Binary Vectors
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Binary Vectors
A transfer DNA (T-DNA) binary system is a pair of plasmids consisting of a T-DNA binary vector and a ''vir'' helper plasmid. The two plasmids are used together (thus ''binary'') to produce genetically modified plants. They are artificial vectors that have been derived from the naturally occurring Ti plasmid found in bacterial species of the genus '' Agrobacterium'', such as '' A. tumefaciens''. The binary vector is a ''shuttle vector'', so-called because it is able to replicate in multiple hosts (e.g. '' Escherichia coli'' and ''Agrobacterium''). Systems in which T-DNA and ''vir'' genes are located on separate replicons are called T-DNA binary systems. T-DNA is located on the binary vector (the non-T-DNA region of this vector containing origin(s) of replication that could function both in ''E. coli'' and ''Agrobacterium'', and antibiotic resistance genes used to select for the presence of the binary vector in bacteria, became known as vector backbone sequences). The replicon con ...
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Plasmids
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 intern ...
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Selectable Marker
A selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a transfection or other procedure meant to introduce foreign DNA into a cell. Selectable markers are often antibiotic resistance genes (''An antibiotic resistance marker is a gene that produces a protein that provides cells expressing this protein with resistance to an antibiotic.''). Bacteria that have been subjected to a procedure to introduce foreign DNA are grown on a medium containing an antibiotic, and those bacterial colonies that can grow have successfully taken up and expressed the introduced genetic material. Normally the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are considered useful selectable markers for ''E. coli' ...
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Genetics
Genetics is the study of genes, genetic variation, and heredity in organisms.Hartl D, Jones E (2005) It is an important branch in biology because heredity is vital to organisms' evolution. Gregor Mendel, a Moravian Augustinian friar working in the 19th century in Brno, was the first to study genetics scientifically. Mendel studied "trait inheritance", patterns in the way traits are handed down from parents to offspring over time. He observed that organisms (pea plants) inherit traits by way of discrete "units of inheritance". This term, still used today, is a somewhat ambiguous definition of what is referred to as a gene. Trait inheritance and molecular inheritance mechanisms of genes are still primary principles of genetics in the 21st century, but modern genetics has expanded to study the function and behavior of genes. Gene structure and function, variation, and distribution are studied within the context of the cell, the organism (e.g. dominance), and within the ...
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PGreen
The pGreen plasmids are vectors for plant transformation. They were first described in 2000 as components of a novel T-DNA binary system. The supporting web page provides supplementary information and ongoing support to researchers to request their plasmid resources. As these plasmids have been taken up by the research community, the plasmids have been developed, expanding the resources available to the community. Researchers are encouraged to contribute to this research community by submitting their vector sequence to genbank and providing a description of the plasmid on the site. pGreenI and pGreenII pGreen is the original pGreen plasmid. pGreenII features plasmid backbone modification to improve plasmid stability. T-DNA regions No transformation selection pGreenII 0000: minimal T-DNA with Left and Right border, ''lacZ'' gene for blue/white selection during cloning multiple cloning site derived from pBluescript. pGreenII 62-SK: derived from pGreenII 0000, the ''Lac''Z b ...
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Transformation Efficiency
Transformation efficiency is the efficiency by which cells can take up extracellular DNA and express genes encoded by it. This is based on the competence of the cells. It can be calculated by dividing the number of successful transformants by the amount of DNA used during a transformation procedure. Transformants are cells that have taken up DNA (foreign, artificial or modified) and which can express genes on the introduced DNA. Measurement By measuring the transformation efficiency, we can utilize the information from our experiment to evaluate how effectively our transformation went.This is a quantification of how many cells were altered by 1µg of plasmid DNA. In essence, it is a sign that the transformation experiment was successful. Transformation efficiency should be determined under conditions of cell excess. The number of viable cells in a preparation for a transformation reaction may range from 2×108 to 1011; most common methods of ''E. coli'' preparation yield aroun ...
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EHA101
EHA101 was one of the first and most widely used ''Agrobacterium'' helper plasmid for plant gene transfer. Created in 1985 in the laboratory of Mary-Dell Chilton at Washington University in St. Louis, it was named after the graduate student who constructed it. The EH stands for "Elizabeth Hood" and A for "''Agrobacterium''". The EHA101 helper strain is a derivative of A281, the hypervirulent ''A. tumefaciens'' strain that causes large, fast-growing tumors on solanaceous plants. This strain is used for moving genes of interest into many hundreds of species of plants all over the world. For recalcitrant crops such as maize, wheat, and rice, the EHA helper strains are often employed for gene transfer. These strains are efficient at promoting T-DNA transfer because of the hypervirulence of the vir genes suggesting that a higher success rate can be achieved on these "hard to transform" crops or cultivars. The chromosomal background of EHA101 is C58C1, a cured nopaline strain. The h ...
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PGreen
The pGreen plasmids are vectors for plant transformation. They were first described in 2000 as components of a novel T-DNA binary system. The supporting web page provides supplementary information and ongoing support to researchers to request their plasmid resources. As these plasmids have been taken up by the research community, the plasmids have been developed, expanding the resources available to the community. Researchers are encouraged to contribute to this research community by submitting their vector sequence to genbank and providing a description of the plasmid on the site. pGreenI and pGreenII pGreen is the original pGreen plasmid. pGreenII features plasmid backbone modification to improve plasmid stability. T-DNA regions No transformation selection pGreenII 0000: minimal T-DNA with Left and Right border, ''lacZ'' gene for blue/white selection during cloning multiple cloning site derived from pBluescript. pGreenII 62-SK: derived from pGreenII 0000, the ''Lac''Z b ...
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Origin Of Replication
The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by semiconservative replication prior to cell division to ensure each daughter cell receives the full complement of chromosomes. Material was copied from this source, which is available under Creative Commons Attribution 4.0 International License This can either involve the replication of DNA in living organisms such as prokaryotes and eukaryotes, or that of DNA or RNA in viruses, such as double-stranded RNA viruses. Synthesis of daughter strands starts at discrete sites, termed replication origins, and proceeds in a bidirectional manner until all genomic DNA is replicated. Despite the fundamental nature of these events, organisms have evolved surprisingly divergent strategies that control replication onset. Although the specific replication o ...
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Transgene
A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the phenotype of an organism. ''Transgene'' describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism's genetic code. In general, the DNA is incorporated into the organism's germ line. For example, in higher vertebrates this can be accomplished by injecting the foreign DNA into the nucleus of a fertilized ovum. This technique is routinely used to introduce human disease genes or other genes of interest into strains of laboratory mice to study the function or pathology involved with that particula ...
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Genetically Modified Plant
Genetically modified plants have been engineered for scientific research, to create new colours in plants, deliver vaccines, and to create enhanced crops. Plant genomes can be engineered by physical methods or by use of ''Agrobacterium'' for the delivery of sequences hosted in T-DNA binary vectors. Many plant cells are pluripotent, meaning that a single cell from a mature plant can be harvested and then under the right conditions form a new plant. This ability can be taken advantage of by genetic engineers; by selecting for cells that have been successfully transformed in an adult plant a new plant can then be grown that contains the transgene in every cell through a process known as tissue culture. Research Much of the advances in the field genetic engineering has come from experimentation with tobacco. Major advances in tissue culture and plant cellular mechanisms for a wide range of plants has originated from systems developed in tobacco. It was the first plant to be geneti ...
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Antimicrobial Resistance
Antimicrobial resistance (AMR) occurs when microbes evolve mechanisms that protect them from the effects of antimicrobials. All classes of microbes can evolve resistance. Fungi evolve antifungal resistance. Viruses evolve antiviral resistance. Protozoa evolve antiprotozoal resistance, and bacteria evolve antibiotic resistance. Those bacteria that are considered extensively drug resistant (XDR) or totally drug-resistant (TDR) are sometimes called "superbugs".A.-P. Magiorakos, A. Srinivasan, R. B. Carey, Y. Carmeli, M. E. Falagas, C. G. Giske, S. Harbarth, J. F. Hinndler ''et al''Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria... Clinical Microbiology and Infection, Vol 8, Iss. 3 first published 27 July 2011 ia Wiley Online Library Retrieved 28 August 2020 Although antimicrobial resistance is a naturally-occurring process, it is often the result of improper usage of the drugs and management of the infections. Antibiotic resistance is a major subset o ...
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Transfer DNA
The transfer DNA (abbreviated T-DNA) is the transferred DNA of the tumor-inducing (Ti) plasmid of some species of bacteria such as ''Agrobacterium tumefaciens'' and ''Agrobacterium rhizogenes(actually an Ri plasmid)''. The T-DNA is transferred from bacterium into the host plant's nuclear DNA genome. The capability of this specialized tumor-inducing (Ti) plasmid is attributed to two essential regions required for DNA transfer to the host cell. The T-DNA is bordered by 25-base-pair repeats on each end. Transfer is initiated at the right border and terminated at the left border and requires the ''vir'' genes of the Ti plasmid. The bacterial T-DNA is about 24,000 base pairs long and contains plant-expressed genes that code for enzymes synthesizing opines and phytohormones. By transferring the T-DNA into the plant genome, the bacterium essentially reprograms the plant cells to grow into a tumor and produce a unique food source for the bacteria. The synthesis of the plant hormones auxin ...
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