What is a GMO? "genetically modified organism (GMO)" defines an organism in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination Typically plant modifications.

Which foods contain GM product? USDA Approval for GM food crops Corn Soy Papaya alphalfa Canola Potato Chicory Rice Squash Sugarbeet Tomatoes Approval does not necessarily mean these crops are distributed Database of GM crops: www.cera-gmc.org/?action=gm_crop_database& Which foods contain GM product? Bolded crops are widely grown & sold in US.

Which foods contain GM product? HT = herbicide tolerant In January 2011, despite protests from organic groups and public health advocates, Agriculture Secretary Tom Vilsack announced that the USDA had approved the unrestricted planting of genetically modified alfalfa In the United States, the FDA Center for Food Safety and Applied Nutrition reviews summaries of food safety data developed and voluntarily submitted by developers of engineered foods, in part on the basis of comparability to conventionally-produced foods. There are no specific tests required by FDA to determine safety. FDA does not approve the safety of engineered foods[citation needed], but after its review, acknowledges that the developer of the food has asserted that it is safe. http://en.wikipedia.org/wiki/Genetically_modified_food Sources: 1996-1999 Fernandez and McBride, 2000-2004: USDA, National Agriculture Statistics Service, Acreage. http://en.wikipedia.org/wiki/Genetically_modified_food

Why test for GMO’s? Legislation Export What about unlabeled food? US: food labeled “GM-Free” <5% GM EU: food labeled “GM” if >1% GM Japan: food labeled “GM” if >5% No requirement to identify food as GMO in Canada or US. Required to note nutritional information not whether GMO (US and Canada) Export What about unlabeled food?

Why have GM crops? Growing human population Loss of farmable land Remediation of soil Enrich nutrient content Pigs that have reduced phosphates in their feces Pigs with omega three fa

Desirable Traits Pest Resistance Herbicide Tolerance Viral Resistance Drought Resistance Increased Nutritional Value Improved Fruit Altered Ripening Pest resistance = Bacillus thuringiensis (Bt), cry delta endotoxin (protein); Herbicide resistance = RoundUp Ready, glyphosate tolerance, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), from agrobacterium. Drought resistance = C4 plants, stomata that close to minimize water loss. Increased Nutritional Value = Golden Rice, beta carotene, pigs with omega 3 fa. Improved fruit = Flavr Savr tomatoes, delayed softening. Altered Ripening = malin (ethylene precursor; plant hormone).

Opponents argue • Creation of super pests • Creation of super weeds Loss of biodiversity Biotechnology companies control agriculture Health concerns ---allergy to protein produced

Method for Genetic Modification of Crops Choose desirable trait Clone the gene Engineer the gene Transform gene into plant Backcross GM plant into high yield crops

Choose desirable trait Pest Resistance: Bt crops Bacillus thuringiensis protein is a delta endotoxin which kills corn borers HerbicideTolerance: Round Up Ready crops Agrobacterium tumifaciens protein with resistance to Round Up herbicide (glyphosate) Bacillus thuringiensis Maize line Bt11 was developed through a specific genetic modification to be resistant to attack by European corn borer (ECB; Ostrinia nubilalis), a major insect pest of maize in agriculture. The novel variety produced the insecticidal protein, Cry1Ab, derived from Bacillus thuringiensis subsp. kurstaki (B.t.k.) HD-1 strain. Delta-endotoxins, such as the Cry1Ab protein expressed in Bt11, act by selectively binding to specific sites localized on the brush border midgut epithelium of susceptible insect species. Following binding, cation-specific pores are formed that disrupt midgut ion flow and thereby cause paralysis and death. Cry1Ab is insecticidal only to lepidopteran insects, and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins. (www.agbios.com) Delta endotoxin crystal

Transform gene into plant Isolate plant cells Grow undifferentiated callus Transform cells Take plant leaf sample, break up and develop callus on media. Callus is mass of undifferentiated cells. Transform using gene gun or other methods. Grow callus on selective media for transformants, transfer to growth media…becomes plant. Other methods, electroporation, agrobacterium, whiskers. Totipotent property of plants. Select cells Grow transgenic plant Redifferentiate callus

How to test for GMOs ELISA: PCR: Test for presence of proteins expressed from genetic modifications Pro: Quick, cheap, low tech Con: Crop specific, protein stability PCR: Test for presence of inserted foreign DNA Pro: ID different GM crops, DNA stability Con: Expensive, timely ELISA used only for fresh food (cheaper, faster). Photosystem II gene that all plants have: use as test for viable plant DNA.

How to test for GMOs Test for GMOs by PCR: Grind food Extract DNA from sample Test sample DNA for viable plant DNA Test sample DNA for genetic modifications

PCR Developed in 1983 by Kary Mullis, Still ongoing lawsuits with reard to the Taq plymerase

Which foods yield viable plant DNA? Very Reliable 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 Wheat flour Soy flour Meatballs and burgers containing soy protein Potato chips Soy-based protein drinks/powders

Why use CaMV 35S and NOS? CaMV 35S – Sequence for the promoter of 35S 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 About 65% of food crops use 35S promoter, by adding NOS detection, can detect about 80% of GM foods. 35S promoter drives expression of 35S RNA transcript of CaMV. 2 main transcripts of CaMV. 19S and 35S. 19S codes for proteins. 35S is reverse transcribed to make the virion DNA in the cytoplasm of the infected cell for the productions of new virus particles. Nopaline is an amino acid derivative, derived from arginine, produced in wound sites by tumor inducing (Ti) plasmid from A.tumefaciens.Nopaline provides food for A.tumefaciens and therefore gives selective advantage to A. tumefaciens. Nopaline also induces conjugation to transfer Ti plasmid into other bacteria. NOS genes are on the T-DNA that is transfered into the plant genome by the Ti plasmid

Extract DNA from food

Why these steps? Grinding food to release DNA InstaGene chelates divalent ions (e.g. Mg2+) necessary for DNA degrading enzymes (e.g. DNases) Only 50 μl of food transferred otherwise InstaGene is overwhelmed (~ 5 mg of original material) Boiling releases DNA from food into the InstaGene solution Pellet InstaGene and food debris because InstaGene inhibits PCR reaction (Taq needs Mg++) Why these steps? Mg++ Mg++ Mg++ Mg++ Mg++ Mg++ Mg++ Mg++ InstaGene

Set up PCR reactions

The PCR Reaction What do you need? What is needed for PCR? The PCR Reaction What do you need? Template - the DNA to be amplified Primers - 2 short specific pieces of DNA whose sequence flanks the target sequence Forward Reverse Nucleotides - dATP, dCTP, dGTP, dTTP Magnesium chloride - enzyme cofactor Buffer - maintains pH & contains salt Taq DNA polymerase – thermophillic enzyme from hot springs

The PCR Reaction How does it work? Heat (94oC) to denature DNA strands Cool (59oC) to anneal primers to template Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA Repeat 40 cycles

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

Engineer the gene Ti plasmid GO Bt gene ori Ti genes STOP Engineer the gene Bt gene Add promoter and terminator, streamline genes by removing introns. Ti plasmid from Agrobacterium. ori Ti plasmid Ti genes Antibiotic resistance

Backcross GM plant into high yield crops YYgg x yyGG YyGg YYgG YygG YYgg Yygg YYgg x YyGg GM plant = yyGG High yield plant = YYgg Repeatedly backcross GM plant to high yield plant to reintroduce hybrid traits (genome). YYgG YYgg YYGg YYGG YYgG x YYgG

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

GMO Investigator Kit Lab Extensions Independent studies Data Mining/Bioinformatics for specific genes E.g. Design primers to the cry genes in Bt corn Testing for blended foods

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 InstaGene matrix transferred to PCR reactions

GMO Investigator Kit contents Bio-Rad certified Non-GMO food InstaGene Master Mix GMO primers Plant PSII primers GMO & PSII positive control DNA PCR MW Ruler DPTPs, microtubes, PCR tubes, foam floats Manual Not Included but required: Thermal cycler Water bath/heat block Electrophoresis Module (agarose, TAE buffer & Fast Blast DNA stain) Electrophoresis equipment & power supply 2-20 ul pipettes & barrier tips

In laboratory cultures natural competence is usually tightly regulated and often triggered by nutritional shortages or adverse conditions. competence is the ability of a cell to take up extracellular ("naked") DNA from its environment