Plasmids are circular DNA found in bacteria that replicate independently of the host’s DNA. Recombinant plasmids are used to clone genes and express large amounts of known genes. Transformation and electroporation introduce DNA into cells. Recombinant plasmids require a selectable marker, origin of replication, and special sequences for restriction enzymes. New kits use engineered bacterial strains for easy purification of the protein expressed by the gene.
A plasmid is a circular piece of DNA found in many bacteria. The most notable feature of plasmids is that they replicate independently of the host’s main DNA. Often a plasmid is used in recombinant cloning technology to clone newly isolated genes. It is also very common to use a recombinant plasmid to express large amounts of a known gene to obtain RNA or protein from it. Such recombinant gene expression has been indispensable to the biotech industry.
Recombinant plasmids were first developed in the laboratory rat of the bacterial world, Escherichia coli. Many other types of bacteria can host such plasmids. These self-replicating DNA fragments can naturally transfer between different types of bacteria. Despite this, it was sometimes difficult to introduce the recombinant plasmids into other types of bacteria.
The main procedure for introducing DNA into other cells is known as transformation, in which bacteria are treated with chemicals that make them more likely to take up foreign DNA. Another technique involves shocking the bacteria with an electric current. This is known as electroporation.
The reasons for creating a recombinant plasmid vary. Often when DNA is first isolated from a particular tissue or organism, it is transformed into plasmids to create a library. Then DNA can be extracted from single colonies. Next, they can be screened by DNA sequencing to determine what types of genes are present, if the sequences are present in a database. Sometimes genes with unknown functions are cloned.
In other cases, the gene product is well known, but researchers wish to express large quantities of it for further study. The gene can be cloned into recombinant plasmids which are overexpression vectors. They are specially designed to produce large quantities of RNA or protein. This has been especially valuable for recombinant human proteins, which were previously often only available from cadavers, making studying the function of a particular gene very difficult.
Several factors are involved in constructing a plasmid that can be used in molecular cloning. The plasmid must have a selectable marker. This makes it possible to select a cell with the gene. Normally, the population of cells that lack the gene with the marker far exceeds the amount of cells that carry it. Generally a recombinant plasmid has resistance to an antibiotic, or it can grow in the absence of a particular amino acid.
Such a plasmid needs an origin of replication so that it can start synthesizing its recombinant DNA. Additionally, a recombinant plasmid requires a series of special sequences for a restriction enzyme to cleave DNA to allow a gene to be inserted into the cloning vector. There are a large number of highly specialized restriction enzymes for specific DNA sequences that must be present where the gene begins and ends.
Traditional strains of bacteria have been used for decades for DNA cloning. Additionally, there are new kits that use specially engineered bacterial strains to facilitate overexpression of the gene product. They combine the technology for cloning a gene with a method that allows easy purification of the protein expressed by the gene once it has been cloned into recombinant plasmid.
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