Word: plasmids
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Dates: during 1970-1979
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...special trick to make the experiment work," Gilbert says. The insulin gene is inserted into a plasmid at a site before the end of another gene, which codes for a protein known as penicillinase. The insulin and penicillinase proteins are synthesized in a fused form when the genes are read together...
...genes will be read and translated into protein. So Doty and other researchers are interested in determining what DNA sequence changes accompany changes in production levels of the proteins. "One has to have a probe that is either highly specific or easily labeled," according to Doty. This is where plasmids and genetic engineering comes in. Once a DNA segment is inserted into a bacterial plasmid, the researchers can grow up a supply of it "overnight." This may be used to identify complementary copies of RNA and to determine where sequence changes occur...
Genes from other organisms [3] are inserted into the DNA of E. coli bacteria which copies and decodes DNA rapidly. A ring of DNA--a plasmid--which is transferred between bacteria, is used for the incorporation procedure. It is easily isolated from a bacterial cell [1], cut open [2] and used as a receptor for a foreign gene [4]. The plasmid then carries the inserted DNA into a cell [5] where many copies can be "cloned...
After the plasmids are separated from the chromosomal DNA in a centrifuge, they are placed in a solution with a chemical catalyst called a restriction enzyme. This enzyme cuts through the plasmids' DNA strips at specific points. It leaves overlapping, mortise-type breaks with "sticky" ends. The opened plasmid loops are then mixed in a solution with genes-also removed by the use of restriction enzymes-from the DNA of a plant, animal, bacterium or virus. In the solution is another enzyme called a DNA ligase, which cements the foreign gene into place in the opening of the plasmids...
Finally, the chimeras are placed in a solution of cold calcium chloride containing normal E. coli bacteria. When the solution is suddenly heated, the membranes of the E. coli become permeable, allowing the plasmid chimeras to pass through and become part of the microbes' new genetic structure. When the E. coli reproduce, they create carbon copies of themselves, new plasmids -and DNA sequences-and all. Thus they become forms of life potentially different from what they had been before-imbued with characteristics dictated not only by their own E. coli genes but also by genes from an entirely different...