Recombinant DNA Technology Biotechnology Recombinant DNA Technology 6/1/2018

Bellwork Explain what genetic engineering means to you? 6/1/2018

Objectives Define recombinant DNA Create a paper model of a recombinant plasmid and identify the key components 6/1/2018

Key Terminology Recombinant DNA molecule Chimera DNA ligase Plasmid Vector Transformation Chromosomal DNA

Overview Recombinant DNA Technology involves using methods to create new DNA molecules New DNA molecules made by piecing together DNA from different organisms This recombinant DNA (rDNA) is known as a chimera Goat boy

Examples of Recombinant DNA Technology rhInsulin, marketed by Eli, Lilly and Company Recombinant human growth hormone (Nutropin, marketed by Genentech, Inc) Recombinant “activase” (marketed Genentech, Inc)-dissolves blockages in blood vessels

Significance of rDNA These proteins normally are produced in very small amounts or are synthesized at the wrong time in the human body By being able to make these products in large amounts in other organisms, like bacteria. Can be used to help individuals that are deficient in these proteins

Plasmid DNA A small circular double stranded DNA found in bacteria and yeast Separate from the chromosomal DNA Chromosomal DNA - controls growth and division of organism

Characteristics of Plasmid DNA Not involved in regulating growth and division of organism. Consider autonomous in that it has control over its replication Presence of plasmids in bacteria often present to protect against humans medicines by carrying antibiotic resistance genes Prevents antibiotics from killing the bacteria

Why is a Plasmid called a Vector ? Because the plasmid can replicate and carry recombinant DNA into organism of interest

Making a Recombinant DNA Molecule Step 1: Isolation of human DNA encoding Insulin. Isolate Human DNA Cut DNA into fragments with restriction enzymes Identify the DNA fragment that encodes for the protein, insulin

Making a Recombinant DNA Molecule Step 2: Place DNA molecule into a cloning vector (commonly plasmid DNA) Cut plasmid with same restriction enzyme used to make DNA fragments that contain insulin Ligate fragments into the plasmid with DNA ligase DNA ligase is an enzyme that “glues” the DNA fragments together to create the recombinant DNA molecule

Picture of Cloning Vector

Transforming Bacteria with Recombinant DNA Step 3: The recombinant DNA molecule is taken up by the Bacterium by transformation When a bacterial cell takes up foreign DNA (e.g. plasmid vector containing human insulin), it is said to be transformed Bacteria then allowed to reproduce to make lots of cells containing the recombinant DNA molecule

PROPERTY OF PIMA COUNTY JTED, 2010 Think-Pair-Share Why is the plasmid with the human insulin DNA considered to be a recombinant DNA molecule or chimera? 3. Think-pair-share teacher presents a question teacher gives wait time for student to form answer teacher instructs students to share their answer with a partner teacher calls on non-volunteers to share with the class PROPERTY OF PIMA COUNTY JTED, 2010 3 15

Identify the Bacteria with the Recombinant DNA molecule Must eliminate bacteria cells that do not carry the recombinant plasmid Accomplished by growing bacteria on media containing an antibiotic Antibiotic –chemicals that prevent the growth and survival of bacteria How does the antibiotic work?

Antibiotic Resistance

Bacterium Takes Up Cloning Vector Bacterium takes up cloning vector will now become ampicillin resistance. In other words, will not be killed by the presence of ampicillin in the growth media The bacteria without the cloning vector will be killed because do not contain the ampicillin resistant gene.

PROPERTY OF PIMA COUNTY JTED, 2010 Think-Pair-Share You have transformed bacteria with the plasmid containing the human insulin gene. Only 10% of the bacteria take up the recombinant DNA molecule. Predit which petri plate will have more bacteria growth, the one + ampicillin or the one – ampicillin. Explain your reasoning. 3. Think-pair-share teacher presents a question teacher gives wait time for student to form answer teacher instructs students to share their answer with a partner teacher calls on non-volunteers to share with the class PROPERTY OF PIMA COUNTY JTED, 2010 3 21

PROPERTY OF PIMA COUNTY JTED, 2010 12 Word Summary 48 37 18 21 14 13 23 In 12 words or less, list key concepts you learned today 26. 12 Word Summary in 12 words or less, summarize the most important aspects from today's lesson teacher uses a strategy to check all PROPERTY OF PIMA COUNTY JTED, 2010 22 22

Q & A: Recombinant DNA Why is it useful to create bacteria that contain a new gene? What is the purpose of restriction enzymes in this process? What is the purpose of DNA Ligase? Describe the 3 steps for making recombinant DNA. Explain the specific steps you would use to create bacteria with a human growth factor gene.