1. Irene is 10 years old and in the last few weeks, she suddenly experienced extreme tiredness, weight loss, and increased thirst. Her parents were concerned, so they took her to the doctor. Dr. Ross took Irene’s blood to test for her blood sugar. The results of the test indicated that she was a diabetic. When the doctor shared the results, Irene broke into tears. The combination of her sudden poor health and the news of her diabetes was too much for her to handle. “I don’t understand. What is diabetes and how can I get better?” The doctor felt bad for Irene. She said, “Irene, I can explain how this happened, and how we can fix it.” What is Irene’s problem? Irene is diabetic Objective: Understand How Humans Benefit from Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing

2. People with diabetes may not have enough insulin or may not be able to use it properly. Insulin is a hormone that controls the level of blood sugar (also called glucose) in your body. The sugar then builds up in the blood and overflows into the urine, passing out of your body unused. This deprives you of an important source of energy. Objective: Understand How Humans Benefit from Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing What is Diabetes? A disease in which a person has high blood sugar, because the body does not produce enough insulin

3. All people with type 1 diabetes, and some people with type 2, need to take insulin to help control their blood sugar levels. Type 1 diabetes means your body doesn’t make any insulin. Type 2 diabetes means your body either doesn’t make enough insulin or doesn’t use it properly. The goal of taking insulin is to keep your blood sugar level in a normal range as much as possible so you’ll stay healthy. Over time, high blood sugar levels can cause serious health problems such as blindness and kidney failure. Objective: Understand How Humans Benefit from Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing How Can Irene Get Better? Irene needs to take insulin

4. The first successful insulin preparations came from cows (and later pigs). In the 1980's technology had advanced to the point where we could make human insulin. The technology which made this approach possible was the development of recombinant DNA techniques. In simple terms, the human gene which codes for the insulin protein was cloned (copied) and then put inside of bacteria. A number of tricks were performed on this gene to make the bacteria want to use it to constantly make insulin. Big vats of bacteria now make tons of human insulin. From this, pharmaceutical companies can isolate pure human insulin Objective: Understand How Humans Benefit from Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing How Can We Make Insulin? The human gene which codes for the insulin protein was put inside of bacteria

5. Objective: Understand the Main Steps in Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing, restriction enzyme Step #1 Remove DNA from human cell. Use restriction enzymes to cut a segment of DNA that contains a gene of interest, for example, the gene regulating insulin production.

6. What is a plasmid? A small, circular, DNA molecule present in Bacteria Objective: Understand the Main Steps in Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing, restriction enzyme Step #2 Remove a plasmid from a bacterium and treated with the same restriction enzyme.

7. Step #3 Binds the insulin gene with the opened plasmid to form a recombinant plasmid. Objective: Understand the Main Steps in Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing, restriction enzyme

8. Step #4 The recombinant plasmid is re-inserted back into the bacterium. Objective: Understand the Main Steps in Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing, restriction enzyme

9. Step #5: Bacteria clone into a large number of identical daughter cells. Objective: Understand the Main Steps in Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing, restriction enzyme

10. Step #6: The recombinant plasmid replicates as part of the bacteria’s DNA. Objective: Understand How Humans Benefit from Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing

11. Step #7 Insulin is produced by bacteria and extracted for human use. Objective: Understand How Humans Benefit from Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing

12. Objective: Understand the Main Steps in Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing, restriction enzyme Describe the picture above following the numbered sequence. Assure to include these terms: restriction enzymes, DNA, plasmid, gene, recombinant plasmid, replication, cloning, insulin.

13. Human Cell Gene for human insulin Recombinant DNA Gene for human insulin Sticky ends DNA recombination DNA insertion Bacterial Cell Plasmid Bacterial chromosome Bacterial cell containing gene for human insulin Figure 13-9 Making Recombinant DNA Gene for human insulin Recombinant DNA Gene for human insulinSticky ends Bacterial cell containing gene for human insulinPlasmid DNA recombinationDNA insertion Objective: Understand How Humans Benefit from Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing

14. Bacteria clone into a large number of identical daughter cells Bind the insulin gene with the opened plasmid to form a recombinant plasmid. Extract the DNA from a human cell. Insulin is produced by bacteria and extracted for human use. Remove the plasmid from a bacterium. The recombinant plasmid is re-inserted back into the bacterium. The recombinant plasmid replicates as part of the bacteria’s DNA. Treat the bacterial plasmid with the same restriction enzyme. Use restriction enzymes to isolate a segment of (DNA) that contains the insulin gene. Objective: Understand How Humans Benefit from Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing 1 8 6 9 7 5 3 4 2

15. Objective: Understand How Scientists Manipulate DNA New Words: E. coli, Ampicillin, GFP, plasmid, antibiotic Do Now: List 3 things you learned in the Harlem DNA Lab http://www.dnalc.org/harlemdnalab/BacterialTrans.html

16. Complete the table below: What do you have in the dish? What is your hypothesis? What are the results? How do you explain it? +GFP Non transformed bacteria LB (food) Non transformed bacteria LB (food) Ampicillin Transformed bacteria LB (food) GFP Transformed bacteria LB (food) Ampicillin GFP Transformed bacteria grow in the form of green colonies Hypothesis is true Transformed bacteria grow in the form of green colonies LB/Amp Non transformed bacteria spread across the entire plate No growth All bacteria grow across the entire plate Less green colonies LB/Amp LB Same as expected in hypothesis Non transformed bacteria feed on LB Nothing stop them from growing Only transformed bacteria can survive and grow in the presence of antibiotic ampicillin Non transformed bacteria killed by antibiotic ampicillin Non transformed and transform bacteria feed on LB Nothing stop them from growing Objective: Understand How Scientists Manipulate DNA New Words: E. coli, Ampicillin, GFP, plasmid, antibiotic.