How are genetically modified organisms created Bacteria Yeast

How are genetically modified organisms created? Bacteria Yeast Animals Worms Flies Mammals Plants

Ways of inserting foreign DNA into cells

Engineering E. coli to produce a foreign protein

Synthesis of c. DNA using reverse transcriptase Complementary DNA (c. DNA) has no introns, and can be expressed in bacteria to make eukaryotic proteins.

Genetic engineering of animals

Agrobacterium-mediated transformation

Choose desirable trait Bacillus thuringiensis • Pest Resistance: Bt crops §Bacillus thuringiensis protein is a delta endotoxin kills corn borers • Herbicide. Tolerance: Round Up Ready crops §Agrobacterium tumifaciens protein with resistance to Round Up herbicide (glyphosate) Delta endotoxin crystal

Clone the gene Bacillus thuringiensis Delta endotoxin crystal Bt gene Ti plasmid Ti genes Antibiotic resistance ori

Transform gene into plant Isolate plant cells Grow undifferentiated callus Transform cells Select cells Redifferentiate callus Grow transgenic plant

Why have GM crops? • Growing human population • Loss of farmable land • Remediation of soil • Enrich nutrient content

Desirable Traits • Pest Resistance • Herbicide Tolerance • Viral Resistance • Drought Resistance • Increased Nutritional Value • Improved Fruit • Altered Ripening

Opponents argue • Creation of super pests • Creation of super weeds • Loss of biodiversity • Biotechnology companies control agriculture • Health concerns

Which foods contain GM product? US Approval for GM food crops • Corn • Soy • Papaya • Canola • Potato • Chicory • Rice • Squash • Sugarbeet • Tomatoes Approval does not necessarily mean these crops are distributed Database of GM crops: www. agbios. com

Which foods contain GM product?

Which foods yield viable plant DNA? Very Reliable Less Reliable Very Difficult / Not Possible Fresh corn Veggie sausages Veggie burgers Oil Fresh papaya Tortilla chips Fried corn snacks Salad dressing Corn bread mix Flavored tortilla chips Popcorn Cereal (eg cornflakes) Corn meal Puffed corn snacks Fries Soy flour Meatballs and burgers containing soy protein Soy-based protein drinks/powders Potato chips Wheat flour

Why test for GMO’s? • Legislation – US: food labeled “GM-Free” <5% GM – EU: food labeled “GM” if >1% GM – Japan: food labeled “GM” if >5% • Export • What about unlabeled food?

Kit Controls • Bio-Rad certified non-GMO food –Verify PCR is not contaminated • GMO positive control DNA –Verify GMO-negative result is not due to PCR reaction not working properly • Primers to universal plant gene (Photosystem II) –Verify viable DNA was extracted

Why amplify a plant gene? To confirm that viable DNA was extracted and that negative GM result isn’t due to a non-viable template. Use highly conserved chloroplast gene from Photosystem II – part of the light reaction of photosynthesis.

Why use Ca. MV 35 S and NOS? Ca. MV 35 S – Sequence for the promoter of 35 S transcript of the Cauliflower mosaic virus. Used because it functions in every plant cell NOS- Sequence for nopaline synthase terminator from soil bacterium Agrobacterium tumefacians Used because it evolved to be recognized in most plants

Extract DNA from food

Why these steps? Mg++ • Grinding food to release DNA • Insta. Gene chelates divalent ions (e. g. Mg 2+) necessary for DNA degrading enzymes (e. g. DNases) Mg++ Insta. Gene Mg++ • Only 50 μl of food transferred otherwise Insta. Gene is overwhelmed (~ 5 mg of original material) • Boiling releases DNA from food into the Insta. Gene solution • Pellet Insta. Gene and food debris because Insta. Gene inhibits PCR reaction (Taq needs Mg++)

Set up PCR reactions

Analysis of Results 1 2 3 4 5 6 7 GMO positive 1: non-GMO food with plant primers 2: non-GMO food with GMO primers 3. Test food with plant primers 1 4: Test food with GMO primers 5: GMO positive template with plant primers 6: GMO positive template with GMO primers 7: PCR MW Ruler GMO negative 2 3 4 5 6 7

Trouble shooting • False Positives – Contamination-sterile technique; 10% bleach to clean pipette barrels, mortars & pestles, bench tops; barrier tips for all steps. • False Negatives – No DNA extracted – Possible food type or possibly primers do not work on that plant species – Insta. Gene matrix transferred to PCR reactions