Genetic Engineering. First, the nucleus of human cells are burst. Human cell. Nucleus. Genetic Engineering. The chromosomes are cut up into small fragments and the required gene identified. Fragment containing required gene. Chromosome fragments. Genetic Engineering.
First, the nucleus of human cells are burst
The chromosomes are cut up into small fragments and the required gene identified.
Fragment containing required gene
Next the fragments are spread out and the required one isolated.
Segment with required gene
Bacterial cell wall
Structure of a typical bacterium
Plasmids are loops of DNA separate from the main chromosome. They carry genes for things like antibiotic resistance. This makes them very useful to theGenetic engineer.
In the above plasmid, the YELLOW gene is one that gives the bacterium resistance to one antibiotic (eg Penicillin).
The GREEN gene gives resistance to a different antibiotic (eg Tetracycline)
By using special enzymes, we can make a cut in the midst of ONE of theseantibiotic resistance genes.In this example, we will cut open the ‘T’ gene
Prepared human gene
Next, we introduce the prepared HUMAN gene to the mixture. If all goes according to plan, the human gene will fit into the cut in the plasmidso that the green ‘T’ gene will no longer work correctly.
No P or T gene
P and T
Genes intactGenetic Engineering
As plasmids are extremely small, we cannot tell by looking which ones have gotthe human gene in the right place. We need to use a ‘shotgun’ approach andincubate thousands of plasmids with hundreds of bacterial cells
Cell with P and T intact
Cell with neither P or T
Some cells will take up the recombinant plasmid, some will take up original plasmids, others will take up no plasmds at all or ones without antibioticresistance genes.
An agar plate containing Penicillin is used to allow only those cells which havetaken up a suitable plasmid to survive and divide. These cells must have resistanceto Penicillin
Next, these colonies are sub-cultured onto agar containing tetracycline. Only cells resistant to BOTH antibiotics will be able to grow. We are interested in those cells which WON’T grow in the presence of Tetracycline
This colony will probably have the correct plasmid to produce the product from thehuman gene. Cells from this colony will be grown on a large scale and the mediumanalysed for the presence of the product from the human gene, eg growth hormone