The term plasmid was first introduced by the American molecular biologist JoshuaLederberg in 1952. Plasmid is an extra chromosomal DNA molecule that is capable of replicating independently from the chromosomal DNA and is generally circular and double-stranded. Plasmids occur naturally in bacteria and in some eukaryotic organisms, and their size varies from 1 to over 1,000 kilobase pairs. Plasmids of more than 50 kb might be characterized as large plasmids, whereas plasmids used as tools in molecular genetics are often smaller than 10 kb.
Plasmids that are used in genetic engineering are called vectors. The gene to be replicated is inserted into copies of a plasmid that contain genes for making cells resistant to particular antibiotics. Then the plasmids are inserted into bacteria by a process called transformation. The inserted genes express and the resulting proteins break down antibiotics. Thus the antibiotics act as filter to select only the modified bacteria. Now these bacteria can be grown in large numbers, harvested and lysed to isolate the plasmid of interest. Another major use of plasmids is to produce large amount of proteins of interest by growing bacteria containing a plasmid harbouring genes for that protein. This is a cheap and easy way of mass-producing proteins such as insulin or even antibiotics.
PLASMID ISOLATION
The techniques for plasmid isolation are based on the fact that plasmids usually occur in the covalently closed circular configuration within the host cells. After gentle cell lysis all intracellular macromolecules must be eliminated, while plasmid DNA is purified. The smaller a plasmid is the easier is its isolation.
Alkaline Lysis: In this method, bacteria are lysed in a solution that contains sodium dodecylsulfate (SDS) that denatures proteins and breaks the cell membrane. Then sodiumhydroxide (NaOH) is added to make the solution alkaline and to cause the plasmid and the genomic DNA to denature into single strands. The solution is then brought to a neutral pH by adding potassiumacetate. The genomic DNA being very large is unable to reanneal into double-strands and hence forms a precipitate. However, the plasmid DNA being much smaller, reanneals into double strands when potassium acetate is added, and therefore remains in dissolved state in the solution. Potassium acetate also precipitates the SDS and most bacterial proteins and lipids that are bound to it. The contents are then spun in a centrifuge and the supernatant that contains the plasmids is removed from the precipitate containing genomic DNA.
Boiling Lysis: This method is similar to alkaline lysis except that high temperatures are used to denature the plasmid and genomic DNAs. The bacteria are placed in boiling water bath which lyses cells and denatures DNA. The solution is then cooled, causing the plasmid DNA to reanneal and remain in solution while the genomic DNA precipitates. The solution is then spun in a centrifuge and the supernatant liquid containing plasmid DNA is removed. This technique appears to be older and used much less frequently than the alkaline lysis.
TYPES OF PLASMIDS
F-Plasmids or F-factors
These plasmids play a major role in conjugation in bacteria. They are in the form of circular DNA molecules containing about 99,159 base pairs. One region of the plasmid contains genes involved in regulation of the DNA replication (repgenes), while the other region contains transposable elements (IS3 Tn 1000, IS3 and IS2 genes), which give it an ability to function as episome. The third larger region, called the tra region, consists of tragenes which promote the transfer of plasmids during conjugation. Example is F-plasmid of E. coli.
R-Plasmids
These are the most widespread and well studied group of plasmids that confer resistance to antibiotics. R-plasmids typically have genes that code for enzymes that are able to destroy and modify antibiotics. Some R-plasmids possess only a single resistance gene whereas others can have as many as eight. Plasmid R 100, for example, is a 94.3 kilobase-pair plasmid that carries resistance genes for sulfonamides, streptomycin, spectinomycin, chloramphenicol, tetracyclin etc. Many R-plasmids are conjugative and possess drug-resistant genes in the form of transposable elements, they play an important role in medical microbiology as their spread through natural populations can have profound consequences in the treatment of bacterial infections. For example, pAMP plasmid that has 4539 base pairs, has a gene ampr that confers resistance to the antibiotic ampicillin; pKAN plasmid has 4207 base pairs and the gene kanr that confers resistance to the antibiotic kanamycin.
Virulence-Plasmids
These plasmids confer pathogenesity on the host bacterium and make the bacterium better able to resist the host defence system or produce toxins. For example, Ti-plasmid of Agrobacterium tumefaciens induces crown gall disease in angiosperm plants. Similarly, enterotoxilgenic strains of E. coli cause traveller’s diarrhoea because of plasmid that code for an enterotoxin which induces extensive secretion of water and salts into the bowel.
Col-Plasmids
These plasmids carry genes that confer to the host bacterium ability to kill other bacteria by secreting a type of protein called bacteriocin, which often kills cells by creating channels in the plasma membrane thus increasing its permeability. They also degrade DNA or RNA or attack peptidoglycan and weaken the cell-wall. Bacteriocins act only against closely related strains of bacteria. For example, Col E1 plasmid of E. coli codes for the synthesis of bacteriocin called colicin, which kills other susceptible strains of E. coli. Col plasmids of some E. coli strains code for the synthesis of bacteriocin, namely cloacin that kills Enterobacter species. Lactobacillus bacteria produce bacteriocin called NisinA, which strongly inhibits the growth of a wide variety of gram (+) bacteria and hence is used as a preservative in food industry.
Metabolic Plasmids
Metabolic plasmids, also called degradative plasmids, possess genes coding for enzymes that degrade unusual substances such as aromatic compounds, pesticides and sugars, e.g. TOL plasmid of Pseudomonas putida. However, some metabolic plasmids occurring in certain strains of Rhizobium induce nodule formation in legumes, where they carry out fixation of atmospheric nitrogen.
USES OF PLASMIDS
The earliest use of plasmids in pharmaceutical manufacturing is the use of recombinant DNA technology to modify Escherichia coli bacteria to produce human insulin, which was performed at Genentech in 1978. Genentech researchers produced artificial genes for each of the two protein chains that comprise the insulin molecule. The artificial genes were then recombined into plasmids and inserted into Escherichiacoli, which were induced to produce 100,000 molecules of chain A or chain B human insulin. The two protein chains were then combined to produce insulin molecules.
In 1979, scientists at Genentech produced human growth hormone by inserting DNA fragment coding for human growth hormone into a plasmid that was implanted in Escherichiacoli. The gene that was inserted into the plasmid was created by reverse transcription of the mRNA found in pituitary glands to complementary DNA.
Using recombinant DNA technology, blood clotting factor IX was produced using transgenic Chinese hamster ovary cells in 1986. Plasmids containing the Factor IX gene, along with plasmids with a gene that codes for resistance to methotrexate, were inserted into Chinese hamster ovary cells via transfection, which produced significant quantities of Factor IX, which was shown to have substantial coagulation properties. In 1992, the factor VIII was also produced using transgenic Chinese hamster ovary cells.
Many human genes have been cloned in E. coli and in yeast, which has made it possible to produce large quantities of human proteins in vitro. Cultured cells of E. coli and yeast or mammalian cells transformed with a human gene are being used to manufacture more than 100 products for human therapy today. For example, Insulin for diabetics; factorVIII for males suffering from haemophilia A; factorIX for haemophilia B; human growthhormone; erythropoietin for treating anaemia; several interferons; several interleukins; granulocyte-macrophage colony-stimulating factor for stimulating the bone marrow after a bone marrow transplant; tissue plasminogen activator for dissolving blood clots; adenosinedeaminase for treating severe immunodeficiency; parathyroidhormone; several monoclonal antibodies; hepatitis B surface antigen to vaccinate against the hepatitis B virus; C1 inhibitor used for the treatment of hereditary angioneurotic oedema.