Agenda – Applying DNA knowledge to diabetes Warm-up: Attractive & professional thank-you notes Diabetes, Insulin, and rDNA Review Recombinant DNA Plasmids Recombinant Paper Plasmid

Recombinant DNA project Notes on what you have learned – due tomorrow Electronic presentations http://prezi.com/ezf2bged4bia/genetic-engineering-and-biotechnology/Genetic Engineering and Biotechnology by Dong Min Kim on Prezi

Diabetes and Biotechnology Biotechnology solutions Recombinant DNA

Questions- What is diabetes? Why is there growing concern about diabetes? What is the role of insulin? What is the role of biotechnology in the treatment of diabetes?

Biotechnology & Diabetes Treatment Read Calorie-Coated Diabetes

Beta Cell in Pancreas

Pancreas, thymus

Cell Biology Of Insulin Response

Cell Biology Of Insulin Response

Cell Biology Of Insulin Response

Insulin Gene is on the 11th Chromosome (short arm) It contains 153 bases. A small, simple protein

Recombinant DNA with insulin gene inserted Recombinant DNA: A desired gene to the plasmid. Bacteria is transformed to accept the plasmid.

Chromosome & Plasmid

Interesting facts about plasmids What do plasmids do in bacteria? Why are plasmids used in recombinant DNA? In nature?

Interesting facts about plasmids What do plasmids do in bacteria? Why are plasmids used in recombinant DNA? Extra genes Not essential for living Benefit for survival under certain conditions In nature, some plasmids Antibiotic genes Antibiotic resistant genes Bacteria can be transformed by adding circular DNA but not linear Exonuclease: Breaks down genes in a linear process Outside to the inside

Making a Recombinant DNA Product What are the steps needed? Paper plasmids - Construction of the pAMP and pKAN Plasmids Questions

Genetic Engineering –Recombinant DNA How? Identify a molecule produced by a living organism Isolate the instructions (DNA sequence = gene) Put the instructions into another cell or organism Allow the cell to replicate Harvest the desired product

The Vector (E. coli bacteria).

What is needed to produce a product with recombinant DNA?

Recombinant DNA Insulin Identify a insulin gene in humans Use restriction enzymes to isolate the gene in a DNA fragment DNA fragment is added to another DNA source = vector such as plasmids of bacteria or yeast Recombinant DNA is placed in a host cell As the host cell divides (replicates), the rDNA also replicates Harvest, purify, test & market

What we need to learn about: Bacteria (Vector) Copying cells - Mitosis Proteins How are enough insulin is made? PCR

Recombinant DNA & Cloning

What is DNA cloning? When DNA is extracted from an organism, all its genes are obtained In gene (DNA) cloning a particular gene is copied (cloned)

Why Clone DNA? e.g. gene defects related to specific diseases A particular gene can be isolated and its nucleotide sequence determined Control sequences of DNA can be identified & analyzed Protein/enzyme/RNA function can be investigated Mutations can be identified e.g. gene defects related to specific diseases Organisms can be ‘engineered’ for specific purposes e.g. insulin production, insect resistance and more

How is DNA cloned?, I DNA is extracted- here from blood Blood sample DNA is extracted- here from blood Restriction enzymes, e.g. EcoR I, Hind III, etc., cut the DNA into small pieces Different DNA pieces cut with the same enzyme can join, or recombine. DNA Restriction enzymes

DNA Cloning, II Bacterial plasmids (small circular DNA additional to a bacteria’s regular DNA) are cut with the same restriction enzyme A chunk of DNA can thus be inserted into the plasmid DNA to form a “recombinant” molecule

DNA cloning, III The recombinant plasmids are then mixed with bacteria which have been treated to make them “competent”, or capable of taking in the plasmids This insertion is called transformation

DNA Cloning, IV The plasmids have naturally occurring (or inserted) genes for antibiotic resistance Bacteria containing plasmids with these genes will grow on a medium containing the antibiotic- the others die, so only transformed bacteria survive

DNA Cloning, V The transformed bacterial cells form colonies on the medium Each cell in a given colony has the same plasmid (& the same DNA) Cells in different colonies have different plasmids (& different DNA fragments)

Screening, I Screening can involve: Phenotypic screening- the protein encoded by the gene changes the color of the colony Using antibodies that recognize the protein produced by a particular gene

Screening, II 3. Detecting the DNA sequence of a cloned gene with a probe (DNA hybridization)

rDNA project Choice of format: Presentation: Equivalent to Test Grade PowerPoint Presentation with notes Globster (electronic poster) Museum boxes Presentation: Process to make rDNA Process before rDNA Benefits Disadvantages or bioethical issues Regulation Equivalent to Test Grade

Additional Resources http://www.accessexcellence.org/AE/AEC/CC/restriction.php Discovery and Applications of REs - http://videos.howstuffworks.com/hsw/22635-discoveries-with-bill-nye-restriction-enzymes-video.htm http://tools.neb.com/NEBcutter2/ http://www.accessexcellence.org/AE/AEC/CC/activity1.php