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It is a small DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently.
They are commonly found as small circular double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms .
In nature, plasmids often carry genes that can benefit the survival of the organism, for example resistance to antibiotics.
Although chromosomes are large and contain all the genetic information essential for living under normal conditions, plasmids are usually very small and contain only additional genes that may be useful to the body in certain situations or particular conditions.
Artificial plasmids are widely used as vectors in molecular cloning, serving to drive replication of recombinant DNA sequences within host organisms.
In the laboratory, plasmids can be introduced into a cell by transformation.
Plasmids are considered replicons, units of DNA capable of autonomously replicating within a suitable host. However, plasmids, like viruses, are generally not classified.
Plasmids are transmitted from one bacterium to another (even another species) primarily through conjugation.
This transfer of genetic material from host to host is a horizontal gene transfer mechanism, and plasmids are considered part of the mobilome.
Unlike viruses (which coat their genetic material in a protective protein layer called a capsid), plasmids are “naked” DNA and do not encode the genes necessary to encapsulate genetic material and transfer it to a new host.
However, some classes of plasmids encode the “sexual” conjugative pilus necessary for their own transfer. The size of the plasmid ranges from 1 to more than 200 kbp, and the number of identical plasmids in a single cell can range from one to thousands in some circumstances.
The relationship between microbes and plasmid DNA is neither parasitic nor mutualistic, since each implies the presence of an independent species that lives in a deleterious or commensal state with the host organism.
In contrast, plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage in a given environmental state.
Plasmids can carry genes that provide resistance to natural antibiotics in a competitive ecological niche.
Either the proteins produced can act as toxins under similar circumstances, or they allow the body to use particular organic compounds that would be advantageous when nutrients are in short supply.
History
The term plasmid was introduced in 1952 by the American molecular biologist Joshua Lederberg to refer to “any extrachromosomal inherited determinant.”
The term includes any bacterial genetic material that exists extrachromosomally during at least part of its replication cycle.
But because that description includes bacterial viruses, the notion of plasmid was refined over time to understand genetic elements that reproduce autonomously.
Later, in 1968, it was decided that the term plasmid should be adopted as the term for extrachromosomal genetic element, and to distinguish it from viruses, the definition was narrowed down to genetic elements that exist exclusively or predominantly outside the chromosome and can replicate so autonomous.
Properties and characteristics
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 the replicon.
A typical bacterial replicon can 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.
The smaller plasmids make use of the host’s replicative enzymes to make copies of themselves, while the larger plasmids can carry specific genes for the replication of those plasmids.
Some types of plasmids can also insert into the host chromosome, and these integrating plasmids are sometimes called episomes in prokaryotes.
Plasmids almost always carry at least one gene. Many of the genes carried by a plasmid are beneficial to host cells, for example:
They allow the host cell to survive in an environment that would otherwise be lethal or restrictive to growth.
Some of these genes encode antibiotic resistance or heavy metal resistance traits.
While others can produce virulence factors that allow a bacterium to colonize a host and overcome its defenses.
Or they have specific metabolic functions that allow bacteria to utilize a particular nutrient, including the ability to break down recalcitrant or toxic organic compounds.
Plasmids can also provide bacteria with the ability to fix nitrogen.
However, some plasmids have no observable effect on the phenotype of the host cell or their benefit to host cells cannot be determined, and these plasmids are called cryptic plasmids.
Naturally occurring plasmids vary greatly in their physical properties. Their size can range from very small mini-plasmids of less than 1 kilobase pair (Kbp) to very large megaplasmids of several megabase pairs (Mbp).
At the high end, little can differentiate between a megaplasmid and a minichromosome.
Plasmids are generally circular; however, examples of linear plasmids are also known. These linear plasmids require specialized mechanisms to replicate their ends.
Plasmids can be present in an individual cell in varying numbers, ranging from one to several hundred.
The normal number of plasmid copies that can be found in a single cell is called the copy number and is determined by how the initiation of replication is regulated and the size of the molecule.
Larger plasmids tend to have lower copy numbers.
Such single copy plasmids have systems that actively attempt to deliver one copy to both daughter cells. These systems, which include the parABS system and the parMRC system, are often referred to as the partition system or partition function of a plasmid.
Classifications and types
Plasmids can be classified in several ways. Plasmids can be broadly classified into conjugative plasmids and non-conjugative plasmids.
Conjugative plasmids contain a set of transfer genes or TRAs that promote sexual conjugation between different cells.
In the complex conjugation process, the plasmid can be transferred from one bacterium to another through the sexual pili encoded by some of the TRA genes.
Non-conjugative plasmids are unable to initiate conjugation, therefore they can be transferred only with the help of conjugative plasmids.
An intermediate class of plasmids is mobilizable and carries only a subset of the genes necessary for transfer.
Plasmids can also be classified into incompatibility groups. A microbe can harbor different types of plasmids, however, 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 quickly be lost from the cell. Therefore, different plasmids can 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 therefore cannot be kept together in a single cell.
Another way to classify plasmids is by function:
Fertility F-plasmids : containing TRA genes. They are able to conjugate and result in the expression of sexual pili.
Cabbage plasmids : which contain genes that code for bacteriocins, proteins that can kill other bacteria.
Degradative plasmids : which allow the digestion of unusual substances, eg. toluene and salicylic acid.
Cartoon vector
Artificially constructed plasmids can be used as vectors in genetic engineering. A wide variety of plasmids are commercially available for such uses.
The gene to be replicated is normally inserted into a plasmid that normally contains a number of characteristics for use.
These include a gene that confers resistance to particular antibiotics (ampicillin is most often used for bacterial strains).
An origin of replication to allow bacterial cells to replicate plasmid DNA and a suitable site for cloning (called a multiple cloning site).
Cloning
Plasmids are the most commonly used bacterial cloning vectors.
These cloning vectors contain a site that allows inserting DNA fragments, for example, a multiple cloning site or a polylinker having several commonly used restriction sites to which DNA fragments can be ligated.
These plasmids contain a selectable marker, generally an antibiotic resistance gene, which confers on bacteria an ability to survive and proliferate in a selective growth medium containing the particular antibiotics.
Cells after transformation are exposed to selective media, and only cells containing the plasmid can survive.
In this way, the antibiotics act as a filter to select only the bacteria that contain the plasmid DNA.
The vector may also contain other marker genes or reporter genes to facilitate selection of the plasmid with the cloned insert.
Bacteria containing the plasmid can then be cultured in large numbers, harvested, and the plasmid of interest can then be isolated using various plasmid preparation methods.
A plasmid cloning vector is typically used to clone DNA fragments up to 15 kbp.
To clone longer lengths of DNA, lambda phages with lysogeny genes, cosmids, bacterial artificial chromosomes, or yeast artificial chromosomes are deleted.
Protein production
Another important use of plasmids is to produce large amounts of proteins.
Gene therapy
The plasmid can also be used for gene transfer into human cells as a potential treatment in gene therapy so that it can express the missing protein in the cells.
Some gene therapy strategies require the insertion of therapeutic genes into preselected 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 zinc finger nuclease could help deliver a therapeutic gene to a specific site to avoid cell damage, cancer-causing mutations, or an immune response.
Disease models
Plasmids were historically used to genetically engineer rat embryonic stem cells in order to create models of rat genetic diseases.
The limited efficacy of plasmid-based techniques precluded their use in creating more accurate human cell models.
However, advances in adeno-associated virus recombination techniques and zinc finger nucleases have enabled the creation of a new generation of isogenic human disease models.
Episodes
The term episome was introduced by François Jacob and Élie Wollman in 1958 to refer to extrachromosomal genetic material that can replicate autonomously or integrate into the chromosome.
Since the term was introduced, however, its use has changed as plasmid has become the preferred term for autonomous replication of extrachromosomal DNA.
At a 1968 symposium in London, some participants suggested dropping the term episome, although others continued to use the term with a change in meaning.
Today, some authors use episomes in the context of prokaryotes to refer to a plasmid that is capable of integrating into the chromosome.
Integrating plasmids can be replicated and stably maintained in a cell through multiple generations, but always at some stage exist as an independent plasmid molecule.
In the context of eukaryotes, the term episomes is used to refer to a non-integrated closed extrachromosomal circular DNA molecule that can replicate in the nucleus.
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, which can arise during artificial gene amplifications or in disease processes (eg, transformation of cancer cells).
Episomes in eukaryotes behave similarly to plasmids in prokaryotes in that the DNA is stable and replicates with the host cell.
Viral cytoplasmic episomes (as in poxvirus infections) can also occur.
Some episomes, such as herpesviruses, replicate in a rolling circle mechanism, similar to the bacterial phage virus. Others replicate through a bidirectional replication mechanism (Theta-like plasmids).
In either case, the episomes remain physically separate from the host cell chromosomes.
Several cancer viruses, including Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus, remain dormant, chromosomally distinct episomes in cancer cells, where the viruses express oncogenes that promote proliferation of cancer cells.
In cancers, these episomes passively replicate along with the host’s chromosomes when the cell divides.
When these viral episomes initiate lytic replication to generate multiple virus particles, they generally activate innate cellular immunity defense mechanisms that kill the host cell.
Plasmid maintenance
Some plasmids or microbial hosts include an addiction system or a postsegregational kill (PSK) system, such as the hok / sok (kill / suppressor) system of plasmid R1 in Escherichia coli.
This variant produces both a long-lasting poison and a short-lived antidote.
Various types of plasmid addiction systems (toxin / antitoxin, metabolism-based, ORT systems) have been described in the literature and used in biotechnical (fermentation) or biomedical (vaccine therapy) applications.
Finally, overall productivity could be improved.
In contrast, virtually all plasmids used biotechnologically (such as pUC18, pBR322, and derived vectors) do not contain toxin and antitoxin addiction systems and therefore must be kept under antibiotic pressure to avoid loss of plasmids.
Yeast plasmids
Yeasts naturally harbor various plasmids.
Notable among these are the 2 μm plasmids (small circular plasmids often used for genetic engineering of yeast) and the linear pGKL plasmids from Kluyveromyces lactis, which are responsible for the killer phenotypes.
Other types of plasmids are often related to yeast cloning vectors including:
- Yeast integrating plasmid (YIp), yeast vectors that depend on integration into the host chromosome for survival and replication, and are generally used when studying the functionality of a single gene or when the gene is toxic.
- It is also related to the URA3 gene, which encodes an enzyme related to the biosynthesis of pyrimidine nucleotides (T, C).
- Yeast Replicative Plasmid (YRp), which carries a chromosomal DNA sequence that includes an origin of replication. These plasmids are less stable, as they can be lost during budding.
Plasmid DNA extraction
As mentioned above, plasmids are often used to purify a specific sequence, as they can be easily purified from the rest of the genome.
There are several methods for isolating plasmid DNA from bacteria, the archetypes of which are miniprep and maxiprep / bulkprep.
The former can be used to quickly discover if the plasmid is correct in any of several bacterial clones.
The yield is a small amount of impure plasmid DNA, which is sufficient for restriction digest analysis and for some cloning techniques.
In the latter, much larger volumes of bacterial suspension are produced from which a maxi-preparation can be carried out. In essence, it is a miniprep to scale followed by additional purification.
This results in relatively large amounts (several hundred micrograms) of very pure plasmid DNA.
Conformations
The open circular nicked DNA has a strand cut.
The relaxed circular DNA is fully intact with both strands uncut, but has been enzymatically relaxed (supercooling removed).
This can be modeled by letting a twisted extension cord unwind and relax and then plug it into itself.
The supercoiled (or covalently closed circular) DNA is completely intact with both strands uncut, and with an integral twist, resulting in a compact shape. This can be modeled by twisting an extension cord and then plugging it into itself.
Supercoiled denatured DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact; this may be the result of excessive alkalinity during plasmid preparation.
The migration rate for small linear fragments is directly proportional to the voltage applied at low voltages.
At higher voltages, the larger fragments migrate at continuously increasing but different rates. Therefore, the resolution of a gel decreases with increasing voltage.
At a specified low voltage, the rate of migration of small linear DNA fragments is a function of their length.
Large linear fragments (more than 20 kb or less) migrate at a certain fixed rate regardless of length.
This is because the molecules “respect”, and most of the molecule follows the leading end through the gel matrix.
Restriction digests are frequently used to analyze purified plasmids. These enzymes specifically break DNA into certain short sequences.
The resulting linear fragments form ‘bands’ after gel electrophoresis. It is possible to purify certain fragments by cutting the gel bands and dissolving the gel to release the DNA fragments.
Due to its tight conformation, supercoiled DNA migrates faster through a gel than linear or open circular DNA.
Bioinformatics and design 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 predict restriction enzyme cleavage sites, and plan manipulations.
Examples of software packages that handle plasmid maps are ApE, Clone Manager, GeneConstructionKit, Geneious, Genome Compiler, LabGenius, Lasergene, MacVector, pDraw32, Serial Cloner, VectorFriends, Vector NTI, and WebDSV.
This software helps to perform full experiments in silico before conducting wet experiments.
Plasmid Collections
Many plasmids have been created over the years, and researchers have distributed plasmids to plasmid databases, such as the nonprofit Addgene and BCCM / LMBP.
One can find and request plasmids from those databases for further research.
The researcher also usually uploads plasmid sequences to the National Center for Biotechnology Information (NCBI) database. Using the National Center for Biotechnology, specific plasmid database sequences can be searched.
