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Transformation (Genetics)Transformation is the genetic alteration of a cell resulting from the introduction, uptake and expression of foreign genetic material (DNA or RNA). This is a common technique in molecular biology. The effect was first demonstrated in 1944 by Oswald Avery, Collin Macleod, and Maclyn McCarty, who first demonstrated gene transfer in Streptococcus pneumoniae. Oswald, Avery and McCarty call the uptake and incorporation of DNA by bacteria transformation. More genererally the term is used to describe mechanisms of DNA and RNA transfer in molecular biology. For example the production of transgenic plants like genetically modified maize requires the insertion of new genetic information into the maize genome using an appropriate mechanism for DNA transfer. Historical context Transfer mechanisms Bacterial Bacterial transformation is comparatively easy and simple. These methods can be used to genetically engineer bacteria: Heat shocking (42C for 90-120 seconds) and exposure of cells to divalent cations such as CaCl2 makes the cell walls permeable to plasmid DNA. A plasmid DNA will usually contain an antibiotic resistance gene which is placed in a bacterial strain, that has no antibiotic resistance. Therefore, only transformed bacteria can grow on a media with the antibiotic (this is known as a selection medium). One example of this is putting in a plasmid that contains the encoding for the protein -lactamase, which makes bacteria resistant to ampicillin. This is called the bla gene. The bacterial colony is then treated with ampicillin, thus weeding out those bacteria who did not take up the plasmid with the bla gene. Another selection medium is bioluminescence, using a gene taken from jellyfish. A second way of bacterial transformation is Conjugation, effictively bacterial sex, the exchange of genetic material occurs through the transfer of plasmid DNA. This is a natural way for bacteria to exchange genetic material. Electroporation is a way to make holes in cells with an electric field of 100-200V. Now plasmid DNA can enter the cell through these holes. Natural repair mechanisms will close these holes afterwards. Transduction, is the transfer of genetic material by a bacteriophage, viruses that infect bacteria. Yeasts and Fungi These methods are currently known to transform yeasts: - High Efficency Transformation according to Gietz, R. D. and R. A. Woods. 2002 TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.
- Two Hybrid System Protocol: The two-hybrid system involve the use of two different plasmids in a single yeast cell. One plasmid contains a cloned gene or DNA sequence of interest while the other plasmid contains a library of genomic or cDNA. http://www.umanitoba.ca/faculties/medicine/biochem/gietz/2HS.html
- Rapid Transformation Protocol allows for transformation with any yeast cell source. See Gietz/Wood above.
- Frozen Yeast Protocol allows you to prepare frozen yeast cells that are competent for transformation after thawing.
Plants A number of mechanisms are available to transfer DNA into an organism, these include: - Agrobacterium mediated transfomation is the easiest and most simple plant transformation. Plant tissue, often leaves, are cut in small pieces, eg. 10x10mm and soaked for 10 minutes in an agrobacterium. Some cells along the cut will be transformed by the bacterium, that inserts its DNA into the cell. Placed on selectable rooting and shooting media, the plants will regrow. Unfortunately, many plants are not transformable by this method.
- Particle bombardment: Coat small gold or tungsten particles with DNA and shoot them into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. This method also allows transformation of plant plastids. The transformation efficiency is lower then in agrobacterial mediated transformation, but most plants can be transformed with this method.
- Electroporation: make holes in cell walls using electricty, that allows DNA to enter.
- Viral transformation: Package your genetic material into a virus and let it deliver the genetic material to its host cell.
Animals - Microinjection: use a thin needle and inject the DNA directly in the core of embryonic cells.
- Viral transformation: Package your genetic material into a virus and let it deliver the genetic material to its host cell.
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