Plasmid

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A plasmid is a small DNA molecule inside a cell that is physically separated from the chromosomal DNA and can replicate independently. They are most commonly found as small circular DNA molecules, double-stranded in bacteria; However, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that can be beneficial to the survival of organisms, such as antibiotic resistance. While 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 organisms in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning, serves to encourage replication of recombinant DNA sequences in host organisms. In the laboratory, plasmids can be incorporated into cells through transformation.

Plasmids are considered replicas , units of DNA capable of independently replicating within the appropriate host. However, plasmids, such as viruses, are generally not classified as life. Plasmids are transmitted from one bacterium to another (even from other species) mostly through conjugation. Transfer of the host-to-host genetic material is one of the horizontal gene transfer mechanisms, and plasmids are considered part of the mobilome. Unlike viruses (which wrap their genetic material in layers of protective proteins called capsid), plasmids are "naked" DNA and do not encode genes necessary to encapsulate genetic material to be transferred to a new host. However, some plasmid classes encode the conjugative "sex" pilots required for their own transfer. The size of the plasmid varies from 1 to more than 200 kbp, and the number of identical plasmids in one cell can range from one to thousands in some circumstances.

The relationship between microbial and plasmid DNA is not parasitic or mutualistic, since each implies the existence of independent species living in adverse or commensal conditions with host organisms. In contrast, plasmids provide a mechanism for the transfer of horizontal genes in microbial populations and usually provide selective advantages under given environmental conditions. Plasmids can carry genes that provide resistance to antibiotics that occur naturally in a competitive environment, or the resulting proteins can act as toxins under similar circumstances, or allow the organism to use certain organic compounds that will benefit when the nutrients are scarce.

Video Plasmid

History

The term was introduced in 1952 by American molecular biologist Joshua Lederberg to refer to "the determinant of extrachromosomal offspring." The initial use of this term includes any genetically existing bacterial material extrachromosomally for at least part of the replication cycle, but because the description includes a bacterial virus, the idea of ​​a plasmid is enhanced over time to form autonomous genetic elements that reproduce. Then in 1968, it was decided that the term plasmid should be adopted as a term for extrachromosomal genetic material, and to distinguish it from viruses, the definition is narrowed down into genetic elements that exist exclusively or primarily outside the chromosomes and can replicate autonomously.

Maps Plasmid

Properties and characteristics

In order for plasmids to replicate independently within cells, they must have a stretch of DNA that can act as the origin of replication. The self-replicating unit, in this case a plasmid, is called a replicon. A typical bacterial replica may consist of a number of elements, such as genes for a specific replica plasmid replication initiation protein (Rep), a repeating unit called the iteron, DnaA box, and adjacent AT-rich areas. Smaller plasmids use host replication enzymes to make copies of themselves, while larger plasmids can carry specific genes for replication of the plasmid. Several types of plasmids may also be incorporated into the host chromosome, and these integrative plasmids are sometimes referred to as episodes in prokaryotes.

Plasmids almost always carry at least one gene. Many of the genes carried by plasmids are beneficial to host cells, for example: enabling host cells to survive in environments that otherwise would kill or limit growth. Some of these genes encode the properties of antibiotic resistance or resistance to heavy metals, while others can produce virulence factors that allow bacteria to colonize the host and overcome their defenses, or have certain metabolic functions that allow bacteria to utilize certain nutrients, including the ability to degrade compounds organic recalcitrant or toxic. Plasmids can also provide bacteria with the ability to repair nitrogen. Some plasmids, however, have no observable effect on the host cell phenotype or their benefits to host cells can not be determined, and this plasmid is called a cryptic plasmid.

Plasmids that occur naturally vary in their physical properties. Its size can range from a very small mini-plasmid with less than 1 kilobase pairs (Kbp), to a very large megaplasmid of several megabase pairs (Mbp). At the upper end, little can distinguish between megaplasmids and minicromosomes. Plasmids are generally circular, but linear plasmid samples are also known. This linear plasmid requires a special mechanism to mimic the tip.

Plasmids can be present in individual cells in varying amounts, ranging from one to several hundreds. The normal number of copies of plasmids that can be found in one cell is called the number of copies, and is determined by how replication initiation is regulated and the size of the molecule. Larger plasmids tend to have lower number of copies. Plasmids of low copies that exist only as one or more copies in each bacterium are, in cell division, in danger of being lost in one of the separating bacteria. Such one-copy plasmids have systems that attempt to actively distribute copies to both child cells. This system, which includes the parABS system and the parMRC system, is often referred to as the partition system or partition function of a plasmid.

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Classification and type

Plasmids can be classified in several ways. Plasmids can be broadly classified into conjugate plasmids and non-conjugate plasmids. Conjugative plasmids contain a set of transfers or tra genes that promote sexual conjugation between different cells. In a complex conjugate process, plasmids can be transferred from one bacterium to another via sex pili encoded by several tra genes (see figure). Non-conjugate plasmids are not capable of initiating conjugation, then they can be transferred only with the help of a conjugate plasmid. The middle class of plasmids can be mobilized, and carry only a portion of the genes needed for transfer. They can eradicate a conjugate plasmid, transferring at high frequencies just in front of it.

Plasmids can also be classified into groups of discrepancies. A microbe can store different types of plasmids; however, different plasmids can only exist in one bacterial cell if they are compatible. If two plasmids are not compatible, one or the other will disappear quickly from the cell. Therefore different plasmids can be assigned to different groups of discrepancies depending on whether they can co-exist together. Incompatible plasmas (included in the same group of incompatibilities) usually share the same replication or partition mechanisms that can not be stored together in a single cell.

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

• Fertility F-plasmid, which contains the tra genes. They are able to conjugate and produce sex pili expressions.
• Resistance (R) plasmids, which contain genes that provide resistance to antibiotics or toxins. Historically known as the R-factor, before plasmid properties are understood.
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• Degradative Plasmid, which allows the digestion of unusual substances, eg. toluene and salicylic acid.
• The virulence of plasmids, which turns bacteria into pathogens.

Plasmids can belong to more than one functional group.

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Vector

Artificially created plasmids can be used as vectors in genetic engineering. These plasmids serve as important tools in biotech genetics and laboratories, where they are typically used to clone and amplify (make multiple copies) or express specific genes. Various plasmids are commercially available for such use. The gene to be replicated is usually inserted into a plasmid that usually contains a number of features to use. These include genes that provide resistance to certain antibiotics (ampicillin most commonly used for bacterial strains), the origin of replication to allow bacterial cells to replicate plasmid DNA, and sites suitable for cloning (referred to as multiple cloning sites).

Cloning

Plasmid is the most commonly used bacterial cloning vector. This cloned vector contains sites that allow DNA fragments to be inserted, such as multiple cloning sites or polylinkers that have some commonly used restriction sites where DNA fragments can be ligated. Once the desired gene is inserted, the plasmid is fed into the bacteria through a process called transformation. This plasmid contains selectable markers, usually antibiotic resistance genes, which give bacteria the ability to survive and proliferate in selective growth mediums containing certain antibiotics. Cells after transformation are exposed to selective media, and only cells containing plasmids can survive. In this way, antibiotics act as a filter to select only bacteria containing plasmid DNA. Vectors may also contain other marker genes or reporter genes to facilitate the selection of plasmids with cloned inserts. Plasma-containing bacteria can then be grown in large quantities, harvested, and an attractive plasmid can then be isolated using a variety of plasmid preparation methods.

Plasmid cloning vectors are typically used to clone DNA fragments up to 15 kbp. To clone longer DNA lengths, lambda phage with the removed lysogeny gene, cosmids, bacterial-made chromosomes, or yeast-made chromosomes are used.

Protein production

Another major use of plasmids is to make large amounts of protein. In this case, researchers grow bacteria containing plasmids that store the desired genes. Just as bacteria produce proteins to provide antibiotic resistance, it can also be induced to produce large amounts of protein from the inserted gene. This is a cheap and easy way to mass-produce protein gene codes for, for example, insulin.

Gene therapy

Plasmids can also be used for the transfer of genes into human cells as a potential treatment in gene therapy so as to express inadequate proteins in cells. Some gene therapy strategies require the insertion of a therapeutic gene at a previously selected chromosome target site in the human genome. The vector plasmid is one of many approaches that can be used for this purpose. Zinc nuclease finger (ZFNs) offers a way to cause the breaking of the site-specific double strand into the DNA genome and leads to homologous recombination. Plasmids that encode ZFN can help deliver therapeutic genes to certain sites so cellular damage, cancer-causing mutations, or immune responses are avoided.

The disease model

Plasmids have historically been used to genetically engineer mouse embryonic stem cells to create models of mouse genetic disease. The limited efficiency of the plasmid-based technique precludes its use in the creation of more accurate human cell models. However, the development of the associated Adeno virus recombination techniques, and nucleation of Zinc fingers, has enabled the creation of a new generation of isogenic human disease models.

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Episomes

The term episome was introduced by FranÃÆ'§ois Jacob and ÃÆ'â € lie Wollman in 1958 to refer to an extra-chromosomal genetic material that can replicate autonomously or become integrated into the chromosome. Since the term was introduced, however, its use has shifted as plasmid has become the preferred term for autonomous replication of extrachromosomal DNA. At the 1968 symposium in London some participants suggested that the term episome was abandoned, though others continued to use the term with a shift in meaning.

Today some authors use episome in the context of prokaryotes to refer to plasmids capable of integrating into chromosomes. The integrative plasmids can be replicated and stable maintained in cells through several generations, but always at some stage they exist as independent plasmid molecules. In the context of eukaryotes, the term episomes is used to mean circular molecules of closed, non-integrated extrachromosomal that can be replicated in the nucleus. Viruses are the most common example of this, such as herpes virus, adenovirus, and polyomavirus, but some are plasmids. Other examples include fragmentary chromosome fragments, such as multiple minute chromosomes, which can arise during the amplification of an artificial gene or in a pathological process (eg, cancer cell transformation). Episodes in eukaryotes behave similarly to plasmids in prokaryotes because DNA is maintained stable and replicated with host cells. Episodes of cytoplasmic virus (as in poxvirus infections) may also occur. Some episodes, such as the herpes virus, replicate in a revolving circular mechanism, similar to a bacterial phag virus. Others replicate via a two-way replication mechanism ( Theta type plasmid). In both cases, the episode remains physically separate from the stem cell chromosome. Some cancer viruses, including Epstein-Barr virus and Kaposi-related herpes virus, are preserved as latent episodes, different chromosomes in cancer cells, in which the virus expresses oncogenes that promote the proliferation of cancer cells. In cancer, these episodes passively replicate together with the master's chromosome when the cell divides. When this episode begins lytic replication to produce some virus particles, they generally activate the innate cellular immune defense mechanism that kills the host cell.

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Wasmid maintenance

Some plasmids or microbial hosts include addictive systems or postreggregational killer systems (PSKs), such as the killer killer system of plasmid R1 in Escherichia coli . This variant produces a long-lived toxin and a short antidote. Several types of plasmid addiction systems (toxins/antitoxins, metabolic-based systems, ORTs) are described in the literature and used in biotech applications (fermentation) or biomedicine (vaccines). The daughter cells that keep the plasmid copies survive, while the child cells that fail to inherit the plasmid die or suffer from reduced growth rates due to the lingering toxins of the stem cells. Finally, overall productivity can be improved.

In contrast, almost all biotechnologically used plasmids (such as pUC18, pBR322 and inherited vectors) do not contain toxic antioxidant addiction systems and therefore need to be kept under antibiotic pressure to avoid the loss of plasmids.

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Plasmid yeast

Yeast naturally stores various plasmids. Important among them are 2 Âμm plasmids - small circular plasmids often used for genetic engineering yeasts - and linear pGKL plasmids from Kluyveromyces lactis , which are responsible for killer phenotypes.

Other types of plasmids are often associated with yeast cloning vectors that include: Yeast integrative plasmid (YIp), a vector yoke that relies on integration into the host chromosome for survival and replication, and is usually used when studying solo gene functionality or when the gene is toxic. It is also connected to the URA3 gene, which encodes enzymes associated with pyrimidine nucleotide biosynthesis (T, C);

Plasmid Replicative Yeast (YRp) , which transports a chromosome DNA sequence that includes the origin of the replication. This plasmid is less stable, because they can get lost during the beginner.

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Plasmid DNA Extraction

As mentioned above, plasmids are often used to purify certain sequences, as they can be easily cleared from the rest of the genome. For its use as a vector, and for molecular cloning, plasmids often need to be isolated.

There are several methods to isolate plasmid DNA from bacteria, the arketipe which is miniprep and maxiprep / bulkprep . The former can be used to immediately find out whether the plasmid is correct in one of several bacterial clones. The result is a small amount of impure plasmid DNA, sufficient for analysis by digesting restrictions and for some cloning techniques.

In the latter, larger volumes of bacterial suspensions are grown from which maximal preparation can be performed. In essence, this is an enhanced miniprep followed by additional purification. This produces a relatively large amount (several hundred micrograms) of very pure plasmid DNA.

In recent years, many commercial devices have been made to perform plasmid extraction at various scales, purity, and degree of automation. Commercial services can prepare plasmid DNA at prices quoted below $ 300/mg in milligrams and $ 15/mg in grams (beginning 2007).

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Conform

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

• Nicked open-circular The DNA has one truncated strand.
• relaxed circular DNA is fully intact with both pieces cut, but has been enzymatically relaxed (supercoils omitted). This can be modeled by letting the extension cable bend loose and relax, then enter it yourself.
• Linear The DNA has a free edge, either because the two strands have been cut or because the DNA is linear in vivo . This can be modeled with a power extension cord that is not plugged in to itself.
• Supercoiled (or covalently closed-circular ) The DNA is fully intact with both pieces cut, and with integral twist, resulting in a compact shape. This can be modeled by rotating the extension cable and then installing it yourself.
• Supercoiled denatured DNA is like supercoiled DNA , but having an unpaired area makes it a bit less compact; this may result from excessive alkalinity during the preparation of the plasmid.

The migration rate for small linear fragments is directly proportional to the voltage applied to the low voltage. At higher voltages, larger fragments migrate at an increasing but different rate. Thus, the gel resolution decreases with increasing stress.

At the specified low voltage, the migration rate of small linear DNA fragments is a function of its length. Large linear fragments (more than 20 kb or more) migrate at a certain fixed rate regardless of length. This is because the 'resperate' molecules, with most of the molecules following the leading edge through the gel matrix. Restriction digestion is often used to analyze purified plasmids. These enzymes typically break down DNA in certain short sequences. The resulting linear fragment forms a 'band' after gel electrophoresis. It is possible to purify certain fragments by cutting the bands from the gel and dissolving the gel to release the DNA fragments.

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

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Software for bioinformatics and design

The use of plasmids as a technique in molecular biology is supported by bioinformatics software. These programs record the plasmid DNA vector sequence, help predict restriction enzyme clearance sites, and plan for manipulation. Examples of software packages that handle plasmid maps are ApE, Clone Manager, GeneConstructionKit, Geneious, Genome Compilers, LabGenius, Lasergene, MacVector, pDraw32, Serial Cloner, VectorFriends, Vector NTI, and WebDSV. This software helps to conduct the entire experiment in silico before doing a wet experiment.

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Plasmid collection

Source of the article : Wikipedia