Genetically Engineering Lab

rDNA/ Genetic Engineering Lab

Introduction: Diabetics require extra insulin to live a normal life. The insulin they need has been obtained from cow and pig sources in the past. This type of insulin is very expensive and often can carry proteins, which cause allergic reactions. Recently, scientists have perfected a method to combine the human gene for insulin with bacterial DNA. This type of DNA is called Recombinant DNA. Once the gene for insulin is combined with the bacterial DNA the bacteria can then produce human insulin. This insulin is cheaper to produce and is relatively free of allergens (proteins which may cause allergic reactions)

Bacteria often contain small circular pieces of DNA called plasmids. In addition to their regular DNA, these plasmids frequently carry genes for antibiotic resistance. They are also easily separated from bacteria and easily reabsorbed into bacteria. Because of these properties, scientists use plasmids as a vector (carrier) for the insulin gene. Once the plasmids are absorbed into bacteria, their DNA, including the added insulin DNA, will be translated into products by the bacteria in which they reside. The bacteria which absorbed the plasmid are identified by their altered resistance to antibiotics and grown in large quantities. The insulin they produce is purified and marketed.

This method of genetic engineering is a window to the future. Many genetically engineered organisms have been created so far such as oil slick digesting bacteria and bacteria which confer insect resistance to their plant hosts. The future of recombinant DNA also holds the promise of correcting genetic disorders in humans through gene therapy (insertion of normal genes to overcome detrimental products or lack of products from a mutated gene). The Human Genome Project has paved the way for this type of gene therapy by sequencing the entire human DNA.

Purpose: In this activity you will construct a simple model of the process of genetic recombination (gene transfer) which demonstrates the insertion of an insulin gene into a bacterial plasmid.

Materials: Lab packet (including DNA sheet) Scissors Tape Method: 1. Color the gene for insulin, the gene for tetracycline resistance, and the gene for ampicilin resistance using different colors for each. Make a key to indicate which color was used for each gene. 2. Cut out the DNA strips. Assemble the plasmid DNA in a circle by taping the ends together. Leave the insulin gene flat. 3. Draw a diagram of the circular plasmid DNA including base pairs. Color the areas of antibiotic resistance using the color key created in step 1 of this lab. 4. Restriction enzymes are used to cut DNA at specific known points in the base pair sequence. These enzymes don’t just chop the DNA molecule in half; they cut into one strand and then cut between base pairs before cutting the DNA creating a “sticky end”. The enzyme used to cut open the plasmid DNA is also used to cut out the insulin gene from a segment of human DNA 5. a. Diagram the 4 base pair sequence of your restriction enzyme. b. Indicate the cutting line you need to make in the plasmid and human DNA by drawing a dotted line 6. Match your restriction enzyme to 2 locations on foreign DNA and 1 location on the plasmid. Draw a line where to cut using the restriction enzyme as a template. Cut your DNA at the base sequence that you matched to the restriction enzyme site. Be sure to cut in the direction of the arrow. You will use both sides of restriction enzyme to make cuts above and below insulin gene and only one cut to open the plasmid. You should have “sticky ends” on both the plasmid and the human DNA 7. Once the “sticky ends” are matched up between the insulin gene and the plasmid DNA, a ligase enzyme joins the pieces. 8. Insert the insulin gene in the plasmid by matching up the sticky ends and taping the pieces together. You now have recombinant DNA! 9. Draw a diagram of the recombinant plasmid. Indicate the insulin gene placement using the color key created in step 1 of this lab.

Analysis Questions :

Directions: Answer the following on a separate piece of paper. 1. What two types of enzymes are required to make recombinant DNA? 2. You used scissors and tape in this lab. a. Which enzyme was represented by the action of the scissors to cut open the plasmid and to isolate the insulin gene? b. Which enzyme was represented by taping together the “sticky ends”? 3. Why do scientists use the same enzyme to remove the insulin and cut the plasmid open? 4. When you cut the plasmid with your “enzyme” did you cut into either of the genes for antibiotic resistance? If yes, which one(s)? 5. a. Will the bacteria that absorbed the recombinant plasmid be able to survive in an environment that contains tetracycline? Ampicillin? b. Why or why not? Honors supplement:

Directions: Answer the following on a separate piece of paper. 1. How do you think scientists use resistance to antibiotics to identify bacteria which contain recombinant plasmids? 2. Humans are very different from bacteria. Why do you think it is possible to combine their DNA and have bacteria successfully manufacture human protein? 3. Some recombinant bacteria provide beneficial traits to crops, improving yields. Once a genetically engineered bacteria is released into the environment, it is impossible to eradicate. a. Could such organisms have an impact on important, natural, nutrient cycles or food webs? b. Should genetically engineered bacteria be released to the environment? Provide support for your answer. Cite any sources used.