2. 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.

3. 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:
Which foods contain GM product?
Bolded crops are widely grown & sold in US.

4. 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.
Sources: Fernandez and McBride, : USDA, National Agriculture Statistics Service, Acreage.

5. 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?

6. 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

7. 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).

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

9. 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

10. 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. (
Delta endotoxin crystal

11. 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

12. 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.

13. 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

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

15. 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

16. 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

17. Extract DNA from food

18. 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

19. Set up PCR reactions

21. 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

22. 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

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

24. 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

25. 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

26. 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

27. 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

28. 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

29. 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

31. 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