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Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. This takes place through a pilus. It is a parasexual mode of reproduction in bacteria.
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.
An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins.
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.
Agrobacterium tumefaciens is the causal agent of crown gall disease in over 140 species of eudicots. It is a rod-shaped, Gram-negative soil bacterium. Symptoms are caused by the insertion of a small segment of DNA, from a plasmid into the plant cell, which is incorporated at a semi-random location into the plant genome. Plant genomes can be engineered by use of Agrobacterium for the delivery of sequences hosted in T-DNA binary vectors.
Agrobacterium is a genus of Gram-negative bacteria established by H. J. Conn that uses horizontal gene transfer to cause tumors in plants. Agrobacterium tumefaciens is the most commonly studied species in this genus. Agrobacterium is well known for its ability to transfer DNA between itself and plants, and for this reason it has become an important tool for genetic engineering.
The transfer DNA is the transferred DNA of the tumor-inducing (Ti) plasmid of some species of bacteria such as Agrobacterium tumefaciens and Agrobacterium rhizogenes . 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.
In genetic engineering, a gene gun or biolistic particle delivery system is a device used to deliver exogenous DNA (transgenes), RNA, or protein to cells. By coating particles of a heavy metal with a gene of interest and firing these micro-projectiles into cells using mechanical force, an integration of desired genetic information can be introduced into desired cells. The technique involved with such micro-projectile delivery of DNA is often referred to as biolistics, short for "biological ballistics".
A tumour inducing (Ti) plasmid is a plasmid found in pathogenic species of Agrobacterium, including A. tumefaciens, A. rhizogenes, A. rubi and A. vitis.
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.
Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene delivery must reach the genome of the host cell to induce gene expression. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.
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.
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.
Acetosyringone is a phenolic natural product and a chemical compound related to acetophenone and 2,6-dimethoxyphenol. It was first described in relation to lignan/phenylpropanoid-type phytochemicals, with isolation from a variety of plant sources, in particular, in relation to wounding and other physiologic changes.
Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.
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.
Transient expression, more frequently referred to "transient gene expression", is the temporary expression of genes that are expressed for a short time after nucleic acid, most frequently plasmid DNA encoding an expression cassette, has been introduced into eukaryotic cells with a chemical delivery agent like calcium phosphate (CaPi) or polyethyleneimine (PEI). However, unlike "stable expression," the foreign DNA does not fuse with the host cell DNA, resulting in the inevitable loss of the vector after several cell replication cycles. The majority of transient gene expressions are done with cultivated animal cells. The technique is also used in plant cells; however, the transfer of nucleic acids into these cells requires different methods than those with animal cells. In both plants and animals, transient expression should result in a time-limited use of transferred nucleic acids, since any long-term expression would be called "stable expression."
Patricia C. Zambryski is a plant and microbial scientist known for her work on Type IV secretion and cell-to-cell transport in plants. She is also professor emeritus at the University of California, Berkeley.
The root inducing (Ri) -plasmid of Rhizobium rhizogenes is a plasmid capable of undergoing horizontal gene transfer of its transfer DNA (T-DNA), upon contact with a plant host. The T-DNA of the Ri-plasmid affects the plant host in such a way, that gene expression is altered, especially in regard to phytohormonal balances, metabolism and certain phenotypical characteristics.
References
- ↑ Lee LY, Gelvin SB (February 2008). "T-DNA binary vectors and systems". Plant Physiology. 146 (2): 325–32. doi:10.1104/pp.107.113001. PMC 2245830 . PMID 18250230.
- 1 2 Hoekema A, Hirsch PR, Hooykaas PJ, Schilperoort RA (May 1983). "A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid". Nature. 303 (5913): 179–180. Bibcode:1983Natur.303..179H. doi:10.1038/303179a0. S2CID 4343344.
- ↑ "As I remember, the "binary" refers to the function of interest being divided into two parts encoded by two separate plasmids rather than two bacterial hosts: we used the term "shuttle vectors" to refer to the multiple host property." (P. R. Hirsch, personal communication to T. Toal, Feb 27, 2013)
- 1 2 3 Slater A, Scott N, Fowler M (2008). Plant Biotechnology the genetic manipulation of plants. New York: Oxford University Press Inc.
- ↑ Bevan M (November 1984). "Binary Agrobacterium vectors for plant transformation". Nucleic Acids Research. 12 (22): 8711–21. doi:10.1093/nar/12.22.8711. PMC 320409 . PMID 6095209.
- ↑ Hajdukiewicz P, Svab Z, Maliga P (September 1994). "The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation". Plant Molecular Biology. 25 (6): 989–94. doi:10.1007/BF00014672. PMID 7919218. S2CID 9877624.
- 1 2 Xiang C, Han P, Lutziger I, Wang K, Oliver DJ (July 1999). "A mini binary vector series for plant transformation". Plant Molecular Biology. 40 (4): 711–7. doi:10.1023/a:1006201910593. PMID 10480394.
- ↑ "List of legacy pCAMBIA vectors – Cambia" . Retrieved 2020-08-10.
- ↑ Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (April 2000). "pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation". Plant Molecular Biology. 42 (6): 819–32. doi:10.1023/a:1006496308160. PMID 10890530.
- 1 2 Lee S, Su G, Lasserre E, Aghazadeh MA, Murai N (May 2012). "Small high-yielding binary Ti vectors pLSU with co-directional replicons for Agrobacterium tumefaciens-mediated transformation of higher plants". Plant Science. 187: 49–58. doi:10.1016/j.plantsci.2012.01.012. PMID 22404832.
- 1 2 Pasin F, Bedoya LC, Bernabé-Orts JM, Gallo A, Simón-Mateo C, Orzaez D, García JA (October 2017). "Multiple T-DNA Delivery to Plants Using Novel Mini Binary Vectors with Compatible Replication Origins". ACS Synthetic Biology. 6 (10): 1962–1968. doi:10.1021/acssynbio.6b00354. PMID 28657330.
- ↑ Hellens R, Mullineaux P, Klee H (October 2000). "Technical Focus:a guide to Agrobacterium binary Ti vectors". Trends in Plant Science. 5 (10): 446–51. doi:10.1016/s1360-1385(00)01740-4. PMID 11044722.
- ↑ "pGreen on the Web". www.pgreen.ac.uk.