2. GMO Investigator Kit Is your food genetically modified?

3. GMO Investigator Kit Instructors
Sherri Andrews, Ph.D.
Curriculum and Training Specialist
Bio-Rad Laboratories
Essy Levy, M.Sc.

4. Why teach GMO testing? Inquiry-based Real-world test
Environmental Science
Plant Physiology
Genetics and biotechnology
Bioinformatics/Data Mining
Standards-based
Why teach GMO: most critical; introduce contemporary topics. Disecting a frog/cutting edge (joke)
Connection to the real world science.

6. GMO Investigator Kit Advantages
Extract and amplify DNA from different food samples
Perform genuine diagnostic procedures
Use PCR and electrophoresis to find GMO foods
Sufficient materials for 8 student workstations
Complete the activity in three 45 minute lab sessions
Laboratory extensions: Real-Time PCR

7. GMO Workshop Time Line Introduction to GM foods
DNA extraction of food products
Set up PCR reactions
Electrophorese PCR products
Analysis and interpretation of results
Teachers, we realize you are multitasking. We have a full/ action packed workshop. Introduce the materials
Hand outs/OH and resources
Labs in a box, easy to store, all inclusive, curricula. We
Will go through a lot of materials but you always have the resources to go back to and reference.
Great way to combine a lot of different skills all in one.

8. GMO Investigator Procedures Overview

9. What is a GMO? "genetically modified organism (GMO)"
an organism in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination
What is a GMO: Good place to start with your students.
Remember this is an active PPT file

10. 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:
Which foods contain GM product?
Bolded crops are widely grown & sold in US.
Go to the agbio.com web site, extensive and will give you a lot of information on the individual plants
The intent of the kit is to provide a basis of knowledge so that the students get the information (for debate etc..)

11. Which foods contain GM product?
80% of soy crop in GMO
This brings up the question… how much of the of food in your pantry contain GMO
Sources: Fernandez and McBride, : USDA, National Agriculture Statistics Service, Acreage.

12. 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
You can search which foods/students projects/ will contain GM. BioRad scientists have already figured out which foods yield better results
DNA is very very stable… Jurassic park DNA 65M years, think about the torture a dorito Corn chip has gone through and you cans still get DNA out of it!!!!

13. 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%
Export
What about unlabeled food?
The business of Food Import/export

14. 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.
There is also ELISA test for GMO.

15. 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
We’re going to constantly talk about this 2 step porcess. 1-did we get DNA form the sample? 2- is this sample genetically modified? (you can tell from the DNA!)

16. 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
This kit has 3 different levels of controls. This is a great solid way to check the preparations.
Mung bean control…
Get the kids to pick their food, get the kids excited about making decisions on their labs.
Identify the tubes… what has what, the prep tubes and how these are labeled
NG- non GMO
+ - positive control
Tube 3 will have the tube with your sample.
Discuss, primer for PS II: this is the control that will verify if we really have DNA (plant DNA)

17. 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.
Specifically the 32 kDa psbA gene
This gives you a chance to go back to that part of the course when we talk about chloroplasts (blast from the chloroplasts)
Highly conserved…

18. 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

19. Laboratory Quick Guide

20. Extract DNA from food
Take out the quick guides: How many of you teach English Language learners. Point out the images…
Walk through the Quick Guide:
We already did the first few steps, the preparation of the NG (mung bean)
Step 7: repeat the same for the chip (or chosen food) so we will start from step #7
Through the magic of biorad. We found a chip that weighs exactly 1 gram (wow!)
Using the disposable pipette you will add 5mls of h2O. (introduce the pipette: graduated Vol.)
One person should be grinding while the other person adds the H2O.
What is the total ml of h2o that you should add? 10ml
Do I use a new tip/pipette: let them make some their own choices(you decide) and then bring it up at the end. When in doubt, change.
Something to think about… we have been very cavalier in this work shop, in your class you might want to use gloves. What food would you start with if you were to do this with your students? Start with the non-Gmo
When you do your flicking, make sure that all that little grainy material is mixed up (chelex)
Review use of pipette: why use filtered tips
Talk about master mix:
Preparation of PCR reactions: The smart people at BR made this color coded: Green for plant DNA; red for GMO
Prep samples and add to the thermocycler
Kirk is on!

21. Volumetric Measurements

22. 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++)
Mg++
Mg++
Mg++
Mg++
Mg++
Mg++
Mg++
Quick trick of pipette baggies.
Talk about the chelex beads, why is it important…
What is going on and how does the chelex extraction work. Why is it usefull and when is it a problem
DNA is very stable but you are not going to find the entire DNA, it will be chopped up even M of years latter. (JP/fill in with frog DNA)
Mg++
InstaGene

23. Set up PCR reactions
What are the things we need to do a PCR. Again mentioned the color codes. (Method to the madness)

24. 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
What is PCR? Find a needle in a hay stack and make a hay stack out of a needle.
Freetos (no DNA survives) what are the dNTP’s; we need to put back the Mg from the reaction since we moved it w the chelex
The term Taq.
Include the flash, PCR reaction.

25. Polymerase Chain Reaction
PCR Animation
In flash. Explain why 3’ vs. 5’ volunteer people elbow #5 and hand is Pho, tush is 3’end use your students to do the entire helix… great way to demonstrate anti-II and restriction digest, and also how each dNTP binds to the next one (great activity for the Monks)
Brain is working on how to fit it in. make sure to back off and go back and draw it. Build up drawing.
These primers are designed specific to the sequences… it does not find all GMO’s only about 80%, only the GMOs that were generated using those promoters etc…

26. 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
Reads 3-5 and writes 1-3. Importance of the 3’. Like AZT….terminate nucleotides.
You need new primers. Millions of copies of primers. Etc… 3rd cycle is the first time we see product only. So now, each cycle you duplicate. Now go home and teach mom/dad. All this is on-line. Flash animations etc…

27. Why have GM crops? Growing human population Loss of farmable land
Remediation of soil
Enrich nutrient content
Not a good idea to do this as the 1st PCR. But it may be that you’d like to teach it in context (this context)
Bee crash in CA? Are all the bees being toxified by CA GMOs, huge pop decline. Why GMO’s. when you challenge nature with something new, what does nature do? NATURAL SELECTION, there is always going to be a few that will hold on. People are naïve to think that scientists don’t think of these things, but it’s a choice. Do we need to feed the world now? Or do we need to give a higher consideration to the “pushed” natural selection caused by “hurring” up this directed evolution.

28. Desirable Traits Pest Resistance Herbicide Tolerance Viral Resistance
Drought Resistance
Increased Nutritional Value
Improved Fruit
Altered Ripening
Kirk’s MONSATO experience
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).

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

30. 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
What do you need to do to make a GMO? It takes about 12 years to generate (breed) a new species of corn in the traditional way. (do we have that time? As a company? As world population? What about the effects?

31. Choose desirable trait
Pest Resistance: Bt crops
Bacillus thuringiensis protein is a delta endotoxin 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. (
Technically safe to eat, so only those organisms that eat corn only would be affected by this endotoxin
How does round-up work? It block the ability to manufacture a specific AA, therefore (like in JP/Lysine) will kill off the plant by lacking that essential AA.
Delta endotoxin crystal

32. Clone the gene Ti plasmid Bacillus thuringiensis
Delta endotoxin crystal
Bt gene
How can we
ori
Ti plasmid
Ti genes

33. 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

34. 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.
Helios: bind the plasmids to Au and shoot it into the plant. Shoot, grow and see if its genetically modified,
Select cells
Grow transgenic plant
Redifferentiate callus

35. 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).
Then you check if you have the modification, but if you do not have a high yield, you can continue the crossing with a high yield plant so that you have a final GM/high yield plant!
NOW: change to loading, loading dye, buffer, %, X etc…
YYgG
YYgg
YYGg
YYGG
YYgG x YYgG

36. 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
4: Test food with GMO primers
5: GMO positive template with plant primers
6: GMO positive template with GMO primers
7: PCR MW Ruler 1 2 3 4 5 6 7
Explicitly say that DNA is not “that long” what determines the with of the band is the size of the well (not the length of the fragment)
Did we get DNA? Mung beans (bean sprouts)
Are they genetically modified? No
There are plenty of controls in this experiments.
Middle lanes: Is the test food GMO?
GMO positive control
Most things have the coliflower mosaic promoter.
GMO negative

37. GMO Investigator Kit Lab Extensions
Independent studies
Data Mining/Bioinformatics for specific genes
E.g. Design primers to the cry genes in Bt corn
Quantitative Real-Time PCR
High school kids can do their own Independent studies.
Poster from Kirk!
Dialysis paper sandwich. Topperware, rubberband, gel, lots of h2O, 2nd film, sandwich, this is good for 3% gel. For the 1% gel use the gel-bond.

38. 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

39. 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