What is the role of a plasmid in genetic engineering?

Answer 1

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.

• A plasmid can be considered as a suitable vehicle for cloning, because

  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.

• Ex: for plasmid cloning vectors are pBr322,pACYC18,pUC,pUN121.

Answer 2

A plasmid is a small DNA molecule that is physically separate from, and can replicate independently of, chromosomal DNA within a cell. In nature, they carry genes that may benefit survival of the organism and can frequently be transmitted from one bacterium to another by horizontal gene transfer.

Answer 3

Plasmids are DNA molecules, usually circular, that are independent of the chromosomal DNA. In bacteria these can replicate independently as well.

In genetic engineering, plasmids are called Vectors, and are used to isolate and multiply a specific gene. which, because they are independent of chromosomal DNA, can be transferred into other organisms.

Answer 4

Biotechnology is shortened as biotech. It is the use of microorganisms as a living factories in the manufacturing of important product. Plasmid is a small extrachromosomal genetic materia that can replicate on its own. Bacteria plasmid is used in the manupulation of DNA, it can confer on bacteria a property such as drug resistance.

Answer 5

First you either find restriction sites within the gene, or add some to the ends using custom primers. Then you digest the gene with restriction enzymes specific to those sites. You can choose to make them different if the gene's orientation in the vector is important. You must also digest the vector with the same enzymes. You mix the two together in a ratio by molarity (I think?). 3:1 ratio of insert to vector is common. Then you ligate those puppies together using DNA ligase. You then will probably have to run them on a gel to find plasmids with single insertion and gel extract them.

Answer 6

Plasmids are typically used to introduce foreign DNA into a host cell. This can be done a multitude of ways, one of which is electroporation.

Answer 7

the way moves

Answer 8

by smiling