First locate the gene of interest and organism to insert the gene into, which was the insulin gene in this lab. Then, obtain a plasmid from bacteria and what is its antibiotic resistance. In this lab, our plasmid was resistant to ampicillin.
Next, find a restriction enzyme that makes one cut in the plasmid and one above and below the gene of interest so it can be taken out and inserted into the plasmid. Restriction enzymes cut DNA wherever they find a specific sequence. It cuts using "sticky ends" so that the DNA base pairs can easily bond together. We used Hin dIII because it made only one cut in the plasmid and the closest cuts above and below the insulin gene. If the enzyme had made two cuts in the plasmid, then the DNA would break apart, resembling two unattached semicircles.
Then, add ligase to stick the ends of the gene into the plasmid. After that, plate bacteria on petri dish with the antibiotic mixed. We could use ampicillin since the plasmid we chose to insert the insulin gene into was resistant to it, and only those with ampicillin resistance would survive; therefore, those would be the ones with the insulin gene. We would not use tetracycline or kanamycin because our genetically modified bacteria would be killed because they are not resistant to these antibiotics. Finally, we would transfer the bacteria to a "broth" so they will multiply quickly and then extract and purify the protein the inserted gene produced.
This process is important in our everyday lives because it provides people who cannot produce a certain protein with a way to get it into their bodies, for example diabetic patients cannot produce enough insulin, but because of recombinant DNA they can take insulin injections or pills. This process could also be used to make vaccines and make crops resistant to pesticides or enhance their flavor and nutritional content.
Next, find a restriction enzyme that makes one cut in the plasmid and one above and below the gene of interest so it can be taken out and inserted into the plasmid. Restriction enzymes cut DNA wherever they find a specific sequence. It cuts using "sticky ends" so that the DNA base pairs can easily bond together. We used Hin dIII because it made only one cut in the plasmid and the closest cuts above and below the insulin gene. If the enzyme had made two cuts in the plasmid, then the DNA would break apart, resembling two unattached semicircles.
Then, add ligase to stick the ends of the gene into the plasmid. After that, plate bacteria on petri dish with the antibiotic mixed. We could use ampicillin since the plasmid we chose to insert the insulin gene into was resistant to it, and only those with ampicillin resistance would survive; therefore, those would be the ones with the insulin gene. We would not use tetracycline or kanamycin because our genetically modified bacteria would be killed because they are not resistant to these antibiotics. Finally, we would transfer the bacteria to a "broth" so they will multiply quickly and then extract and purify the protein the inserted gene produced.
This process is important in our everyday lives because it provides people who cannot produce a certain protein with a way to get it into their bodies, for example diabetic patients cannot produce enough insulin, but because of recombinant DNA they can take insulin injections or pills. This process could also be used to make vaccines and make crops resistant to pesticides or enhance their flavor and nutritional content.
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