Recombinant DNA Lab: Thinking Like a Biotechnician

In this lab, we wanted to better understand the techniques of recombinant DNA technology and how bacteria can be used to mass produce a protein product, such as insulin for diabetics. We were given paper strips of cell DNA that had to be attached end to end in order to build a model. We also created a plasmid, which is circular DNA that is found in bacteria. Plasmids are naturally resistant to a certain antibiotic; in our case, the plasmid was naturally resistant to the antibiotic tetracycline. 

During the process of transformation, restriction enzymes cut DNA whenever they recognize a specific sequence. It cuts above and below the gene of interest (insulin gene) to "cut out" the gene to be placed into the plasmid. Usually a restriction enzyme makes a jagged cut, creating "sticky ends" which can bond with other DNA. The restriction enzyme also recognizes the same sequence of DNA on the plasmid. In our lab, we used the enzyme Hpa II because the sequence it recognizes matched base pairs on the DNA and one the plasmid and because its restriction site was close to the insulin gene. It cuts the DNA at two sites (above and below the gene) and it cuts the plasmid once (this is where the gene will be inserted. If an enzyme was to cut the plasmid in two places instead of one, part of the plasmid would be removed before the gene was inserted. Now, the enzyme ligase is added, which reattaches sticky ends. At this point, a recombinant plasmid has been created.

Next, you would put the bacteria in a petri dish containing the antibiotic that the plasmid is naturally resistant to. In our case, this would be tetracycline. We wouldn't use any other antibiotic because the plasmid will not be resistant to those. This would test whether the host cells have taken in the plasmid. Only cells with the plasmid will survive. When the bacteria containing the plasmid reproduce, they will begin to produce the gene product (insulin).

This process is vital in our everyday lives for mass producing a protein product that will be useful to us, especially something as important as insulin, which can be used to treat a diabetic patient. Recombinant DNA technologies could also be used for delaying food expiration and making it last longer, as well as resistance to pesticides.