Plasmids are circular DNA found in bacteria and used in genetic engineering, particularly gene cloning. The process involves isolating the gene and plasmids, cutting and inserting the gene into the plasmid, and then inserting the plasmid into a bacterium for replication. This process is used to create human insulin.
Within many different bacteria, small circular pieces of DNA can be found in the cytoplasm. These circles of DNA are known as plasmids and are separated by chromosomal DNA or the DNA that carries the genes for bacterial cells. Several copies of plasmids are often present in the bacterial cell at the same time. Plasmids play a very important role in genetic engineering, especially gene cloning.
When genes are cloned, the process usually takes place inside the bacteria. To get the gene that needs to be cloned into bacteria, a vector is needed. A plasmid is what is used as a carrier, as it can easily pass from one cell to another.
There are a number of steps involved in gene cloning before inserting a plasmid into a host cell. First, the gene that is to be copied must be isolated, as well as the plasmids that are to be used as vectors. Once this is done, the gene must be inserted into the plasmid DNA. The plasmid is then inserted into the bacterial host cell for replication.
To isolate plasmids from bacterial cells, the cells must initially be treated with enzymes to break down the cell walls of the bacteria. The larger chromosomal DNA is separated from the smaller plasmids using a centrifuge. The isolated plasmid DNA is now ready to be inserted into the gene.
Plasmids consist of a circle of double-stranded DNA. To insert the desired gene, the plasmid DNA is cut with restriction enzymes. These enzymes cut DNA only at very specific nucleotide sequences. Once the plasmid DNA has been cut, linker sequences are added to the free ends which are related to the ends of the gene to be inserted. This ensures that the gene fits exactly in the plasmid.
Once the gene has been inserted into the plasmid, it is now ready to be inserted into a living bacterium. Bacteria replicate their plasmids so that a single cell can contain many copies. There can be up to 200 copies of a single plasmid within a bacterium. If the plasmid is introduced into many bacterial cells, many copies of the gene can be produced relatively quickly, particularly as bacterial cells replicate approximately every 20 minutes.
This is the process used to create human insulin. The gene coding for insulin has been isolated and inserted into a plasmid. All of the plasmids containing the insulin gene were then introduced into a bacterium, where they replicated. The bacteria then continued to replicate, so that many millions of cells containing the insulin gene can be created in a very short time. This cloned gene now provides a reliable source for human insulin.
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