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