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Genetically modified foods

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  • Bolded crops are widely grown & sold in US.
  • 80% of soy crop in GMO
  • ELISA used only for fresh food (cheaper, faster). Photosystem II gene that all plants have: use as test for viable plant DNA.
  • Specifically the 32 kDa psbA gene
  • 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
  • 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. Improved fruit = Flavr Savr tomatoes, delayed softening. Altered Ripening = malin (ethylene precursor; plant hormone).
  • 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)
  • Add promoter and terminator, streamline genes by removing introns. Ti plasmid from Agrobacterium.
  • 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.
  • Repeatedly backcross GM plant to high yield plant to reintroduce hybrid traits (genome).
  • C:\Documents And Settings\Louay Labban Uok\Desktop\All\Powerpoints\Gmo Slides 1

    1. 2. GMO Investigator Kit <ul><li>Instructor </li></ul><ul><li>Sherri Andrews, Ph.D. </li></ul><ul><li>North Carolina School of the Arts </li></ul><ul><li>Winston-Salem, NC </li></ul>
    2. 3. Why teach GMO testing? <ul><li>Inquiry-based </li></ul><ul><li>Real-world test </li></ul><ul><li>Environmental Science </li></ul><ul><li>Plant Physiology </li></ul><ul><li>Genetics and biotechnology </li></ul><ul><li>Bioinformatics/Data Mining </li></ul>
    3. 4. GMO Workshop Time Line <ul><li>Introduction to GM foods </li></ul><ul><li>DNA extraction of food products </li></ul><ul><li>Set up PCR reactions </li></ul><ul><li>Electrophorese PCR products </li></ul><ul><li>Analysis and interpretation of results </li></ul>
    4. 5. What is a GMO? <ul><li>&quot;genetically modified organism (GMO)&quot; means an organism in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination </li></ul>
    5. 6. Which foods contain GM product? <ul><li>US Approval for GM food crops </li></ul><ul><li>Corn </li></ul><ul><li>Soy </li></ul><ul><li>Papaya </li></ul><ul><li>Canola </li></ul><ul><li>Potato </li></ul><ul><li>Chicory </li></ul><ul><li>Rice </li></ul><ul><li>Squash </li></ul><ul><li>Sugarbeet </li></ul><ul><li>Tomatoes </li></ul><ul><li>Approval does not necessarily mean these crops are distributed </li></ul><ul><li>Database of GM crops: www.agbios.com </li></ul>
    6. 7. Which foods contain GM product? Sources: 1996-1999 Fernandez and McBride, 2000-2004: USDA, National Agriculture Statistics Service, Acreage.
    7. 8. 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
    8. 9. Why test for GMO’s? <ul><li>Legislation </li></ul><ul><ul><li>US: food labeled “GM-Free” <5% GM </li></ul></ul><ul><ul><li>EU: food labeled “GM” if >1% GM </li></ul></ul><ul><ul><li>Japan: food labeled “GM” if >5% </li></ul></ul><ul><li>Export </li></ul><ul><li>What about unlabeled food? </li></ul>
    9. 10. How to test for GMOs ELISA : 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
    10. 11. How to test for GMOs <ul><li>Test for GMOs by PCR: </li></ul><ul><li>Grind food </li></ul><ul><li>Extract DNA from sample </li></ul><ul><li>Test sample DNA for viable plant DNA </li></ul><ul><li>Test sample DNA for genetic modifications </li></ul>
    11. 12. Kit Controls <ul><li>Bio-Rad certified non-GMO food </li></ul><ul><ul><li>Verify PCR is not contaminated </li></ul></ul><ul><li>GMO positive control DNA </li></ul><ul><ul><li>Verify GMO-negative result is not due to PCR reaction not working properly </li></ul></ul><ul><li>Primers to universal plant gene (Photosystem II) </li></ul><ul><ul><li>Verify viable DNA was extracted </li></ul></ul>
    12. 13. 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.
    13. 14. Why use CaMV 35S and NOS ? CaMV 35S – Sequence for the promoter of 35S transcript of the Ca uliflower m osaic v irus. Used because it functions in every plant cell NOS- Sequence for no paline s ynthase terminator from soil bacterium Agrobacterium tumefacians Used because it evolved to be recognized in most plants
    14. 15. Extract DNA from food
    15. 16. Why these steps? <ul><li>Grinding food to release DNA </li></ul><ul><li>InstaGene chelates divalent ions (e.g. Mg 2+ ) necessary for DNA degrading enzymes (e.g. DNases) </li></ul><ul><li>Only 50 μ l of food transferred otherwise InstaGene is overwhelmed (~ 5 mg of original material) </li></ul><ul><li>Boiling releases DNA from food into the InstaGene solution </li></ul><ul><li>Pellet InstaGene and food debris because InstaGene inhibits PCR reaction (Taq needs Mg ++ ) </li></ul>Mg ++ Mg ++ Mg ++ Mg ++ Mg ++ Mg ++ Mg ++ Mg ++ InstaGene
    16. 17. Set up PCR reactions
    17. 18. Volumetric Measurements 50 μ l
    18. 19. The PCR Reaction What do you need? What is needed for PCR? <ul><li>Template - the DNA to be amplified </li></ul><ul><li>Primers - 2 short specific pieces of DNA whose sequence flanks the target sequence </li></ul><ul><ul><ul><li>Forward </li></ul></ul></ul><ul><ul><ul><li>Reverse </li></ul></ul></ul><ul><li>Nucleotides - dATP, dCTP, dGTP, dTTP </li></ul><ul><li>Magnesium chloride - enzyme cofactor </li></ul><ul><li>Buffer - maintains pH & contains salt </li></ul><ul><li>Taq DNA polymerase – thermophillic enzyme from hot springs </li></ul>
    19. 20. PCR Review PCR Animation
    20. 21. The PCR Reaction How does it work? Heat ( 94 o C ) to denature DNA strands Cool ( 59 o C ) to anneal primers to template Warm ( 72 o C ) to activate Taq polymerase, which extends primers and replicates DNA Repeat 40 cycles
    21. 22. Why have GM crops? <ul><li>Growing human population </li></ul><ul><li>Loss of farmable land </li></ul><ul><li>Remediation of soil </li></ul><ul><li>Enrich nutrient content </li></ul>
    22. 23. Desirable Traits <ul><li>Pest Resistance </li></ul><ul><li>Herbicide Tolerance </li></ul><ul><li>Viral Resistance </li></ul><ul><li>Drought Resistance </li></ul><ul><li>Increased Nutritional Value </li></ul><ul><li>Improved Fruit </li></ul><ul><li>Altered Ripening </li></ul>
    23. 24. Opponents argue <ul><li>• Creation of super pests </li></ul><ul><li>• Creation of super weeds </li></ul><ul><li>Loss of biodiversity </li></ul><ul><li>Biotechnology companies control agriculture </li></ul><ul><li>Health concerns </li></ul>
    24. 25. Method for Genetic Modification of Crops <ul><li>Choose desirable trait </li></ul><ul><li>Clone the gene </li></ul><ul><li>Engineer the gene </li></ul><ul><li>Transform gene into plant </li></ul><ul><li>Backcross GM plant into high yield crops </li></ul>
    25. 26. Choose desirable trait <ul><li>Pest Resistance: Bt crops </li></ul><ul><ul><li>Bacillus thuringiensis protein is a delta endotoxin kills corn borers </li></ul></ul><ul><li>HerbicideTolerance: Round Up Ready crops </li></ul><ul><ul><li>Agrobacterium tumifaciens protein with resistance to Round Up herbicide (glyphosate) </li></ul></ul>Bacillus thuringiensis Delta endotoxin crystal
    26. 27. Clone the gene Ti plasmid ori Bt gene Bacillus thuringiensis Delta endotoxin crystal Ti genes
    27. 28. Engineer the gene Antibiotic resistance Ti plasmid ori Bt gene Ti genes STOP GO
    28. 29. Transform gene into plant Isolate plant cells Grow undifferentiated callus Transform cells Select cells Redifferentiate callus Grow transgenic plant
    29. 30. Backcross GM plant into high yield crops GM plant = yy GG High yield plant = YY gg YY gg x yy GG Y y G g YY gg x Y y G g YY g G Y yg G YY gg Y ygg YY g G x YY g G YY g G YY gg YYG g YYGG
    30. 31. 1 3 2 7 6 5 4 GMO positive GMO negative 7: PCR MW Ruler 1: non-GMO food with plant primers 2: non-GMO food with GMO primers 3: Test food with plant primers 4: Test food with GMO primers 5: GMO positive template with plant primers 6: GMO positive template with GMO primers Analysis of Results 1 3 2 7 6 5 4
    31. 32. GMO Investigator Kit Lab Extensions <ul><li>Independent studies </li></ul><ul><li>Data Mining/Bioinformatics for specific genes </li></ul><ul><ul><li>E.g. Design primers to the cry genes in Bt corn </li></ul></ul><ul><li>Testing for blended foods </li></ul>
    32. 33. Trouble shooting <ul><li>False Positives </li></ul><ul><ul><li>Contamination-sterile technique; 10% bleach to clean pipette barrels, mortars & pestles, bench tops; barrier tips for all steps. </li></ul></ul><ul><li>False Negatives </li></ul><ul><ul><li>No DNA extracted </li></ul></ul><ul><ul><li>Possible food type or possibly primers do not work on that plant species </li></ul></ul><ul><ul><li>InstaGene matrix transferred to PCR reactions </li></ul></ul>
    33. 34. GMO Investigator Kit contents <ul><li>Bio-Rad certified Non-GMO food </li></ul><ul><li>InstaGene </li></ul><ul><li>Master Mix </li></ul><ul><li>GMO primers </li></ul><ul><li>Plant PSII primers </li></ul><ul><li>GMO & PSII positive control DNA </li></ul><ul><li>PCR MW Ruler </li></ul><ul><li>DPTPs, microtubes, PCR tubes, foam floats </li></ul><ul><li>Manual </li></ul><ul><li>Not Included but required: </li></ul><ul><li>Thermal cycler </li></ul><ul><li>Water bath/heat block </li></ul><ul><li>Electrophoresis Module (agarose, TAE buffer & Fast Blast DNA stain) </li></ul><ul><li>Electrophoresis equipment & power supply </li></ul><ul><li>2-20 ul pipettes & barrier tips </li></ul>

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