What Is Biotechnology? ■ Any technique that uses living organisms or substances from those organisms to make or modify ■ a product ■ improve plants or animals ■ to develop microorganisms for specific uses

Genetic Engineering ■ Genetic engineering is taking one or more genes from one organism and ….. ■ Transferring them to another organism ■ Putting them back into the original organism in different combinations

GMOs GMO’s: Genetically Modified Organism ■ Transgenic Organisms: organism that has one or more genes from another organism inserted into its genome ■ Example: Bt Corn ( pest resistant) ■ Recombinant DNA (rDNA): is DNA that contains genes from more than one organism ■ Example: rInsulin

What are the Benefits of Biotechnology? ■ Medicine ■ Human ■ TPA, Dissolve blood clots ■ Cancer Treatments ■ Embryo Screening ■ Veterinary ■ Hybrid Dogs ■ Surrogate Cows ■ Environment ■ Clean up oil spills (bioremediation) ■ Agriculture ■ Bt Corn ( pest resistant) ■ Food products ■ Cheese, Yogurt, Fermented foods, Bread, Alcohol) ■ Industry/manufacturing ■ Better cleaners

Medicine - Mass produce Insulin - TPA to dissolve blood clots - Embryo screening - Stem Cell research - Hybrid animals

Agriculture - Pest resistant crops - Foods with additional nutrition or increased yield - New varieties of foods

Industrial and Environment - Waste treatment - Biofuels - Bioremediation (clean oil spills)

How else has Genetic Engineering benefit us? ■ Adding a clotting agent to the blood that would help dissolve clots. ■ TPA--- DONE!!!!! ■ What if: ■ Cancer free genetics ■ HIV free genetics ■Creating something that was resistant to these diseases??? IS IT POSSIBLE?

Insulin ■ The first commercial product made by genetic engineering (1976). ■ Insulin is the hormone used to control their blood sugar levels in diabetics ■ First sold in 1982, (S. San Francisco) PIGS E. Coli

Biotech Tools Restriction Enzyme: an enzyme that cuts DNA Ligase: an enzyme that pastes DNA back together a re-establishes the ester bond Gene of Interest: Gene that we are going to manipulate; Example Insulin Gene Recipient Genome: Where you are going to put the gene once you’ve got it; Ex. E. coli plasmid Plasmid: Circular piece of DNA that bacteria have in addition to the normal chromosome

Restriction Enzyme Natural function of restriction enzymes: ■ To protect bacteria against viruses. ■ Digest (cut up) foreign viral DNA. ■ Restriction enzymes are isolated from bacteria Restriction Enzyme: an enzyme that CUTS DNA (like scissors)

A closer look…. EcoR1 5’…. ACTGTACGAATTCGCTA….3’ 3’….TGACATGCTTAAGCGAT…. 5’ EcoR 1

A closer look…. EcoR1 AATTCGCTA….3’ GCGAT….5’ 5’….ACTGTACG 3’….TGACATGCTTAA “sticky ends” -can bind with other DNA molecules with the same overhangs

5’….ACTGTACAGATCCGCTA….3’ 3’….TGACATGTCTAGGCGAT….5’ DNA Ligase (acts like glue) DNA ligase If two pieces of DNA with sticky ends and complimentary bases come close to each other, the two segments of DNA will join by hydrogen bonding

HOW IS IT DONE? Steps for genetically engineering Insulin 1. Take a human cell and remove the nucleus 2. Extract the DNA (genome) from the nucleus 3. Locate the Insulin gene 4. Cut out the Insulin gene with a restriction enzyme 5. Take a Bacterial Plasmid and cut open with the same restriction enzyme 6. Place the Insulin gene into the bacterial Plasmid; you now have recombinant DNA. 7. Place the recombinant DNA back into an Bacterial Cell (transformation) 8. The Bacteria will now produce insulin.

A closer look…. HindIII 5’…. ACTGTACAAGCTTGCTA….3’ 3’….TGACATGTTCGAACGAT…. 5’ HindI II

A closer look…. HindIII AGCTTGCTA….3’ ACGAT….5’ 5’….ACTGTACA 3’….TGACATGTTCGA “sticky ends” -can bind with other DNA molecules with the same overhangs

Create Your Own Recombinant DNA ! 1. Using the shorter sequence, create your plasmid, remember they are circular 2. Highlight the gene of interest TAATGGATCCTT 3. Figure our what restriction enzyme to use. GAATTC EcoR1 Sequence CTTAAG GGATCC BamH1 Sequence CCTAGG 4. The plasmid and DNA must be cut with same Restriction Enzyme 5. Create your Recombinant DNA.

■ Gel electrophoresis is a technique that separates charged particles by size and charge using an electrical current. ■ Electro = Flow of Electricity ■ Phoresis = (Greek) meaning to carry across ■ Charged particles can include DNA, RNA, Proteins, and in this case dyes

Gel electrophoresis ■ We can use the fragments made by the restriction digestion to make a DNA fingerprint ■ DNA is negatively charged (due to phosphate backbone) ■ When we pump electrical charge into the buffer, it will make the DNA move towards the positive charge ■ Sorts DNA by length ■ Smaller DNA fragments travel further in the gel

Imagine you are a DNA molecule ■ If you were inside an agarose gel, your environment would resemble a very dense spider web. ■ The smaller the DNA fragment, the easier it is to get through the web. ■ This creates a DNA Fingerprint

Use electrophoresis to sort and compare DNA segments - the smaller the fragment, the further it will travel thru the gel

■ Particles that are negatively charged are attracted to the positive electrode, and positively charged particles are attracted to the negative electrode. Negatively charged particles Positively Charged Particles Positive electrode Negative Electrode

■ As particles move through the gel they encounter resistance. ■ Smaller particles have less resistance and move faster down the gel. ■ Larger particles have more resistance and move slower down the gel.

■ Agarose: a polysaccharide made from seaweed. ■ Dissolves in buffer when heated ■ Cools to a solid gel with a network of crosslinking molecules. 1.5% agarose gel w/ Electron microscope

Crime Scene DNA Who Committed The Crime?

Who committed the crime? ■ How do you know? ■ Suspect #5 ■ Because the band patterns for the DNA fingerprint are the same

Crime Scene Analysis Crime Scene DNA GAATTCTAATGGATCCTT 1. Digest the DNA samples with the restrictions enzymes: EcoR1 and BamH1 2. Restriction Digestion with EcoR1 (GAATTC) 3. Repeat with BamH1 (GGATCC) 4. Creates 3 pieces (1 bp long, 7 bp long, 10 bp long) 5. Plot fragments on Mock Gel (based on fragment sizes)

Let’s Try it Crime Scene DNA GAATTCTAATGGATCCTT G GATCC BamH1 Sequence G AATTC EcoR1 Sequence Creates 3 pieces 1 bp, 10 bp, 7 bp 10 bp 7 bp 1 bp + _