors such as and for fork progression. The time required for plasmid replication depends on the mode and host fork speed; in , forks advance at approximately 500–1000 base pairs per second, yielding a replication time t=Lvt = \frac{L}{v}, where LL is the plasmid length in base pairs and vv is the fork speed—for bidirectional theta replication, this is effectively halved due to two converging forks. is tightly controlled to synchronize with host , often through Rep protein activation by host factors like DnaA-ATP levels, ensuring replication completes before .

Copy Number and Stability

The copy number of a plasmid refers to the average number of plasmid molecules per bacterial cell, which can range from low (1-2 copies, as in the ) to high (50-700 copies, as in pUC vectors). This multiplicity is primarily determined by the strength of the (ori) and the plasmid's incompatibility group, with stronger oris promoting higher initiation rates and thus elevated copy numbers. Incompatibility arises when plasmids share similar replication control elements, such as overlapping ori sequences or regulatory proteins, preventing their stable coexistence in the same cell by interfering with replication or partitioning. Plasmid stability encompasses the long-term retention of the plasmid across cell generations without selective pressure, influenced by segregational and structural factors. Segregational instability occurs due to uneven partitioning of plasmids during , leading to plasmid-free daughter cells, while structural instability results from mutations or rearrangements in the plasmid DNA that impair replication or essential functions. Stability is typically measured by the retention rate, expressed as the percentage of cells harboring the plasmid after a defined number of generations under non-selective conditions, with high-copy plasmids generally exhibiting greater segregational stability due to random distribution approximating binomial partitioning. The steady-state copy number (CN) can be modeled as the ratio of the plasmid replication initiation frequency to the host rate, ensuring balance between plasmid duplication and dilution during growth:
CN=initiation frequencycell division rate\text{CN} = \frac{\text{initiation frequency}}{\text{cell division rate}}
This equilibrium is modulated by regulatory elements, such as RNA-based controls in ColE1-derived plasmids, where the Rom protein stabilizes the inhibitory I-RNA II complex to reduce premature primer formation and thereby lower the initiation frequency and copy number. Environmental factors, particularly nutrient availability, also impact plasmid propagation by altering host metabolism and replication machinery activity; for instance, nutrient limitation can slow cell division rates relative to initiation, potentially increasing copy number, while rich media may enhance dilution and reduce it.

Classifications and Types

Bacterial Plasmids

Bacterial plasmids are extrachromosomal, circular DNA molecules that replicate autonomously in prokaryotic hosts, often conferring adaptive advantages such as antibiotic resistance or metabolic capabilities. They exhibit significant diversity in function and transmission mechanisms, playing crucial roles in bacterial evolution and horizontal gene transfer. In bacteria, plasmids are classified based on their ability to transfer between cells, their encoded traits, and other properties like size and incompatibility. Conjugative plasmids are self-transmissible genetic elements that encode a complete set of genes for conjugation, including the tra operon responsible for forming a pilus that facilitates direct cell-to-cell DNA transfer. A classic example is the F (fertility) plasmid in Escherichia coli, which contains approximately 100 kb of DNA and directs the assembly of F pili to initiate mating pair formation and subsequent plasmid mobilization. These plasmids promote rapid dissemination of beneficial genes across bacterial populations. In contrast, non-conjugative plasmids lack the full conjugation machinery but can be mobilizable if a helper conjugative plasmid is present in the same cell, enabling their transfer via borrowed transfer factors. R-plasmids, a subset often non-conjugative or mobilizable, carry multiple antibiotic resistance genes, contributing to multidrug resistance phenotypes in pathogens like Enterobacteriaceae; for instance, they can encode resistance to up to eight different antibiotics through clustered determinants. Other notable types include cryptic plasmids, which harbor no identifiable phenotypic traits beyond replication and maintenance functions, yet they persist in bacterial populations and may serve as reservoirs for future acquisition. Degradative plasmids, often conjugative, encode catabolic pathways for breaking down compounds, such as pollutants like or ; examples include pNL1 in aromaticivorans F199 that enables metabolism under environmental stress. Bacterial plasmids are further typed by incompatibility groups, where plasmids within the same group (e.g., IncF, prevalent in E. coli and associated with the , or broad-host-range IncP) cannot stably coexist in the same cell due to shared replication control mechanisms. Size-based distinguishes small plasmids (typically <10 kb, often cryptic) from large ones (>50 kb, frequently carrying accessory genes like those in conjugative or R-plasmids). Plasmids are highly prevalent in certain bacterial lineages, with species like often harboring multiple large plasmids that collectively represent 30-50% of the total genome size; for example, Rhizobium etli CFN42 possesses six plasmids totaling approximately 2.15 Mb alongside a 4.38 Mb , underscoring their integral role in symbiotic lifestyles. This abundance highlights plasmids' contribution to genomic plasticity in prokaryotes.

Non-Bacterial Plasmids

Plasmids in represent a significant class of extrachromosomal elements adapted to the unique cellular environments of these organisms, which differ from through features such as ether-linked that enhance stability in extreme conditions. In halophilic , such as those from the Haloferacaceae family, standalone plasmids are prevalent and often carry genes for adaptation to high-salinity environments, including osmoregulatory functions. These plasmids typically range from 5 to 50 kb in size and replicate via rolling-circle or mechanisms, with copy numbers varying based on environmental stress. For instance, in Haloferax volcanii, multiple plasmids coexist, some encoding CRISPR-Cas systems for defense against phages. Ether lipid adaptations in archaeal , characterized by isoprenoid chains linked via bonds to glycerol-1-phosphate, contribute to plasmid maintenance by providing robust barriers that prevent leakage during replication under hypersaline or . Viral plasmids, often termed satellite nucleic acids, are dependent elements that parasitize bacteriophages for replication and packaging in bacterial hosts but exhibit plasmid-like autonomy in their circular DNA or RNA forms. In bacteriophages like P2 and P4, satellite elements such as P4 maintain a circular double-stranded DNA genome of about 9 kb, replicating via a plasmid-specific origin while hijacking the helper phage's structural proteins for virion assembly. These satellites encode their own repressors and partitioning systems to ensure stable inheritance, with over 1,000 such elements identified across diverse phage families using bioinformatic tools. Satellite nucleic acids can also include single-stranded DNA forms that interfere with helper phage lysis, promoting persistent infection. Viroids, considered non-coding RNA plasmids, are small, circular, single-stranded RNAs (246–430 nt) that replicate autonomously in cells without encoding proteins, relying on host RNA polymerases for rolling-circle replication. Unlike typical plasmids, viroids lack genes but induce through RNA motifs that sequester host factors or trigger RNA silencing. The (PSTVd), the first discovered in , exemplifies this, forming rod-like structures via base-pairing and accumulating to high copy numbers in chloroplasts or nuclei. Viroid replication generates multimeric intermediates cleaved by host ribonucleases, mirroring plasmid processing but in an RNA context. Over 30 viroid species are known, classified into Pospiviroidae and Avsunviroidae families based on replication sites. Linear plasmids in certain bacteria, such as species, deviate from the typical circular form found in most prokaryotes and feature terminal proteins covalently attached to 5' ends to resolve replication issues at . In coelicolor, plasmids like SCP1 (31 kb) use a protein-primed initiation mechanism where terminal proteins (Tpg) serve as primers for , synthesizing palindromic 5' overhangs during replication. These proteins, around 20 kDa, are encoded by plasmid genes and essential for telomere maintenance, preventing end degradation. Linear plasmids in often carry biosynthetic gene clusters for antibiotics, such as actinorhodin, and can integrate into the via . This system contrasts with bacterial circular plasmids by enabling larger genomes without circularization constraints. Cryptic plasmids in bacterial symbionts of eukaryotes are small, non-coding or minimally functional elements that persist without obvious phenotypic benefits but may stabilize symbiont populations. In the intracellular symbiont Buchnera aphidicola of , cryptic plasmids like pLE are multicopy (up to 50 per cell) and encode partitioning genes that ensure to host offspring. These plasmids, often under 5 kb, lack antibiotic resistance or virulence factors but harbor insertion sequences that facilitate rearrangements. Similarly, in , a reproductive manipulator of , cryptic plasmids contribute to genome plasticity despite their apparent dispensability. Their prevalence suggests subtle roles in host-symbiont , such as modulating replication rates under nutrient-limited conditions. Plasmids facilitate (HGT) across domains of life, bridging , , and eukaryotes through mechanisms like conjugation or viral packaging. In , plasmids carrying integron-like arrays capture bacterial genes, as seen in species where HGT introduces metabolic pathways from . Cross-domain transfer via plasmids has distributed defense systems, such as restriction-modification enzymes, across prokaryotic lineages, with evidence from metagenomic analyses showing shared plasmid backbones in diverse environments. For example, large plasmids in anaerobic methane-oxidizing (ANME) acquire sulfate reduction genes from bacterial donors, enabling syntrophic consortia. This plasmid-mediated HGT underscores evolutionary connectivity, with rates estimated at 10^-5 to 10^-3 events per generation in microbial communities.

Specialized Variants

Specialized variants of plasmids deviate from the canonical double-stranded DNA structure, encompassing RNA-based entities, single-stranded DNA forms, and hybrid replicons that blur the lines between plasmids and chromosomal elements. These variants often exhibit unique replication strategies and host interactions, enabling them to function in diverse biological contexts such as plant pathology and bacterial genome organization. RNA plasmids, exemplified by viroid-like agents in plants, consist of small, circular single-stranded RNA molecules that replicate autonomously without encoding proteins. The potato spindle tuber viroid (PSTVd), a prototypical example, features a 359-nucleotide circular RNA genome that adopts a rod-like secondary structure with multiple stems and loops. Unlike typical DNA plasmids, PSTVd replication relies on the host's nuclear DNA-dependent RNA polymerase II, which transcribes the viroid RNA in a rolling-circle mechanism, producing multimeric intermediates that are cleaved and ligated into monomeric circles. These RNA entities, while not true plasmids in the bacterial sense, parallel plasmid behavior by maintaining extrachromosomal persistence and propagating vertically and horizontally in infected plant tissues. In , chromids represent hybrid replicons that combine plasmid-like replication origins with chromosomal features, including essential housekeeping genes such as those for rRNA synthesis. Chromids typically range in size from approximately 0.3 to 3.6 megabases and exhibit composition and codon usage akin to the primary , distinguishing them from non-essential plasmids. A notable instance occurs in , where the secondary (ChrII, ~1.07 megabases) functions as a chromid, harboring essential genes like operons while employing a plasmid-type iteron-based replication system regulated by the initiator protein RctB. This hybrid nature allows chromids to maintain stable copy numbers similar to plasmids during , yet they contribute critically to core cellular functions, reflecting an evolutionary intermediate between plasmids and . Geminivirus-associated plasmids in plants further illustrate specialized single-stranded DNA variants, featuring circular ssDNA genomes that replicate via a virus-encoded replication initiator protein (Rep). These genomes, typically 2.5 to 3.0 kilobases in length for monopartite forms, undergo rolling-circle replication in the host nucleus, where the Rep protein nicks the DNA at a conserved origin and recruits host polymerases for elongation. Geminiviruses, such as those in the genus Begomovirus, package this ssDNA into twinned icosahedral virions, facilitating systemic spread in plants and often associating with satellite DNAs that enhance pathogenicity. Integrons serve as mobilizable plasmid elements that capture and express cassettes, particularly resistance genes, through . These structures, often integrated into conjugative or mobilizable plasmids, contain an integrase (intI) and an attI recombination site, enabling the excision and transfer of cassette arrays via mechanisms like conjugation. In , class 1 integrons on plasmids such as IncI or IncN types exemplify this mobility, allowing rapid adaptation to selective pressures by disseminating resistance determinants across bacterial populations. Sequence-based typing methods, such as plasmid multi-locus typing (pMLST), target replication genes (rep) to classify and track these specialized variants, especially resistance-conferring plasmids. pMLST schemes assign types based on alleles of plasmid backbone loci, including group-specific rep genes, facilitating epidemiological of mobilizable elements like integron-bearing plasmids in clinical isolates. This approach has been instrumental in delineating Inc group diversity and monitoring the global spread of resistance plasmids.

Vectors and Applications

Cloning and Recombinant DNA Technology

Plasmids serve as essential vectors in technology, enabling the insertion and propagation of foreign DNA sequences within host cells due to their autonomous replication capability. The pioneering plasmid vector , developed in 1977, was one of the first widely adopted cloning vehicles for , featuring selectable markers for and resistance to facilitate identification of transformed cells. This plasmid includes unique restriction sites within the resistance genes, allowing for insertional inactivation as a screening method, and its compact 4361 structure supports high copy number maintenance in bacterial hosts. The cloning process begins with restriction enzyme digestion of both the plasmid vector and the target DNA fragment to generate compatible sticky or blunt ends, enabling precise joining. Subsequent ligation using covalently links the insert to the linearized plasmid, forming a recombinant that can be introduced into host cells via transformation methods such as heat shock or . To distinguish successful recombinants from non-insert-containing plasmids, blue-white screening exploits the lacZ in vectors like pUC derivatives; insertion into the (MCS) disrupts α-complementation of , preventing hydrolysis of substrate and resulting in white colonies, while intact lacZ yields blue colonies on indicator plates supplemented with IPTG. E. coli remains the primary host for plasmid propagation due to its efficient uptake, rapid growth, and well-characterized , though shuttle vectors incorporate origins of replication and selectable markers compatible with multiple hosts, such as and , to enable transfer and maintenance across species. These vectors, like pRS series for -E. coli shuttling, allow initial in E. coli followed by expression or analysis in alternative organisms without sequence modification. Plasmids are routinely used to construct gene libraries by cloning fragmented genomic or cDNA into vectors, creating collections of clones that represent the entire or for functional screening. Additionally, PCR-amplified products can be directly cloned into linearized plasmids using TA cloning or restriction-ligation, bypassing the need for initial restriction sites and enabling rapid insertion of specific sequences up to several kilobases. Despite their utility, plasmid-based cloning faces limitations related to insert stability, where repetitive or structured DNA sequences may rearrange or delete during propagation, particularly in high-copy vectors. Cloned genes encoding toxic proteins can also impose metabolic burden on the host, leading to reduced growth, plasmid loss, or selection for mutants with inactivated inserts, often necessitating low-copy vectors or alternative hosts to mitigate these issues.

Expression Systems for Protein Production

Plasmid-based expression systems are engineered to drive high-level transcription and translation of inserted genes in host cells, enabling the production of recombinant proteins for research, diagnostics, and therapeutics. These systems typically incorporate strong promoters, regulatory elements, and selection markers to optimize gene expression while maintaining plasmid stability. In bacterial hosts like Escherichia coli, plasmids serve as versatile vectors for rapid, cost-effective protein synthesis, often achieving yields of several grams per liter in optimized conditions. For eukaryotic systems, such as insect cells, plasmid-derived vectors facilitate post-translational modifications essential for protein functionality. Key to these systems are promoters that control gene transcription. The T7 promoter, derived from bacteriophage T7, is widely used in E. coli due to its high activity when induced by T7 RNA polymerase expressed from the host genome, as in the pET vector series. This inducible system minimizes basal expression to prevent toxicity, with induction via isopropyl β-D-1-thiogalactopyranoside (IPTG) in strains like BL21(DE3). The lac promoter, also IPTG-inducible, offers moderate expression levels suitable for proteins prone to inclusion body formation, while the tac promoter—a hybrid of trp and lac—provides stronger constitutive or inducible expression for higher yields. In baculovirus expression vector systems (BEVS), the polyhedrin promoter drives robust expression in insect cells like Sf9, leveraging the virus's lytic cycle for transient high-level production. Expression cassettes on plasmids include ribosome binding sites (RBS) to facilitate initiation and affinity tags for purification. The Shine-Dalgarno serves as an RBS in bacterial systems, optimizing mRNA- interactions for efficient protein synthesis. Histidine tags (His-tags), typically 6-10 residues, enable facile purification via immobilized metal (IMAC), often fused to the N- or of the target protein without significantly impairing function. The pET series exemplifies these elements in E. coli, with modular designs allowing customizable inserts for diverse proteins. For cells, baculovirus plasmids like pAcUW1 integrate transfer vectors with sites to generate recombinant viruses for expression. Yield optimization involves codon usage adaptation to match host tRNA pools, reducing translational pauses and increasing soluble protein output; for instance, recoding genes for E. coli codons can boost expression by 10- to 100-fold. Co-expression of molecular chaperones, such as /GroES or DnaK, assists proper folding and , particularly for eukaryotic proteins in bacterial hosts, mitigating aggregation into . A prominent example is recombinant insulin production in E. coli, where proinsulin is expressed via pET-like plasmids under T7 control, processed to yield therapeutic insulin at industrial scales exceeding 10 g/L. Scaling these systems transitions from shake-flask cultures to bioreactors, where fed-batch in E. coli maintains high cell densities (up to 100 g/L dry weight) and controlled induction for consistent yields. In BEVS, wave bioreactors and stirred-tank systems support insect cell growth to 10^7 cells/mL, enabling multi-gram production of complex glycoproteins. Process monitoring of , oxygen, and metabolites ensures reproducibility from lab (1-10 L) to pilot (100-1000 L) and commercial scales.

Therapeutic and Model Organism Applications

Plasmids play a pivotal role in , particularly through DNA vaccines that encode antigens to elicit immune responses. For instance, , a plasmid-based encoding the , demonstrated 66.6% efficacy in preventing symptomatic in a phase 3 trial involving over 28,000 participants, with a favorable safety profile including mild injection-site reactions. These vaccines are delivered non-virally, often via followed by , which applies electric pulses to enhance cellular uptake and expression of the plasmid DNA. Electroporation-mediated delivery has shown up to 100-fold increased efficiency in muscle tissues compared to naked DNA injection, enabling transient without genomic integration risks. In disease modeling, plasmids facilitate the creation of transgenic models for by introducing oncogenes or tumor suppressors into somatic cells. Seminal studies have used hydrodynamic tail vein injection of oncogenic plasmids, such as those encoding Ras or , to generate liver tumors in mice that recapitulate human progression, allowing evaluation of therapeutic interventions. vectors, which carry genes like (HSV-TK) under tumor-specific promoters, enable conditional cell ablation; upon administration of , the prodrug is converted to a toxic metabolite, selectively killing transduced cells in models of and . CRISPR plasmids encoding endonuclease and guide RNAs (gRNAs) are widely used for precise in therapeutic contexts. These all-in-one plasmids co-express with multiplexed gRNAs from synthetic arrays, enabling simultaneous of multiple loci; for example, Cas12a-mediated with 10 gRNAs targeting a single locus has achieved approximately 60% efficiency in mammalian cells, with strategies to minimize off-target effects. Such systems support applications like correcting mutations in monogenic diseases or engineering immune cells for . Plasmids also drive biosynthetic gene cluster (BGC) expression for engineering, particularly . of actinomycete BGCs, such as the ~106 kb salinomycin cluster in hosts via plasmid-based cloning, yielding 10.3 mg/L of the , facilitating analog production through promoter swaps and gene knockouts. This approach unlocks cryptic BGCs, enhancing yields of compounds like erythromycin derivatives for combating antibiotic resistance. In model organisms, plasmids underpin the yeast two-hybrid (Y2H) system for detecting protein-protein interactions. The method fuses bait and prey proteins to transcriptional activator domains on separate plasmids, activating reporter genes only upon interaction; high-throughput Y2H screens have mapped over 5,000 interactions in the Saccharomyces cerevisiae proteome, revealing networks essential for signaling pathways.

Episomes and Integration

Definition and Distinction from Plasmids

An episome is defined as an extrachromosomal genetic element capable of replicating autonomously in the host cell while also possessing the ability to integrate into and replicate from the host . This dual capability distinguishes episomes from other genetic elements, allowing them to exist in either an independent or integrated state within the host genome. The term "episome" was coined in 1958 by François Jacob and Élie Wollman to describe genetic factors, such as certain bacteriophages or sex factors, that can alternate between autonomous replication in the cytoplasm and insertion into the bacterial chromosome. Historically, this concept emerged from studies on bacterial conjugation and lysogeny, highlighting episomes as dynamic elements that contribute to genetic variability and host adaptation. While all episomes are a of plasmids—circular, molecules that replicate independently— not all plasmids qualify as episomes, as the latter specifically require mechanisms akin to integrase functions for chromosomal integration. This integration potential enables episomes to leverage the host's replication and segregation machinery when inserted, providing a key functional distinction from non-integrating plasmids. Representative examples include the in bacteria, which serves as a conjugative that can integrate into the to form Hfr strains, facilitating high-frequency recombination. Similarly, bacteriophage lambda functions as a temperate , integrating into the host genome during lysogeny via while maintaining autonomous replication in its lytic cycle. In eukaryotes, the Epstein-Barr virus (EBV) genome persists as a multicopy in latently infected human B-cells, associating with host for stable maintenance. Episomes exhibit bidirectional replication control, enabling them to initiate either independently or in coordination with the host upon integration. This property contributes to their higher stability in the integrated state, where they are segregated along with the chromosomal DNA, reducing loss during compared to purely autonomous forms.

Mechanisms of Chromosomal Integration

Plasmids capable of chromosomal integration, often referred to as episomes, employ several molecular mechanisms to insert their DNA into the host , thereby transitioning from an extrachromosomal state to a stable, heritable form. These processes ensure the plasmid's persistence and can facilitate the transfer of genetic material, including antibiotic resistance genes, across bacterial populations. The primary mechanisms include , , and transposon-mediated insertion, each regulated by host and plasmid-encoded factors to balance integration with potential excision. Site-specific recombination is a precise mechanism where plasmid or phage DNA integrates at specific attachment sites on the host , catalyzed by integrase enzymes. In the case of , the Int protein mediates recombination between the phage attachment site (attP, approximately 240 bp) and the bacterial attachment site (attB, 25 bp), forming hybrid attL and attR sites that flank the integrated . This tyrosine recombinase cleaves and religates DNA strands in a intermediate, requiring host factors like integration host factor (IHF) for bending the attP site to facilitate . Similar systems operate in other integrating elements, such as the φC31 phage integrase, which efficiently recombines attP and attB in diverse bacterial hosts. Homologous recombination enables plasmid integration through sequence similarity between the plasmid and chromosome, often involving double-crossover events that replace or insert genetic material without site specificity. In bacteria like Escherichia coli, the RecA protein plays a central role by forming nucleoprotein filaments on single-stranded DNA, promoting strand invasion and exchange during double-strand break repair. A single crossover initially integrates the entire plasmid as a cointegrate, which is unstable and reversible, while a subsequent second crossover resolves it into a stable insertion, effectively duplicating homologous flanking regions. This mechanism is commonly exploited in genetic engineering for targeted chromosomal modifications, though it requires longer homology arms (500–1000 bp) for efficiency. Transposon-mediated integration occurs when insertion sequence (IS) elements or composite transposons on the plasmid mobilize and insert the entire plasmid or portions into the via a cut-and-paste or replicative transposition pathway. IS elements, such as IS26 in clinically relevant plasmids, flank the transposable unit and encode transposases that recognize inverted repeats, excising the segment and reintegrating it at target sites with little sequence preference, often generating short target site duplications. This is prominent in conjugative plasmids and integrative conjugative elements (ICEs), where transposons facilitate cointegration or fusion events that promote . Unlike site-specific methods, transposon insertion can occur at multiple chromosomal loci, increasing genomic plasticity but risking deleterious mutations. Regulation of these integration mechanisms prevents untimely insertion or excision, maintaining autonomy until environmental cues trigger . In , the CI repressor protein binds operator sites (OL and OR) to repress lytic genes from promoters pL and pR while activating its own expression from pRM, favoring integration during the ; DNA damage induces RecA-mediated CI autocleavage, shifting to lytic excision via Xis protein. This bistable switch ensures integration only under favorable conditions, such as nutrient limitation. Similar regulatory circuits in other systems involve accessory proteins that modulate integrase activity or recombination directionality. Integration alters gene dosage by reducing plasmid copy number from multiple per cell to a single chromosomal copy, stabilizing expression and minimizing metabolic burden, though it can amplify integrated genes during replication. This process enhances potential, as integrated elements like prophages or ICEs can excise and mobilize to new hosts, disseminating traits such as factors. However, frequent integration-excision cycles may impose fitness costs through genomic rearrangements.

Plasmid Maintenance

Partitioning and Segregation

Plasmid partitioning and segregation refer to the processes that ensure the of plasmid copies to cells during bacterial , preventing loss and maintaining plasmid persistence in . Low-copy-number plasmids, typically maintained at 1-2 copies per cell, rely on active partitioning systems to achieve high-fidelity segregation, while high-copy-number plasmids, with dozens of copies, depend primarily on passive mechanisms. These strategies are crucial for plasmid survival, as unequal distribution can lead to plasmid-free cells and eventual curing from the . Active partitioning in low-copy plasmids is mediated by tripartite systems such as ParABS or its functional analog SopABC in plasmids like F and P1. In these systems, ParB (or SopB) proteins bind specifically to centromere-like parS sites on the plasmid DNA, forming a complex that acts as a partition unit. ParA (or SopA), an , interacts with the ParB-parS complex and the bacterial , generating a dynamic that "walks" the plasmid towards cell poles through a diffusion-ratchet mechanism, where powers directed movement and ensures one copy is delivered to each daughter cell. This achieves segregation fidelities exceeding 99.9% per , far surpassing random distribution. For high-copy plasmids, such as with approximately 20 copies per cell, segregation occurs via passive within the , where replicated plasmids move without dedicated machinery, relying on the sheer number of copies to ensure both daughters receive at least one. Copy number influences partitioning efficiency, with higher copies reducing loss risk through probabilistic distribution. In contrast, low-copy plasmids employ models around parS sites, where ParB and spreading from the centromere-like creates a partition-competent complex, with probabilistic bridging and release facilitating poleward transport. The fidelity of random segregation can be modeled mathematically; for passive systems, the probability of plasmid loss per generation approximates 2e−n2e^{-n}, where nn is the average copy number per daughter cell, highlighting how even modest copy numbers yield extremely low loss rates (e.g., ≈0.00018%\approx 0.00018\% for n=10n = 10). Defects in partitioning, such as mutations disrupting ParA/ParB interactions, can lead to missegregation and plasmid . One common experimental method to exploit these defects is plasmid curing using sublethal concentrations of antibiotics like quinolones, which interfere with or topology, preferentially eliminating plasmids without killing the host and resensitizing resistant strains.

Host-Plasmid Interactions for Stability

Plasmids interact with their bacterial hosts through various molecular mechanisms to promote their long-term persistence, often by manipulating host to favor cells retaining the plasmid. These interactions include modules that impose lethal consequences on plasmid-free daughter cells, thereby enforcing stability beyond mere partitioning during . Such strategies are crucial in environments without selective pressure for plasmid-encoded traits, where random segregation could lead to loss. Addiction modules, particularly toxin-antitoxin (TA) systems, are prevalent on plasmids and function by encoding a stable paired with a less stable , ensuring that only plasmid-bearing cells survive post-division. In type II TA systems, the neutralizes the while the plasmid is present, but upon plasmid loss, the degrades faster, allowing the to inhibit essential cellular processes like or . A classic example is the ccdAB system on the , where the CcdB inhibits , leading to double-strand breaks and in segregants. Similarly, the system on plasmid R1 encodes a Kis (antitoxin) protein that inhibits the Kid () ribonuclease, with the antitoxin's shorter triggering post-segregational killing. These systems do not prevent plasmid loss but selectively eliminate non-carriers, maintaining population-level stability as demonstrated in low-copy plasmids where TA modules reduce segregant viability by over 90% in cultures. Plasmids also impose a metabolic burden on hosts by diverting resources for replication, transcription, and of plasmid genes, creating fitness trade-offs that can drive co-adaptation. This burden arises from increased demand on cellular machinery, such as ribosomes and pools, often reducing host growth rates by 5-20% depending on plasmid size and copy number. For instance, high-copy plasmids like pUC can halve host division times in nutrient-limited conditions, but over evolutionary time, mutations in both plasmid and host genomes mitigate these costs, enhancing mutual fitness. Restriction-modification (RM) systems encoded on plasmids further aid evasion of host defenses by methylating incoming DNA to protect against nucleases, while countering host RM barriers during . Plasmids like those in IncP groups carry RM variants that modify their own sequences, reducing degradation rates by up to 100-fold upon conjugation into naive hosts. The evolutionary dynamics of these interactions are shaped by (HGT), fostering co-adaptation between plasmids and diverse hosts through gene exchange and selection for compatible modules. TA and RM systems often spread via HGT, with plasmids evolving to minimize burden while maximizing transmission, as seen in metagenomic analyses of bacterial communities where stable plasmids exhibit reduced metabolic costs after host-specific adaptations. This interplay ensures plasmid persistence across generations, balancing host fitness penalties with benefits like antibiotic resistance conveyance.

Plasmids in Eukaryotes

Cytoplasmic Plasmids

Cytoplasmic plasmids in eukaryotes are extrachromosomal DNA elements that replicate independently in the cytosol, bypassing nuclear replication machinery and often relying on their own encoded enzymes for maintenance. These elements are relatively rare compared to prokaryotic plasmids or nuclear episomes, but notable examples occur in certain yeasts, such as the linear double-stranded DNA plasmids pGKL1 (8.9 kb) and pGKL2 (13.4 kb) in Kluyveromyces lactis. These plasmids reside exclusively in the cytoplasm and confer a killer phenotype to host cells, enabling them to secrete a toxin that inhibits the growth of sensitive yeast strains while protecting the host via an immunity protein. The pGKL plasmids are maintained at high copy numbers, typically 50-100 copies each per haploid cell, ensuring stable transmission during despite the absence of nuclear partitioning systems. Replication occurs via a protein-primed mechanism analogous to that of adenoviruses, where a terminal protein covalently attached to the 5' ends serves as a primer for . pGKL2 encodes essential replication proteins, including a and the terminal protein, while pGKL1 relies on these for its own propagation but contributes genes for production. This autonomous replication in the uses viral-like polymerases encoded by the plasmids themselves, independent of host nuclear or cytosolic polymerases. These plasmids exhibit partitioning behaviors that promote equitable distribution to daughter cells, though the precise mechanisms involve less-characterized interactions than those in nuclear plasmids; stability is enhanced by the killer phenotype's selective advantage in mixed populations. Functionally, the pGKL elements are often viewed as selfish genetic parasites with minimal content—primarily replication factors, , and immunity—yet they provide a net benefit to hosts by outcompeting non-killer strains in natural environments. Due to their high copy number and cytoplasmic expression system, derivatives of pGKL1 have been engineered as linear vectors for in , demonstrating utility in similar to applications of other stable episomes.

Nuclear Plasmids

Nuclear plasmids in eukaryotes are molecules that replicate and function within the nucleus, often relying on host replication machinery for maintenance and expression. Unlike cytoplasmic plasmids, they interact closely with nuclear processes, including dynamics and genomic surveillance mechanisms. These plasmids are engineered or derived from viral elements to enable episomal persistence or targeted integration, facilitating studies and therapeutic applications in model organisms and mammalian cells. In , nuclear plasmids are classified based on their replication and stability features. Yeast replicating plasmids (YRp) incorporate an (ARS) derived from chromosomal DNA, allowing replication initiation but resulting in high instability due to frequent loss during , with segregation rates often below 10% per generation. In contrast, yeast episomal plasmids (YEp) are hybrids incorporating the 2-micron circle plasmid's origin and partitioning genes (REP1, REP2, and FLP), enabling high-copy maintenance (typically 20-50 copies per cell) and stable inheritance with loss rates of 0.2-2% per generation, making them suitable for overexpression experiments. Mammalian nuclear plasmids commonly utilize viral origins for replication within the nucleus. The Epstein-Barr virus (EBV)-derived oriP element, in conjunction with the EBNA1 protein, supports episomal replication and segregation in human cells by tethering plasmids to host chromosomes, achieving persistence for over 50 population doublings in dividing cells without integration. Similarly, the SV40 (ori) drives bidirectional replication in cells expressing SV40 , such as HEK293 lines, permitting transient or semi-stable episomal maintenance with copy numbers up to 100 per cell. Integration of nuclear plasmids into the eukaryotic occurs primarily through , where flanking sequences on the plasmid align with chromosomal targets, enabling precise insertion as demonstrated in systems. Alternatively, transposon-based mechanisms, such as those mediated by DNA transposases like or PiggyBac, facilitate non-homologous integration by excising and inserting plasmid segments into random or semi-targeted nuclear sites. These plasmids are widely applied in transient transfection protocols to study gene function, where nuclear delivery via lipofection or allows short-term expression (24-72 hours) of reporter genes or siRNAs in eukaryotic cells, bypassing stable integration for rapid phenotypic analysis. A key challenge for nuclear plasmids is silencing by host defenses, including epigenetic modifications like and histone deacetylation, which reduce expression over time in up to 90% of episomes within weeks, triggered by innate immune sensors recognizing foreign DNA as a .

Organellar Plasmids

Organellar plasmids are elements found within the mitochondria and chloroplasts of eukaryotic cells, distinct from the main organellar genomes due to their autonomous replication and often linear or chimeric structures. These plasmids typically range in size from a few kilobases and play roles in function, , and , reflecting remnants of ancient bacterial endosymbiosis. Unlike bacterial plasmids, organellar variants frequently exhibit integration with the primary or involvement in degenerative processes, such as or sterility. In plant mitochondria, notable examples include the S1 and S2 plasmids in (Zea mays) associated with S-type (CMS-S). These linear plasmids, measuring 6.4 kb (S1) and 5.4 kb (S2), possess terminal inverted repeats that facilitate their replication and maintenance as episomes within the . The presence of S1 and S2 disrupts pollen development, leading to male sterility, a trait exploited in hybrid crop breeding, though spontaneous reversion to fertility correlates with plasmid loss. Chloroplast DNA in the green alga exists in integrated forms that contribute to and stability, promoting genetic exchanges that enhance adaptability to environmental stresses. These elements aid in repairing double-strand breaks and maintaining integrity during vegetative growth and sexual crosses. In fungal mitochondria, such as those of Podospora anserina, senDNA plasmids represent autonomous linear elements linked to . These plasmids arise from excision and amplification of specific segments, forming circular or linear multimers that accumulate over generations, ultimately causing growth arrest and death. SenDNA propagation involves integration back into the , perpetuating the senescence syndrome in this model organism. Replication of organellar plasmids often proceeds via rolling-circle mechanisms or recombination-dependent processes, allowing rapid amplification without reliance on the host's nuclear machinery. In mitochondria, rolling-circle replication generates multimeric intermediates from linear templates, while recombination-dependent modes predominate in algal chloroplasts to resolve heteroplasmic states. These strategies ensure plasmid persistence amid the dynamic, fragmented nature of organelle genomes. Evolutionarily, organellar plasmids trace their origins to bacterial plasmids acquired during the endosymbiotic events that gave rise to mitochondria and chloroplasts from alphaproteobacterial and cyanobacterial ancestors, respectively. Over time, these elements have adapted through gene loss and structural modifications, functioning as parasitic or mutualistic genetic parasites within organelles.

Methods of Study

Isolation and Purification Techniques

The isolation and purification of plasmid DNA from bacterial cells primarily relies on the alkaline lysis method, which exploits the structural differences between plasmid and chromosomal DNA to achieve selective extraction. Developed by Birnboim and Doly in 1979, this technique involves treating harvested bacterial cells with a solution containing sodium hydroxide (NaOH) and sodium dodecyl sulfate (SDS) to denature both plasmid and chromosomal DNA, rendering the chromosomal DNA insoluble and forming a viscous clot due to its larger size and tangling. Neutralization with potassium acetate then renatures the smaller, supercoiled plasmid DNA, which remains soluble in the supernatant, while the chromosomal DNA and cellular debris precipitate out. This selectivity favors supercoiled plasmid conformations, minimizing contamination from other forms like nicked or linear DNA. Alkaline lysis forms the basis for both miniprep and maxiprep protocols, which differ mainly in scale and yield to suit varying experimental needs. Minipreps process small cultures (1–5 mL) to yield 5–50 μg of plasmid DNA, ideal for routine cloning and sequencing, while maxipreps handle larger volumes (100–500 mL) to produce 100–1,000 μg, suitable for applications requiring substantial quantities like transfection or protein expression. Commercial kits, such as those from QIAGEN, enhance these protocols by incorporating modified alkaline lysis followed by anion-exchange chromatography on silica-based columns, which bind plasmid DNA under high-salt conditions and elute it in low-salt buffer for higher purity. These kits typically include RNase A to degrade RNA contaminants during lysis, ensuring cleaner preparations without additional enzymatic steps. For applications demanding ultra-high purity, such as early sequencing or structural studies, cesium chloride (CsCl) gradient ultracentrifugation serves as a classical alternative or complementary method. This technique uses equilibrium density gradient centrifugation in CsCl solutions, often with ethidium bromide as an intercalating dye, to separate plasmid DNA based on buoyant density: supercoiled plasmids band at a lower density (1.58 g/mL) than chromosomal DNA (1.70 g/mL), forming distinct visible bands after 40–72 hours of ultracentrifugation at 100,000–150,000 × g. Extraction of the plasmid band via syringe puncture yields DNA free of proteins, RNA, and genomic fragments, though it is labor-intensive and less common today due to column-based alternatives. To maximize yields and minimize contamination, protocols emphasize gentle handling to avoid shearing genomic DNA, which can co-purify if cells are vortexed excessively after lysis; instead, inversion or slow pipetting is recommended. RNA is routinely eliminated by adding RNase during the lysis step, while genomic DNA contamination is further reduced by ensuring complete precipitation of debris and using optional DNase treatments if needed, though these are rarely required in optimized kits. Typical yields from a 1 L culture via maxiprep reach 500–1,000 μg, but can vary with plasmid copy number and host strain, underscoring the need for empirical optimization. Magnetic bead-based methods, developed in the 1990s and further advanced for automation and high-throughput purification, address limitations in scalability and hands-on time of traditional approaches. These systems use carboxyl-coated paramagnetic beads that bind plasmid DNA under chaotropic salt conditions after alkaline lysis, allowing magnetic separation of bound DNA from contaminants without centrifugation; elution yields comparable purity to column methods but with faster processing (under 30 minutes per sample). Reviews highlight their integration into robotic platforms for processing up to 96 samples simultaneously, reducing genomic DNA carryover through optimized bead ratios and wash buffers.

Structural Analysis and Conformations

Plasmid DNA can exist in several distinct topological conformations following isolation and purification, primarily the supercoiled (SC), open circular (OC), and linear forms. The supercoiled form represents the native, covalently closed circular structure with intertwined strands, resulting from underwinding or overwinding of the double helix. In contrast, the open circular form arises from a single-strand nick, relaxing the superhelical tension, while the linear form results from double-strand breaks or enzymatic digestion. These conformations are critical for assessing plasmid integrity, as they influence replication efficiency, stability, and interactions with host machinery. Agarose gel electrophoresis serves as a primary technique for separating and identifying these conformations based on their differential migration patterns. Under standard conditions without intercalating agents, supercoiled plasmids migrate the fastest due to their compact structure, followed by linear forms, with open circular plasmids exhibiting the slowest mobility owing to their relaxed, extended shape. The addition of (EtBr), an intercalating , alters this mobility by unwinding the DNA helix and relaxing negative supercoils, causing supercoiled forms to migrate more slowly and potentially resolving topoisomers into distinct bands. This topology-dependent mobility allows for quantitative assessment of conformational purity, often visualized post-staining for enhanced sensitivity. For higher-resolution three-dimensional visualization of plasmid , atomic force microscopy (AFM) enables direct imaging of supercoiled structures at the nanoscale, revealing plectonemic interwindings and branch points without the need for staining or labeling. AFM studies of plasmids, such as , demonstrate how supercoiling compacts the molecule into branched, right-handed writhe configurations under physiological conditions. The topological state is quantitatively described by the (Lk), defined as the sum of twist (Tw), the helical turns along the axis, and writhe (Wr), the coiling of the axis itself: Lk=Tw+WrLk = Tw + Wr Supercoiling introduces a linking difference (ΔLk) from the relaxed state, typically negative in bacteria (ΔLk ≈ -0.06 Lk₀), which partitions into changes in twist (ΔTw) and writhe (ΔWr), with negative writhe contributing to the observed compaction and facilitating processes like transcription initiation. To obtain detailed structural maps, sequencing techniques complement topological analyses by providing nucleotide-level resolution of the plasmid backbone and inserts. Sanger sequencing remains a gold standard for targeted verification of plasmid constructs, offering high accuracy (error rate <0.001%) over reads up to 1,000 bp, commonly used to confirm insert orientation and absence of mutations post-cloning. For comprehensive full-plasmid mapping, next-generation sequencing (NGS) methods, including short-read Illumina or long-read Nanopore platforms, enable de novo assembly of entire sequences (up to 20 kb or more), detecting rearrangements, repetitions, and heterogeneity that gel-based methods cannot resolve. These approaches, often applied after purification to ensure high yield, yield consensus maps essential for functional annotation and quality control.

Bioinformatics and Design Tools

Bioinformatics tools play a crucial role in the , , and of plasmids, enabling researchers to predict features, optimize sequences, and model behaviors without extensive wet-lab experimentation. These computational resources facilitate virtual , sequence annotation, and compatibility assessments, streamlining workflows. Widely adopted software such as SnapGene and Benchling provides intuitive interfaces for these tasks, integrating multiple functionalities to support plasmid engineering. For sequence analysis, tools focus on identifying key genetic elements and potential assembly issues. Open reading frame (ORF) prediction is essential for annotating protein-coding regions in plasmid sequences, with NCBI's ORFfinder employing algorithms to scan DNA for potential start and stop codons, translating them into amino acid sequences. Restriction mapping visualizes enzyme cut sites, aiding in cloning strategy planning; for instance, NEBcutter generates comprehensive maps by simulating digests with over 200 enzymes, highlighting fragment sizes and positions. Compatibility checks for assemblies evaluate sequence overlaps or restriction site conflicts, as implemented in SnapGene, which flags incompatible junctions during virtual ligation to prevent errors in multi-part constructs. Synthetic plasmid leverages specialized algorithms for modular assembly and optimization. SnapGene supports virtual by simulating restriction-ligation, Gibson, and other methods, allowing users to design primers and predict outcomes in a graphical interface. Benchling enables collaborative through cloud-based editing, incorporating features for multi-user annotation and automated primer generation for assemblies. For assembly planning, the NEBridge tool from designs overhangs and predicts junction fidelity, optimizing type IIS enzyme-based modular for up to 25 fragments. Codon optimization algorithms adjust synonymous codons to match host preferences, enhancing expression; tools like those in GenScript apply rarity-based scoring and secondary structure predictions to generate variants, as described in mathematical programming approaches that balance usage bias with stability. Databases provide curated resources for sequence retrieval and typing. Addgene's repository hosts over 100,000 plasmids with annotated sequences, enabling BLAST-based searches for similar constructs and facilitating reagent sharing. PlasmidFinder, developed by the Center for Genomic Epidemiology, identifies and types plasmid replicons in whole-genome sequences using a database of 116 reference replicons, achieving high specificity for incompatibility groups via k-mer matching. Simulations of plasmid dynamics often employ (ODE) models to predict copy number, which influences expression levels and stability. For ColE1-like plasmids, ODE-based models simulate replication control through RNAI-RNAII interactions, where the rate of primer formation is governed by equations such as dP/dt = k_s * RNAII - k_d * P * RNAI, balancing synthesis and degradation to estimate steady-state copy numbers around 15-50 per cell. These models, as implemented in tools like those simulating ColE1 regulation, aid in designing origins for desired replication rates without antibiotics.

References

  1. https://www.ncbi.nlm.nih.gov/mesh?Cmd=DetailsSearch&Term=%2522Plasmids%2522%255BMeSH%2BTerms%255D
  2. https://pmc.ncbi.nlm.nih.gov/articles/PMC10433428/
  3. https://pubmed.ncbi.nlm.nih.gov/9473441/
  4. https://pubmed.ncbi.nlm.nih.gov/20805406/
  5. https://pmc.ncbi.nlm.nih.gov/articles/PMC4379921/
  6. https://pmc.ncbi.nlm.nih.gov/articles/PMC2937521/
  7. https://pmc.ncbi.nlm.nih.gov/articles/PMC9263177/
  8. https://pmc.ncbi.nlm.nih.gov/articles/PMC427208/
  9. https://pmc.ncbi.nlm.nih.gov/articles/PMC12307069/
  10. https://www.nature.com/articles/189947a0
  11. https://pubmed.ncbi.nlm.nih.gov/4894690/
  12. https://www.science.org/doi/10.1126/science.1225829
  13. https://www.nobelprize.org/prizes/chemistry/2020/press-release/
  14. https://www.nature.com/articles/s41467-018-03553-1
  15. https://www.nature.com/scitable/definition/plasmid-plasmids-28/
  16. https://pmc.ncbi.nlm.nih.gov/articles/PMC8628078/
  17. https://www.nature.com/articles/292380a0
  18. https://pmc.ncbi.nlm.nih.gov/articles/PMC9453081/
  19. https://blog.addgene.org/plasmid-101-origin-of-replication
  20. https://blog.addgene.org/plasmids-101-what-is-a-plasmid
  21. https://www.royalsocietypublishing.org/doi/10.1098/rstb.2020.0472
  22. https://journals.asm.org/doi/10.1128/microbiolspec.plas-0029-2014
  23. https://journals.asm.org/doi/10.1128/ecosalplus.esp-0026-2019
  24. https://pmc.ncbi.nlm.nih.gov/articles/PMC98921/
  25. https://journals.asm.org/doi/10.1128/microbiolspec.plas-0035-2014
  26. https://www.cell.com/trends/biochemical-sciences/fulltext/S0968-0004(98)01302-4
  27. https://pmc.ncbi.nlm.nih.gov/articles/PMC5910852/
  28. https://www.sciencedirect.com/science/article/pii/S0092867400803591
  29. https://pubmed.ncbi.nlm.nih.gov/1283002/
  30. https://www.nature.com/articles/s41467-025-61202-5
  31. https://www.pnas.org/doi/pdf/10.1073/pnas.79.17.5347
  32. https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-022-01958-3
  33. https://pmc.ncbi.nlm.nih.gov/articles/PMC9720003/
  34. https://pubmed.ncbi.nlm.nih.gov/7680381/
  35. https://pmc.ncbi.nlm.nih.gov/articles/PMC317936/
  36. https://journals.asm.org/doi/10.1128/microbiolspec.plas-0012-2013
  37. https://pmc.ncbi.nlm.nih.gov/articles/PMC7690428/
  38. https://pmc.ncbi.nlm.nih.gov/articles/PMC2839888/
  39. https://pmc.ncbi.nlm.nih.gov/articles/PMC10973873/
  40. https://pmc.ncbi.nlm.nih.gov/articles/PMC419928/
  41. https://pmc.ncbi.nlm.nih.gov/articles/PMC3067620/
  42. https://www.nature.com/articles/s41579-023-00927-w
  43. https://pmc.ncbi.nlm.nih.gov/articles/PMC4244643/
  44. https://www.nature.com/articles/s41467-022-34588-9
  45. https://academic.oup.com/nar/article/51/6/2759/7068371
  46. https://www.nature.com/articles/s41467-025-55897-9
  47. https://pmc.ncbi.nlm.nih.gov/articles/PMC9952643/
  48. https://www.sciencedirect.com/science/article/pii/S0014579304004934
  49. https://genesdev.cshlp.org/content/15/12/1518.short
  50. https://pubmed.ncbi.nlm.nih.gov/11410532/
  51. https://www.frontiersin.org/journals/genetics/articles/10.3389/fgene.2017.00211/full
  52. https://pmc.ncbi.nlm.nih.gov/articles/PMC10055365/
  53. https://www.science.org/doi/10.1126/sciadv.abq6376
  54. https://pubmed.ncbi.nlm.nih.gov/344137/
  55. https://www.addgene.org/protocols/subcloning/
  56. https://www.promega.com/resources/guides/nucleic-acid-analysis/subcloning/
  57. https://blog.addgene.org/plasmids-101-blue-white-screening
  58. https://blog.addgene.org/plasmids-101-shuttle-vectors
  59. https://academic.oup.com/genetics/article-abstract/122/1/19/5998076
  60. https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/cloning/common-applications-strategies.html
  61. https://www.addgene.org/protocols/pcr-cloning/
  62. https://www.neb.com/en-us/faqs/2013/11/01/what-guidelines-are-recommended-for-improving-clone-stability
  63. https://pmc.ncbi.nlm.nih.gov/articles/PMC10467106/
  64. https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2014.00172/full
  65. https://pmc.ncbi.nlm.nih.gov/articles/PMC7159335/
  66. https://www.nature.com/articles/s42003-020-0939-8
  67. https://pmc.ncbi.nlm.nih.gov/articles/PMC6635841/
  68. https://aiche.onlinelibrary.wiley.com/doi/10.1002/btpr.1842
  69. https://www.nature.com/articles/s41598-020-74091-z
  70. https://pmc.ncbi.nlm.nih.gov/articles/PMC7162232/
  71. https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-014-0141-0
  72. https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2019.00420/full
  73. https://www.thelancet.com/journals/laninf/article/PIIS1473-3099(21)00151-9/fulltext
  74. https://pmc.ncbi.nlm.nih.gov/articles/PMC6600476/
  75. https://pmc.ncbi.nlm.nih.gov/articles/PMC6062593/
  76. https://www.spandidos-publications.com/10.3892/etm.2017.5235
  77. https://www.nature.com/articles/s41467-020-15053-x
  78. https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/bit.27928
  79. https://pmc.ncbi.nlm.nih.gov/articles/PMC7120425/
  80. https://pmc.ncbi.nlm.nih.gov/articles/PMC8819515/
  81. https://www.nobelprize.org/uploads/2018/06/jacob-lecture.pdf
  82. https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_%28Kaiser%29/Unit_1%253A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/2%253A_The_Prokaryotic_Cell_-_Bacteria/2.4%253A_Cellular_Components_within_the_Cytoplasm/2.4C%253A_Plasmids_and_Transposons
  83. https://www.sciencedirect.com/topics/neuroscience/f-factor
  84. https://cshmonographs.org/index.php/monographs/article/view/4992
  85. https://www.pnas.org/doi/10.1073/pnas.98.4.1865
  86. https://pmc.ncbi.nlm.nih.gov/articles/PMC3333862/
  87. https://pmc.ncbi.nlm.nih.gov/articles/PMC5710010/
  88. https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.578056/full
  89. https://www.pnas.org/doi/10.1073/pnas.090527097
  90. https://www.nature.com/articles/srep09684
  91. https://journals.asm.org/doi/10.1128/AEM.00056-10
  92. https://pmc.ncbi.nlm.nih.gov/articles/PMC5180612/
  93. https://journals.asm.org/doi/10.1128/mbio.00762-15
  94. https://www.sciencedirect.com/science/article/pii/S0147619X18301513
  95. https://pmc.ncbi.nlm.nih.gov/articles/PMC7984726/
  96. https://www.pnas.org/doi/10.1073/pnas.1111221108
  97. https://pmc.ncbi.nlm.nih.gov/articles/PMC9049610/
  98. https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.00761/full
  99. https://journals.asm.org/doi/10.1128/microbiolspec.plas-0023-2014
  100. https://pmc.ncbi.nlm.nih.gov/articles/PMC11229316/
  101. https://www.embopress.org/doi/10.1038/emboj.2013.34
  102. https://www.sciencedirect.com/science/article/pii/S0042682211004557
  103. https://journals.asm.org/doi/10.1128/ecosalplus.esp-0003-2019
  104. https://portlandpress.com/biochemj/article/412/1/1/44335/Structural-biology-of-plasmid-partition-uncovering
  105. https://pmc.ncbi.nlm.nih.gov/articles/PMC2168934/
  106. https://pmc.ncbi.nlm.nih.gov/articles/PMC4163524/
  107. https://pmc.ncbi.nlm.nih.gov/articles/PMC145886/
  108. https://academic.oup.com/nar/article/24/10/1879/2359463
  109. https://www.sciencedirect.com/science/article/abs/pii/S0378109799003389
  110. https://pubmed.ncbi.nlm.nih.gov/3060367/
  111. https://www.uniprot.org/uniprotkb/P05468/entry
  112. https://academic.oup.com/femsle/article/178/2/201/582997
  113. https://link.springer.com/article/10.1007/BF00326291
  114. https://febs.onlinelibrary.wiley.com/doi/full/10.1046/j.1432-1327.2000.01645.x
  115. https://academic.oup.com/femsyr/article/12/2/197/526456
  116. https://onlinelibrary.wiley.com/doi/full/10.1002/yea.3228
  117. https://pmc.ncbi.nlm.nih.gov/articles/PMC3838108/
  118. https://pubmed.ncbi.nlm.nih.gov/8382635/
  119. https://pmc.ncbi.nlm.nih.gov/articles/PMC330758/
  120. https://pmc.ncbi.nlm.nih.gov/articles/PMC8067914/
  121. https://www.sciencedirect.com/science/article/pii/S2405471222004641
  122. https://pmc.ncbi.nlm.nih.gov/articles/PMC9601623/
  123. https://pmc.ncbi.nlm.nih.gov/articles/PMC8383295/
  124. https://pmc.ncbi.nlm.nih.gov/articles/PMC299425/
  125. https://pubmed.ncbi.nlm.nih.gov/4095194/
  126. https://www.science.org/doi/10.1126/science.209.4460.1021
  127. https://pmc.ncbi.nlm.nih.gov/articles/PMC2927747/
  128. https://pmc.ncbi.nlm.nih.gov/articles/PMC146090/
  129. https://pubmed.ncbi.nlm.nih.gov/2997455/
  130. https://pubmed.ncbi.nlm.nih.gov/6300793/
  131. https://pmc.ncbi.nlm.nih.gov/articles/PMC231631/
  132. https://pmc.ncbi.nlm.nih.gov/articles/PMC1634775/
  133. https://www.nature.com/articles/srep23744
  134. https://academic.oup.com/nar/article/7/6/1513/2380972
  135. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/alkaline-lysis
  136. https://cshprotocols.cshlp.org/content/2018/1/pdb.prot093351.full.pdf
  137. https://blog.addgene.org/selecting-your-plasmid-purification-kit
  138. https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/plasmid-dna/qiagen-plasmid-kits
  139. https://www.qiagen.com/us/knowledge-and-support/knowledge-hub/bench-guide/plasmid/working-with-plasmids/lysis-of-bacterial-cells-for-plasmid-purification
  140. https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/dna-and-rna-purification/plasmid-dna-purification
  141. https://documents.thermofisher.com/TFS-Assets/LED/Application-Notes/D17309~.pdf
  142. https://www.qiagen.com/us/resources/faq/353
  143. https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/dna-and-rna-purification/problems-during-plasmid-dna-preparation
  144. https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-023-09211-w
  145. https://www.researchgate.net/publication/393184887_Systematic_Review_Recent_Developments_in_Magnetic-Nanoparticle-Based_DNA_Extraction
  146. https://www.nature.com/articles/s41598-024-69406-3
  147. https://pmc.ncbi.nlm.nih.gov/articles/PMC519125/
  148. https://www.pnas.org/doi/10.1073/pnas.0601903103
  149. https://www.pnas.org/doi/10.1073/pnas.94.2.496
  150. https://pmc.ncbi.nlm.nih.gov/articles/PMC4480603/
  151. https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-023-05226-y
  152. https://pmc.ncbi.nlm.nih.gov/articles/PMC10996661/
  153. https://www.snapgene.com/
  154. https://www.benchling.com/molecular-biology
  155. https://www.ncbi.nlm.nih.gov/orffinder/
  156. https://www.hsls.pitt.edu/obrc/index.php?page=URL1043859167
  157. https://goldengate.neb.com/
  158. https://www.genscript.com/codon-optimization.html
  159. https://academic.oup.com/bioinformatics/article/36/13/4012/5822879
  160. https://www.addgene.org/
  161. https://pmc.ncbi.nlm.nih.gov/articles/PMC4384007/
  162. https://cge.food.dtu.dk/services/PlasmidFinder/
  163. https://pubmed.ncbi.nlm.nih.gov/24777092/
Add your contribution
Related Hubs
User Avatar
No comments yet.