Recombinant dna technology steps ppt
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Under laboratory conditions, the isolated plasmids are introduced into bacterial host cells via a process called transformation Figure One of two principal means is generally applied to achieve an experimentally feasible efficiency of transformation:.
Plasmids are then introduced into host cells recombinant dna technology steps ppt a transformation procedure involving co-incubation and a subsequent heat shock step. Plasmids can also be introduced into the host cell via electroporation. During this procedure, cells co-incubated with plasmids are exposed to short pulses of electric shock.
Even with the maintenance of the above conditions facilitating the introduction of plasmids into the host cells, only a very small fraction of bacterial cells will stably take up a plasmid. The identification of transformed—i. The most commonly used selection markers confer resistance to certain antibiotics. The most commonly used antibiotics and the enzymes inactivating these antibiotics are listed below. Appearance of plasmid-containing bacterial colonies on an antibiotic-containing agar nutrient plate.
Ampicillin, a penicillin derivative, inhibits one of the enzymes involved in the synthesis if the bacterial cell wall. The enzyme hydrolyses the recombinant dna technology steps ppt ring of penicillin. The hydrolysed penicillin molecule is ineffective. Tetracycline binds to one of the proteins of the 30S ribosomal subunit. This molecule inhibits the translocation of the ribosome during translation. The tetracycline resistance gene tet r encodes a membrane protein that prevents the entry of the antibiotic into the host cell.
Chloramphenicol binds to the 50S ribosomal subunit, thus inhibiting protein synthesis. The chloramphenicol resistance gene cm r encodes the enzyme chloramphenicol acetyl transferase cat. This enzyme is localised in the cytoplasm, and acetylates chloramphenicol by using acetyl coenzyme A. The modified chloramphenicol molecule is unable to bind to the ribosome. Following transformation, bacterial cells are spread onto a nutrient agar plate containing antibiotics according to the resistance gene contained in the plasmid.
Only the plasmid-containing resistant bacteria will recombinant dna technology steps ppt able to grow on the antibiotic-containing agar plate. Ideally, the appearance of separate bacterial colonies can be observed on the agar nutrient plate Figure Theoretically, one colony contains the descendants clones of a single bacterial cell that had taken up the plasmid. Following the selection recombinant dna technology steps ppt plasmid-containing bacterial cells, the next step is the identification of cells harbouring recombinant plasmids, i.
The most widely applied methodologies for the identification of recombinant colonies are the following:. If the foreign DNA segment the insert is inserted within an antibiotic resistance recombinant dna technology steps ppt, the successful insertion will result in the loss of the corresponding antibiotic resistance.
By using this method, the identification of recombinant colonies is not straightforward, as it requires the application of replica techniques. Numerous vectors contain a segment of the E. Bacterial colonies containing recombinant insert-containing plasmids will thus produce white colonies, while the ones lacking the insert will produce blue colonies.
Recombinant bacterial colonies can also be identified by applying a polymerase chain reaction PCR, Chapter The presence of the insert in the plasmid constructs can also be assessed via restriction endonuclease digest of the isolated plasmids. The presence of the insert in the plasmid constructs can also be assessed via sequencing of the affected region. Introduction of recombinant DNA constructs into host cells and the identification of recombinant colonies.
Introduction of plasmids into the host cell and their amplification therein. Isolation of plasmid DNA.