2 GMO Investigator Kit Is your food genetically modified?
3 GMO Investigator Kit Instructors Sherri Andrews, Ph.D.Curriculum and Training SpecialistBio-Rad LaboratoriesEssy Levy, M.Sc.
4 Why teach GMO testing? Inquiry-based Real-world test Environmental SciencePlant PhysiologyGenetics and biotechnologyBioinformatics/Data MiningStandards-basedWhy 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 samplesPerform genuine diagnostic proceduresUse PCR and electrophoresis to find GMO foodsSufficient materials for 8 student workstationsComplete the activity in three 45 minute lab sessionsLaboratory extensions: Real-Time PCR
7 GMO Workshop Time Line Introduction to GM foods DNA extraction of food productsSet up PCR reactionsElectrophorese PCR productsAnalysis and interpretation of resultsTeachers, we realize you are multitasking. We have a full/ action packed workshop. Introduce the materialsHand outs/OH and resourcesLabs in a box, easy to store, all inclusive, curricula. WeWill 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.
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 recombinationWhat 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 cropsCornSoyPapayaCanolaPotatoChicoryRiceSquashSugarbeetTomatoesApproval does not necessarily mean these crops are distributedDatabase 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 plantsThe 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 GMOThis brings up the question… how much of the of food in your pantry contain GMOSources: Fernandez and McBride, : USDA, National Agriculture Statistics Service, Acreage.
12 Which foods yield viable plant DNA? Very ReliableReliableLess ReliableVery Difficult / Not PossibleFresh cornVeggie sausagesVeggie burgersOilFresh papayaTortilla chipsFried corn snacksSalad dressingCorn bread mixFlavored tortilla chipsPopcornCereal (eg cornflakes)Corn mealPuffed corn snacksFriesWheat flourSoy flourMeatballs and burgers containing soy proteinPotato chipsSoy-based protein drinks/powdersYou can search which foods/students projects/ will contain GM. BioRad scientists have already figured out which foods yield better resultsDNA 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% GMEU: food labeled “GM” if >1% GMJapan: food labeled “GM” if >5%ExportWhat 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 modificationsPro: Quick, cheap, low techCon: Crop specific, protein stabilityPCR:Test for presence of inserted foreign DNAPro: ID different GM crops, DNA stabilityCon: Expensive, timelyELISA 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 sampleTest sample DNA for viable plant DNATest sample DNA for genetic modificationsWe’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 contaminatedGMO positive control DNAVerify GMO-negative result is not due to PCR reaction not working properlyPrimers to universal plant gene (Photosystem II)Verify viable DNA was extractedThis 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 labeledNG- non GMO+ - positive controlTube 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 geneThis 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 cellNOS- Sequence for nopaline synthase terminator from soil bacterium Agrobacterium tumefaciansUsed because it evolved to be recognized in most plantsAbout 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
20 Extract DNA from foodTake 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 #7Through 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? 10mlDo 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-GmoWhen you do your flicking, make sure that all that little grainy material is mixed up (chelex)Review use of pipette: why use filtered tipsTalk about master mix:Preparation of PCR reactions: The smart people at BR made this color coded: Green for plant DNA; red for GMOPrep samples and add to the thermocyclerKirk is on!
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 solutionPellet 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 problemDNA 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 reactionsWhat 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 amplifiedPrimers - 2 short specific pieces of DNA whose sequence flanks the target sequenceForwardReverseNucleotides - dATP, dCTP, dGTP, dTTPMagnesium chloride - enzyme cofactorBuffer - maintains pH & contains saltTaq DNA polymerase – thermophillic enzyme from hot springsWhat 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 chelexThe term Taq.Include the flash, PCR reaction.
25 Polymerase Chain Reaction PCR AnimationIn 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 strandsCool (59oC) to anneal primers to templateWarm (72oC) to activate Taq polymerase, which extends primers and replicates DNARepeat 40 cyclesReads 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 soilEnrich nutrient contentNot 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 ResistanceIncreased Nutritional ValueImproved FruitAltered RipeningKirk’s MONSATO experiencePest 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 biodiversityBiotechnology companies control agricultureHealth concerns
30 Method for Genetic Modification of Crops Choose desirable traitClone the geneEngineer the geneTransform gene into plantBackcross GM plant into high yield cropsWhat 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 cropsBacillus thuringiensis protein is a delta endotoxin kills corn borersHerbicideTolerance: Round Up Ready cropsAgrobacterium tumifaciens protein with resistance to Round Up herbicide (glyphosate)Bacillus thuringiensisMaize 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)Technically safe to eat, so only those organisms that eat corn only would be affected by this endotoxinHow 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 crystalBt geneHow can weoriTi plasmidTi genes
33 Engineer the gene Ti plasmid GO Bt gene ori Ti genes STOPEngineer the geneBt geneAdd promoter and terminator, streamline genes by removing introns. Ti plasmid from Agrobacterium.oriTi plasmidTi genesAntibiotic resistance
34 Transform gene into plant Isolate plant cellsGrow undifferentiated callusTransform cellsTake 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 cellsGrow transgenic plantRedifferentiate callus
35 Backcross GM plant into high yield crops YYgg x yyGGYyGgYYgGYygGYYggYyggYYgg x YyGgGM plant = yyGGHigh yield plant = YYggRepeatedly 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…YYgGYYggYYGgYYGGYYgG 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 primers2: non-GMO food with GMO primers3. Test food with plant primers4: Test food with GMO primers5: GMO positive template with plant primers6: GMO positive template with GMO primers7: PCR MW Ruler1234567Explicitly 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? NoThere are plenty of controls in this experiments.Middle lanes: Is the test food GMO?GMO positive controlMost things have the coliflower mosaic promoter.GMO negative
37 GMO Investigator Kit Lab Extensions Independent studiesData Mining/Bioinformatics for specific genesE.g. Design primers to the cry genes in Bt cornQuantitative Real-Time PCRHigh 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 NegativesNo DNA extractedPossible food type or possibly primers do not work on that plant speciesInstaGene matrix transferred to PCR reactions
39 GMO Investigator Kit Contents Bio-Rad certified Non-GMO foodInstaGeneMaster MixGMO primersPlant PSII primersGMO & PSII positive control DNAPCR MW RulerDPTPs, microtubes, PCR tubes, foam floatsManualNot Included but required:Thermal cyclerWater bath/heat blockElectrophoresis Module (agarose, TAE buffer & Fast Blast DNA stain)Electrophoresis equipment & power supply2-20 ul pipettes & barrier tips