A plasmid vector available today is made with a specific host in mind. For example, if you decide to express a gene in a bacteria, there will be plasmids available with features that suit the particular organism that you wish to transform and they will be different from plasmids used to transfect for example, yeast. However, generally, a plasmid will have at the very least an origin of replication recognizable by the desired organism, a promoter upstream of the multiple cloning site that is recognizable by the organisms, and a selection marker such as an antibiotic resistance gene.
The process of expressing a gene from one organism in another host via plasmid vectors begin with the isolation of the gene from the original organism. For the sake of this example, suppose the insulin gene in humans is the gene of interest. First, beta cells from the Islets of Langerhans will have to be lysed and total RNA will be isolated from the cell. Because DNA is filled with many introns that are hard to get rid of, gene isolation from higher eukaryotes almost always start from the mRNA stage because the introns were already sliced out in mRNA processing. The RNA will be then subjected to reverse-transcriptase polymerase chain reaction with primers specific for the insulin gene. The insulin gene will subsequently be selectively amplified and the reaction mixture can then be purified to contain only cDNA of the insulin gene.
With the purified cDNA, a process called molecular cloning is used to get the gene into the plasmid. The plasmid and the gene are both cut with compatible restriction enzymes. The cuts on the plasmid has to be in the multiple cloning site the the cuts on the gene has to be outside of the open reading frame for the cloning to produce an effective vector. (Review molecular Biology for the necessity of promoters and an intact open reading frame) The cut plasmid and gene fragments are then placed together and ligated. The ligated product should theoretically now contain the gene inside the multiple cloning site directly following the promoter. The promoter may express the gene constitutively or it may be inducible, requiring certain conditions to be met before it is turned on.
The plasmid with the cloned insulin gene can now be transformed into competent bacteria hosts (or yeast if desired, however it will not be as efficient). Competence describes the ability of bacteria to take up DNA from its surroundings. The most commonly used host, E. coli, are artificially made to be competent by treatment with a high concentration calcium solution in a cold environment, while others, such as B. subtilis, are naturally competent. All bacteria can be made competent with electroporation but E. Coli is most often used because of its easily satisfied nutrient requirements and very short generation time. The plasmid and competent E. coli is placed together in a cold environment to initiate the uptake of the plasmid into E. coli cells. The mixture is then heat shocked and bacterial growth medium with the necessary selection agent is added to start the incubation process. If the selection marker on the plasmid is an antibiotic resistance gene, for example ampicillin resistance, a medium with ampicillin will be used to incubate the bacterial culture because only the cells that contain the plasmid will be resistant to the antibiotic while cells that have failed to take up the plasmid will die. The cells can then be incubated for as long as needed and split into different cultures if needed because they now contain the plasmid and will express the gene carried on the plasmid.
1. It can be isolated from the cells
2. It possesses a single restriction site for one or more restriction enzymes.
3.Insertion of a linear molecule at one of these sites does not alter its replication properties.
4.Reinsertion of these vectors to the host cell can identified and selectable.
5.They do not occur free in nature and found in bacterial cells.
In genetic engineering, the bacterial cell takes up the plasmid
It is capable of introducing exogenous genes into plant genomes. T-DNA genes are removed from the Ti plasmid and are replaced with the gene of interest.
Virus and plasmid. Both can insert the target gene into the host's genome.
direct selection possible
it is found in the cytoplasm as a simple circle.
In genetic engineering, the bacterial cell takes up the plasmid
The backbone plasmid serves as a vehicle for carrying and replicating foreign DNA in genetic engineering processes. It provides the necessary elements for DNA replication, such as an origin of replication and antibiotic resistance genes, allowing the foreign DNA to be maintained and expressed in the host organism.
A plasmid is considered recombinant when it contains DNA sequences from two different sources that have been artificially combined, often through genetic engineering techniques like restriction enzyme digestion and ligation. This results in a plasmid with modified or additional genetic material compared to its original form.
It is capable of introducing exogenous genes into plant genomes. T-DNA genes are removed from the Ti plasmid and are replaced with the gene of interest.
Virus and plasmid. Both can insert the target gene into the host's genome.
direct selection possible
The suicide plasmid works by integrating into the host cell's genome and disrupting essential genes, leading to cell death. This allows researchers to selectively eliminate cells that have not successfully incorporated the desired genetic modifications.
That sounds like a recombinant DNA molecule, where two different genetic sequences have been combined and inserted into a plasmid. This technique allows for the creation of new genetic constructs with desired traits or functions. It is commonly used in genetic engineering and biotechnology for a variety of applications.
The Ti plasmid is derived from Agrobacterium tumefaciens, which is a plant pathogen. This plasmid is commonly used as a vector to transfer foreign genes into plant cells in genetic engineering applications.
Agrobacterium-mediated transformation using a plasmid containing a gene for GFP
A plasmid containing a gene for human growth hormone can be used in genetic engineering to produce recombinant human growth hormone. This plasmid can be introduced into host cells, such as bacteria, for the production of the hormone on a large scale.
it is found in the cytoplasm as a simple circle.