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Plasmid as Vectors For Plant TransformationBY: Lorato Lekgari | Category: Biotech-Research | Submitted: 2010-07-22 17:53:02
Article Summary: "Plasmids are found in microorganism mainly in bacterial cells. They are naked double stranded DNA molecules that replicate independently of their host's DNA but cannot live outside the host. .."
Plasmids are found in microorganism mainly in bacterial cells. They are naked double stranded DNA molecules that replicate independently of their host's DNA but cannot live outside the host. They are not parasitic and can give desirable traits to their host. The most known of these being resistance to antibiotics that would otherwise kill the organism but the antibiotic resistant genes found in the plasmid contained in the organism will make the organism to be tolerant and thus resist the activity of the antibiotic. They are transferrable from one host to another as long as the host is suitable. This makes them a very important tool in biotechnology.
Plasmids as Transformation Vectors
Plasmids that are used for genetic engineering or transformation of organisms are referred to as vectors. The vectors used for plant transformation have to be expression vectors as the gene they carry have to be expressed and be transcribed to mRNA in order to produce the proteins needed to give plants a desired phenotype or produce the compound of interest. Vectors used for transformation of bacterial cells are referred to as cloning vectors. In order to be used for transformation the gene of interest first has to be inserted into the plasmid. This is done by cutting the gene of interest with restriction enzymes and then the plasmid is also cut to make way for insertion of the gene, also using restriction enzymes. After successfully ligating the gene into the plasmid vector, it is cloned into bacterial (other systems such as fungi can be used) to multiply it. It is then isolated from the bacteria and used for transformation of plants. In some plant transformation techniques, the plasmid is isolated as it is such as in particle bombardment and for Agrobacterium-mediated transformation, the vector is transferred from the host bacteria such as Escherichia coli, where it was multiplied, into Agrobacterium before being used for transformation.
Common Features of Vectors
Vectors are made of different components that allows for it to be used successfully for genetic transformation. These are
Strong promoter: This is necessary fro the gene to be transcribed. The cauliflower mosaic virus 35S (CamV35S) promoter and maize ubiquitin promoter (Ubi) are used usually for dicotyledonous and monocotyledonous plant transformation respectively.
Multiple cloning sites: This is a site with sites where restriction enzymes can be used to cut the vector in order for the gene to be inserted and ligated into the vector.
Origin of replication: To allow the vector to be able to replicate independently within its host thus multiplying the gene inserted into it.
Selectable markers: This site allows for the selection of bacteria that carries the particular vector. Antibiotic resistance genes are usually used, but other markers can be used. To select for bacteria carrying the vector of interest, it is plated onto media supplemented with the antibiotic and the bacteria that carries the plasmid will be able to survive and multiply in this media and the ones that do not harbor the plasmid will not survive in the same media.
These features of the plasmid make it an important part of genetic engineering not only in plants but in other organisms as well. Plasmids survive only in science or research laboratories. They can be manipulated to include multiple numbers of genes of different genes, so that one transfer result in multiple desirable traits in the plant or any other organism transformed by the particular vector.
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