What is Irene’s problem? Objective: Understand How Humans Benefit from Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing 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 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. What is Diabetes? A disease in which a person has high blood sugar, because the body does not produce enough insulin

How Can Irene Get Better? Objective: Understand How Humans Benefit from Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing 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. How Can Irene Get Better? Irene needs to take insulin

Objective: Understand How Humans Benefit from Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing 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 How Can We Make Insulin? The human gene which codes for the insulin protein was put inside of bacteria

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.

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. 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 #3 Bind 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 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 Step #5: Bacteria clone into a large number of identical daughter cells

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

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

Objective: Understand How Humans Benefit from Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing 8 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. 5 1 9 3 6 7 4 2

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.

Objective: Understand the Main Steps in Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing, restriction enzyme Step #1 Extract the DNA from a human cell Use restriction enzymes to isolate a segment of (DNA) that contains the insulin gene

Objective: Understand the Main Steps in Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing, restriction enzyme Step #2 Remove the plasmid from a bacterium Treat the bacterial plasmid with the same restriction enzyme 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 #3 Bind 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 Step #3 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 Step #4: Bacteria clone into a large number of identical daughter cells .

Objective: Understand How Humans Benefit from Bacterial Transformation New Words: Insulin, recombinant DNA, plasmid, gene splicing Step #4: 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 Step #4 Insulin is produced by bacteria and extracted for human use.

Objective: Understand How Scientists Manipulate DNA New Words: E 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

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