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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. In nature, plasmids often carry genes that benefit the survival of the organism and confer selective advantage such as antibiotic resistance. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain only additional genes that may be useful in certain situations or conditions. 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.
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.
Molecular genetics is a branch of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the structure and/or function of genes in an organism's genome using genetic screens.
In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.
Auxotrophy is the inability of an organism to synthesize a particular organic compound required for its growth. An auxotroph is an organism that displays this characteristic; auxotrophic is the corresponding adjective. Auxotrophy is the opposite of prototrophy, which is characterized by the ability to synthesize all the compounds needed for growth.
A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss Physcomitrella patens. Different types of media are used for growing different types of cells.
Kanamycin A, often referred to simply as kanamycin, is an antibiotic used to treat severe bacterial infections and tuberculosis. It is not a first line treatment. It is used by mouth, injection into a vein, or injection into a muscle. Kanamycin is recommended for short-term use only, usually from 7 to 10 days. As with most antibiotics, it is ineffective in viral infections.
In biology, a gene cassette is a type of mobile genetic element that contains a gene and a recombination site. Each cassette usually contains a single gene and tends to be very small; on the order of 500–1,000 base pairs. They may exist incorporated into an integron or freely as circular DNA. Gene cassettes can move around within an organism's genome or be transferred to another organism in the environment via horizontal gene transfer. These cassettes often carry antibiotic resistance genes. An example would be the kanMX cassette which confers kanamycin resistance upon bacteria.
An efflux pump is an active transporter in cells that moves out unwanted material. Efflux pumps are an important component in bacteria in their ability to remove antibiotics. The efflux could also be the movement of heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals, bacterial metabolites and neurotransmitters. All microorganisms, with a few exceptions, have highly conserved DNA sequences in their genome that encode efflux pumps. Efflux pumps actively move substances out of a microorganism, in a process known as active efflux, which is a vital part of xenobiotic metabolism. This active efflux mechanism is responsible for various types of resistance to bacterial pathogens within bacterial species - the most concerning being antibiotic resistance because microorganisms can have adapted efflux pumps to divert toxins out of the cytoplasm and into extracellular media.
Orotidine 5'-phosphate decarboxylase or orotidylate decarboxylase is an enzyme involved in pyrimidine biosynthesis. It catalyzes the decarboxylation of orotidine monophosphate (OMP) to form uridine monophosphate (UMP). The function of this enzyme is essential to the de novo biosynthesis of the pyrimidine nucleotides uridine triphosphate, cytidine triphosphate, and thymidine triphosphate. OMP decarboxylase has been a frequent target for scientific investigation because of its demonstrated extreme catalytic efficiency and its usefulness as a selection marker for yeast strain engineering.
A shuttle vector is a vector constructed so that it can propagate in two different host species. Therefore, DNA inserted into a shuttle vector can be tested or manipulated in two different cell types. The main advantage of these vectors is they can be manipulated in E. coli, then used in a system which is more difficult or slower to use.
Plant transformation vectors are plasmids that have been specifically designed to facilitate the generation of transgenic plants. The most commonly used plant transformation vectors are T-DNA binary vectors and are often replicated in both E. coli, a common lab bacterium, and Agrobacterium tumefaciens, a plant-virulent bacterium used to insert the recombinant DNA into plants.
An origin of transfer (oriT) is a short sequence ranging from 40-500 base pairs in length that is necessary for the transfer of DNA from a gram-negative bacterial donor to recipient during bacterial conjugation. The transfer of DNA is a critical component for antimicrobial resistance within bacterial cells and the oriT structure and mechanism within plasmid DNA is complementary to its function in bacterial conjugation. The first oriT to be identified and cloned was on the RK2 (IncP) conjugative plasmid, which was done by Guiney and Helinski in 1979.
URA3 is a gene on chromosome V in Saccharomyces cerevisiae (yeast). Its systematic name is YEL021W. URA3 is often used in yeast research as a "marker gene", that is, a gene to label chromosomes or plasmids. URA3 encodes Orotidine 5'-phosphate decarboxylase (ODCase), which is an enzyme that catalyzes one reaction in the synthesis of pyrimidine ribonucleotides (a component of RNA).
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.
Plasmid-mediated resistance is the transfer of antibiotic resistance genes which are carried on plasmids. Plasmids possess mechanisms that ensure their independent replication as well as those that regulate their replication number and guarantee stable inheritance during cell division. By the conjugation process, they can stimulate lateral transfer between bacteria from various genera and kingdoms. Numerous plasmids contain addiction-inducing systems that are typically based on toxin-antitoxin factors and capable of killing daughter cells that don't inherit the plasmid during cell division. Plasmids often carry multiple antibiotic resistance genes, contributing to the spread of multidrug-resistance (MDR). Antibiotic resistance mediated by MDR plasmids severely limits the treatment options for the infections caused by Gram-negative bacteria, especially family Enterobacteriaceae. The global spread of MDR plasmids has been enhanced by selective pressure from antimicrobial medications used in medical facilities and when raising animals for food.
Cyanidioschyzon merolae is a small (2μm), club-shaped, unicellular haploid red alga adapted to high sulfur acidic hot spring environments. The cellular architecture of C. merolae is extremely simple, containing only a single chloroplast and a single mitochondrion and lacking a vacuole and cell wall. In addition, the cellular and organelle divisions can be synchronized. For these reasons, C. merolae is considered an excellent model system for study of cellular and organelle division processes, as well as biochemistry and structural biology. The organism's genome was the first full algal genome to be sequenced in 2004; its plastid was sequenced in 2000 and 2003, and its mitochondrion in 1998. The organism has been considered the simplest of eukaryotic cells for its minimalist cellular organization.
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 RK2 Plasmid is a broad-host-range plasmid belonging to the incP incompatibility group It is notable for its ability to replicate in a wide variety of single-celled organisms, which makes it suitable as a genetic engineering tool. It is capable of transfer, replication, and maintenance in most genera of Gram-negative bacteria. RK2 may sometimes be referred to as pRK2, which is also the name of another, unrelated plasmid. Other names for RK2 include R18, R68, RP1, and RP4. These were all separate isolates, and later found to be identical plasmids. The IncP-1 plasmid group of which RK2 is a part has been described as "highly potent, self-transmissible, selfish DNA molecules with a complicated regulatory circuit"
Streptomyces lavendulae is a species of bacteria from the genus Streptomyces. It is isolated from soils globally and is known for its production of medically useful biologically active metabolites. To see a photo of this organism click here.
References
- ↑ "positive selection". Scitable. Nature. Retrieved 29 September 2011.
- ↑ "negative selection". Scitable. Nature. Retrieved 29 September 2011.
- ↑ Callmigration.org: Gene targeting
- ↑ Jang, Chuan-Wei; Magnuson, Terry (20 February 2013). "A Novel Selection Marker for Efficient DNA Cloning and Recombineering in E. coli". PLOS ONE. 8 (2): e57075. Bibcode:2013PLoSO...857075J. doi: 10.1371/journal.pone.0057075 . PMC 3577784 . PMID 23437314.
- ↑ Boeke JD; LaCroute F; Fink GR (1984). "A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance". Mol. Gen. Genet. 197 (2): 345–6. doi:10.1007/bf00330984. PMID 6394957. S2CID 28881589.
- ↑ Goldstein, Daniel A.; Tinland, Bruno; Gilbertson, Lawrence A.; Staub, J.M.; Bannon, G.A.; Goodman, R.E.; McCoy, R.L.; Silvanovich, A. (2005). "Human safety and genetically modified plants: a review of antibiotic resistance markers and future transformation selection technologies". Journal of Applied Microbiology . 99 (1). Society for Applied Microbiology (Wiley): 7–23. doi:10.1111/j.1365-2672.2005.02595.x. ISSN 1364-5072. PMID 15960661. S2CID 40454719.
