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Enterobacteria phage λ is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli. It was discovered by Esther Lederberg in 1950. The wild type of this virus has a temperate life cycle that allows it to either reside within the genome of its host through lysogeny or enter into a lytic phase, during which it kills and lyses the cell to produce offspring. Lambda strains, mutated at specific sites, are unable to lysogenize cells; instead, they grow and enter the lytic cycle after superinfecting an already lysogenized cell.
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. Plasmids often carry useful genes, such as antibiotic resistance and virulence. 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.
A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites. The vector and the foreign DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector. An example is the viral transfer of DNA from one bacterium to another and hence an example of horizontal gene transfer. Transduction does not require physical contact between the cell donating the DNA and the cell receiving the DNA, and it is DNase resistant. Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell's genome.
A cosmid is a type of hybrid plasmid that contains a Lambda phage cos sequence. Often used as cloning vectors in genetic engineering, cosmids can be used to build genomic libraries. They were first described by Collins and Hohn in 1978. Cosmids can contain 37 to 52 kb of DNA, limits based on the normal bacteriophage packaging size. They can replicate as plasmids if they have a suitable origin of replication (ori): for example SV40 ori in mammalian cells, ColE1 ori for double-stranded DNA replication, or f1 ori for single-stranded DNA replication in prokaryotes. They frequently also contain a gene for selection such as antibiotic resistance, so that the transformed cells can be identified by plating on a medium containing the antibiotic. Those cells which did not take up the cosmid would be unable to grow.
Phage display is a laboratory technique for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. In this technique, a gene encoding a protein of interest is inserted into a phage coat protein gene, causing the phage to "display" the protein on its outside while containing the gene for the protein on its inside, resulting in a connection between genotype and phenotype. The proteins that the phages are displaying can then be screened against other proteins, peptides or DNA sequences, in order to detect interaction between the displayed protein and those of other molecules. In this way, large libraries of proteins can be screened and amplified in a process called in vitro selection, which is analogous to natural selection.
A DNA construct is an artificially-designed segment of DNA borne on a vector that can be used to incorporate genetic material into a target tissue or cell. A DNA construct contains a DNA insert, called a transgene, delivered via a transformation vector which allows the insert sequence to be replicated and/or expressed in the target cell. This gene can be cloned from a naturally occurring gene, or synthetically constructed. The vector can be delivered using physical, chemical or viral methods. Typically, the vectors used in DNA constructs contain an origin of replication, a multiple cloning site, and a selectable marker. Certain vectors can carry additional regulatory elements based on the expression system involved.
Filamentous bacteriophages are a family of viruses (Inoviridae) that infect bacteria, or bacteriophages. They are named for their filamentous shape, a worm-like chain, about 6 nm in diameter and about 1000-2000 nm long. This distinctive shape reflects their method of replication: the coat of the virion comprises five types of viral protein, which are located in the inner membrane of the host bacterium during phage assembly, and these proteins are added to the nascent virion's DNA as it is extruded through the membrane. The simplicity of filamentous phages makes them an appealing model organism for research in molecular biology, and they have also shown promise as tools in nanotechnology and immunology.
M13 is one of the Ff phages, a member of the family filamentous bacteriophage (inovirus). Ff phages are composed of circular single-stranded DNA (ssDNA), which in the case of the m13 phage is 6407 nucleotides long and is encapsulated in approximately 2700 copies of the major coat protein p8, and capped with about 5 copies each of four different minor coat proteins. The minor coat protein p3 attaches to the receptor at the tip of the F pilus of the host Escherichia coli. The life cycle is relatively short, with the early phage progeny exiting the cell ten minutes after infection. Ff phages are chronic phage, releasing their progeny without killing the host cells. The infection causes turbid plaques in E. coli lawns, of intermediate opacity in comparison to regular lysis plaques. However, a decrease in the rate of cell growth is seen in the infected cells. The replicative form of M13 is circular double-stranded DNA similar to plasmids that are used for many recombinant DNA processes, and the virus has also been used for phage display, directed evolution, nanostructures and nanotechnology applications.
Φ6 is the best-studied bacteriophage of the virus family Cystoviridae. It infects Pseudomonas bacteria. It has a three-part, segmented, double-stranded RNA genome, totalling ~13.5 kb in length. Φ6 and its relatives have a lipid membrane around their nucleocapsid, a rare trait among bacteriophages. It is a lytic phage, though under certain circumstances has been observed to display a delay in lysis which may be described as a "carrier state".
A genomic library is a collection of overlapping DNA fragments that together make up the total genomic DNA of a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA. In order to construct a genomic library, the organism's DNA is extracted from cells and then digested with a restriction enzyme to cut the DNA into fragments of a specific size. The fragments are then inserted into the vector using DNA ligase. Next, the vector DNA can be taken up by a host organism - commonly a population of Escherichia coli or yeast - with each cell containing only one vector molecule. Using a host cell to carry the vector allows for easy amplification and retrieval of specific clones from the library for analysis.
Fosmids are similar to cosmids but are based on the bacterial F-plasmid. The cloning vector is limited, as a host can only contain one fosmid molecule. Fosmids can hold DNA inserts of up to 40 kb in size; often the source of the insert is random genomic DNA. A fosmid library is prepared by extracting the genomic DNA from the target organism and cloning it into the fosmid vector. The ligation mix is then packaged into phage particles and the DNA is transfected into the bacterial host. Bacterial clones propagate the fosmid library. The low copy number offers higher stability than vectors with relatively higher copy numbers, including cosmids. Fosmids may be useful for constructing stable libraries from complex genomes. Fosmids have high structural stability and have been found to maintain human DNA effectively even after 100 generations of bacterial growth. Fosmid clones were used to help assess the accuracy of the Public Human Genome Sequence.
Bacteriophage MS2, commonly called MS2, is an icosahedral, positive-sense single-stranded RNA virus that infects the bacterium Escherichia coli and other members of the Enterobacteriaceae. MS2 is a member of a family of closely related bacterial viruses that includes bacteriophage f2, bacteriophage Qβ, R17, and GA.
P1 is a temperate bacteriophage that infects Escherichia coli and some other bacteria. When undergoing a lysogenic cycle the phage genome exists as a plasmid in the bacterium unlike other phages that integrate into the host DNA. P1 has an icosahedral head containing the DNA attached to a contractile tail with six tail fibers. The P1 phage has gained research interest because it can be used to transfer DNA from one bacterial cell to another in a process known as transduction. As it replicates during its lytic cycle it captures fragments of the host chromosome. If the resulting viral particles are used to infect a different host the captured DNA fragments can be integrated into the new host's genome. This method of in vivo genetic engineering was widely used for many years and is still used today, though to a lesser extent. P1 can also be used to create the P1-derived artificial chromosome cloning vector which can carry relatively large fragments of DNA. P1 encodes a site-specific recombinase, Cre, that is widely used to carry out cell-specific or time-specific DNA recombination by flanking the target DNA with loxP sites.
In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.
The CTXφ bacteriophage is a filamentous bacteriophage. It is a positive-strand DNA virus with single-stranded DNA (ssDNA).
Ff phages is a group of almost identical filamentous phage including phages f1, fd, M13 and ZJ/2, which infect bacteria bearing the F fertility factor. The virion is a flexible filament measuring about 6 by 900 nm, comprising a cylindrical protein tube protecting a single-stranded circular DNA molecule at its core. The phage codes for only 11 gene products, and is one of the simplest viruses known. It has been widely used to study fundamental aspects of molecular biology. George Smith and Greg Winter used f1 and fd for their work on phage display for which they were awarded a share of the 2018 Nobel Prize in Chemistry. Early experiments on Ff phages used M13 to identify gene functions, and M13 was also developed as a cloning vehicle, so the name M13 is sometimes used as an informal synonym for the whole group of Ff phages.
Spiroplasma phage 1-R8A2B is a filamentous bacteriophage in the genus Vespertiliovirus of the family Plectroviridae, part of the group of single-stranded DNA viruses. The virus has many synonyms, such as SpV1-R8A2 B, Spiroplasma phage 1, and Spiroplasma virus 1, SpV1. SpV1-R8A2 B infects Spiroplasma citri. Its host itself is a prokaryotic pathogen for citrus plants, causing Citrus stubborn disease.
Bacteriophage AP205 is a plaque-forming bacteriophage that infects Acinetobacter bacteria. Bacteriophage AP205 is a protein-coated virus with a positive single-stranded RNA genome. It is a member of the family Fiersviridae, consisting of particles that infect Gram-negative bacteria such as E. coli.
References
- ↑ Wilson, K.; Walker, J. (2010). Principles and Techniques of Biochemistry and Molecular Biology. 7th ed. New York: Cambridge University Press. p. 751.
- ↑ Barbas, Carlos F.; Burton, Dennis R.; Scott, Jamie K.; Silverman, Gregg J. (2001). Phage Display: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press. ISBN 978-087969740-2.
- ↑ Melnikov, Anatolij A.; Tchernov, Alexander P.; Fodor, Istvan; Bayev, Alexander A. (April 1984). "Lambda phagemids and their transducing properties". Gene. 28 (1): 29–35. doi:10.1016/0378-1119(84)90084-2. PMID 6234200.
- 1 2 Reece, Richard J. (25 June 2004). Analysis of Genes and Genomes. Wiley. ISBN 978-0-470-09157-9.
- ↑ Lund, Paul E.; Hunt, Ryan C.; Gottesman, Michael M.; Kimchi-Sarfaty, Chava (2010). "Pseudovirions as Vehicles for the Delivery of siRNA". Pharmaceutical Research. 27 (3): 400–420. doi:10.1007/s11095-009-0012-2. PMC 2831147 . PMID 19998056.
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