Testing Foods for Introduced Genes

1
GMODetecting Genetically Modified Foods

• Testing Foods for Introduced Genes

2
GMOs

• Genetically modified organisms have DNA that has
  been modified through genetic engineering
• GM foods were first put on the market in the
  early 1990s fueling a revolution in agriculture
• Unlike organisms developed through the
  conventional genetic modification of selective
  breeding (plant breeding and animal breeding) or
  mutation breeding.

3
GMOs

• Organisms that have been genetically modified by
  the insertion of foreign genetic material.
• For plantsthe gene may come from another plant,
  or from another species, or from another
  kingdom.
• Introduced DNA codes for a protein that gives the
  GMO an advantage over the wild type

4
GMO-Animals

• Animal products have been proposed or produced
• Pig engineered to produce omega-3 fatty acids.
• A breed of pigs that are able to absorb plant
  phosphorus more efficiently and as a consequence
  the phosphorus content of their manure is reduced
  
• Sheep that express antibodies in milk

5
GMOs - Plants

• Genetically modified plant products soybean,
  corn, canola, and cotton seed oil.
•  Genes encode
• herbicide resistance,
• insect resistance,
• drought tolerance,
• frost tolerance,
• delayed fruit ripening and other traits.

6
GMO Plants

• Flavr Savr tomato
• the first commercially grown genetically
  engineered food granted a license for human
  consumption
• Produced by the Californian
• company Calgene 1992
• Sold in 1994, and was only available for a few
  years before production ceased

7
Flavor-sav Vs Normal

• More resistant to rotting and softening by adding
  an antisense gene which interferes with the
  production of the enzyme polygalacturonase (see
  RNA interference).
• Softening makes the tomato more susceptible to
  being damaged by fungal infections.

• Wide Variety of Tomatoes
• Picked BEFORE they are ripe.still very firm
• Artificially ripen with ethylene gas
• Easier handling and shelf life

8
Flavr Savr

• Flavr Savr tomatoes could be allowed to ripen on
  the vine, without compromising their shelf-life
  because the inserted gene slowed the softening of
  tomatoes
• Vine-ripe fruits have better flavor and could be
  harvested like green tomatoes without greater
  damage to the tomato itself.

9
BT-Corn

• Bt corn is a variant of maize, genetically
  altered to express the bacterial Bt toxin
• Poisonous to insect pests.
• In the case of corn, the pest is the European
  Corn Borer

10
BT-Corn

• A gene from a microorganism Bacillus
  thuringiensis inserted into the corn genome.
• The gene codes for a protein toxin that forms a
  crystalline productthe product is eaten
  perforates the larval digestive tract.
• The pores allow naturally occurring enteric
  bacteria such as E. coli and Enterobacter to
  infect the insect causing death

11
BT-Corn

• In 2001, 176 varieties of bt corn were
  voluntarily withdrawn from the list of approved
  varieties by the United States Environmental
  Protection Agency
• It was found to have little or no Bt expression
  in the ears and was not found to be effective
  against second generation corn borers.
• (Current status of Bt Corn Hybrids, 2005)

12
BT-Corn

• The Bt endotoxin is not harmful to humans, other
  mammals, birds, fish, or beneficial insects
• Concerns for effects of Bt corn on nontarget
  insects
• A threat to monarch caterpillars?
• 1999 Monarch increased by 30, despite Bt corn
  accounting for 30 of all corn grown in the USA
  that year.
• The beneficial effects of Bt corn on Monarch
  populations can be attributed to reduced
  pesticide use

13
Round-up Ready

• Roundup is the brand name of a systemic,
  broad-spectrum herbicide
• Produced by Monsanto
• The active ingredient glyphosate

14
Round up Ready

• Glyphosate is the most used herbicide in the USA
  , and Roundup is the number one selling herbicide
  worldwide since at least 1980.
• Monsanto also produces seeds which grow into
  plants genetically engineered to be tolerant to
  glyphosate which are known as Roundup Ready
  crops. The genes contained in these seeds are
  patented.

15
Roundup Ready Crops

• In 1996, genetically modified Roundup Ready
  soybeans resistant to Roundup became commercially
  available, followed by Roundup Ready corn in 1998
  
• Current Roundup Ready crops include soy, maize
  (corn), canola, sugar beet, and cotton, with
  wheat, and alfalfa still under development.

16
No Till Farming

• Using Round-Up eliminates ALL plantsexcept
  those that are genetically modified
• No need to till (plow-turn over) the fields.
• Preserves the top soil

But plow-based farming in this region cultivated
an unexpected yield the loss of fertile topsoil
that literally blew away in the winds
17
Glyphosate

• The main active ingredient of Roundup is a simple
  amino acid analogue
• In the early 1970s, it was discovered that
  glyphosate, inhibits the activity of an enzyme
  necessary for making certain amino acids.
• These amino acids are needed for protein
  synthesis in plants

ITS LIKE an amino acid but NOT an amino acid
Stops protein synthesis!!!
18
Glyphosate inhibits EPSPS

• T he enzyme is
• 5-enolpyruvylshikimate-
• 3-phosphate synthase
• (abbreviated EPSPS)
• Plays a key role in the biochemical pathway that
  makes aromatic amino acids phenylalanine,
  tyrosine, and tryptophan.
• This enzyme is only present in plants and
  microorganisms, such as bacteria and fungi.
• Not present in animals and humans

19
Glyphosate Resistance Not Found in plants

• In spite of extensive spraying with Roundup no
  resistant plant species had been detected.
• Monsantoconsidered the economic advantage of
  resistant plants
• Spray extensively with Roundup---kills weeds but
  not crops---if crops are resistant to glyphosate.
  
• NO NEED TO TILL THE SOIL

20
Bacteria also make amino acids

• The target of glyphosate, EPSP synthase is also
  present in bacteria
• A search for resistant bacteria was undertaken.
• The idea is that if a glyphosate-resistant enzyme
  from bacteria could be transferred to plants it
  might make the plants resistant to the herbicide.

Bacteria evolve much faster than plants
21
Glyphosate Resistance Found!

• The C4 strain of Agrobacterium
• A species of bacteria that was found growing in
  the waste-fed column at a factory that made
  glyphosate.
• The EPSP synthase enzyme from this bacterium (C4
  EPSP synthase) was almost completely insensitive
  to glyphosate

22
Agrobacterium tumefaciens

• This bacterium infects plants and injects DNA
  from a plasmid into plant cells
• Injected DNA enters the nucleus and becomes
  incorporated into the plant chromsomes.
• Under normal circumstances Agrobacterium
  tumefaciens causes gall tumors in plants

23
Roundup Ready Cloning

• The C4 EPSP bacterial gene was cloned and
  inserted into a bacterial plant vector in order
  to prepare for cloning into plants.
• The Monsanto C4 EPSP cloning vectors first
  patented September 13, 1994

24
Roundup Ready Cloning

• A plasmid vector that will work in E. coli
• Needs also characteristics that allow the plasmid
  to work in Agrobacterium tumefaciens.
• Needs a promoter..to turn on the gene in plants!

25
Roundup Ready Cloning

• 35S Resistance
• 5
  aaaaaaaaaaa 3

• A plant promoter (P-35S) is inserted at the 5'
  end.
• This promoter is the 35S promoter from
  cauliflower mosaic virus (CaMV).
• The 3' end of the gene is modified by inserting
  the polyadenylation site (NOS 3') from the
  nopaline synthase gene of the tumor-inducing (Ti)
  plasmid from Agrobacterium tumefaciens.

26
Roundup Ready Transformation

• Agrobacterium tumefaciens. infects plants and
  injects DNA into plant cells where it enters the
  nucleus and becomes incorporated into the plant
  chromsomes.
• The recombinat DNA is transferred and no tumors
  are formed.

27
Roundup Ready

• Roundup Ready soybean was the first crop plant
  produced by Monsanto.
• Today, 90 of the soybean crop in the USA
  consists of Roundup Ready plants.
• You can't buy soybean products that don't come
  from genetically modified plants.

28
GMO
29
How to make a Genetically Modified Plant

• Isolate gene that direct cells to make protein of
  interest
• (From bacteria in the sewers of the chemical
  plant making RR)
• Attach the gene to the promoter that works in
  plant
• (Califlower mosaic virus 35S)
• Insert the promoter-gene and a gene for
  selectable marker into plant cells
• Agrobacterium tumefaciens

• Allow the genetically altered cells to grow into
  plants.

30
How to Detect a GMO

• Isolate DNA from plant tissue and food products.
   
• (PCR) is used to assay for evidence of
• the 35S promoter that
• drives expression of the glyphosate resistance
  gene and many other plant transgenes.   

LOOK FOR THE GENETIC DIFFERENCE??
31
35S PROMOTER INDICATES GMO

• Herbicide resistance correlates with an insertion
  allele the 35S promoter that is readily
  identified by electrophoresis on an agarose
  mini-gel.
•  

• Amplification of tubulin, a protein found in all
  plants, provides evidence of amplifiable DNA in
  the preparation, while tissue from wild-type and
  Roundup Ready soy plants are positive controls
  for the 35S promoter.  

32
Control for PCR

• Tubulin is one of several members of a small
  family of globular proteins
• The most common members of the tubulin family are
  a-tubulin and ß-tubulin, the proteins that make
  up microtubules.
• Microtubules are assembled from dimers of a- and
  ß-tubulin.
• Tubulin was long thought to be specific to
  eukaryotes

33
Two PCR reactions are performed for each plant or
food sample.

•  One primer set amplifies the 35S promoter from
  cauliflower mosaic virus.
• The presence of a 35S product is diagnostic for
  the presence of a transgene.
• The 35S promoter is used to drive expression of
  the glyphosate (Roundup) resistance gene or Bt
  gene in edible crops.  

• A second primer set amplifies a fragment of a
  tubulin gene and controls for the presence of
  plant template DNA.
•  
• Since the tubulin gene is found in all plant
  genomes, the presence of a tubulin product
  indicates amplifiable DNA in the sample isolated.
   
• Tubulin is a housekeeping gene

34
Results of a GMO Test
35
PCR to Detect GMO

• The following primer sets were used in the
  experiment
• 5'-CCGACAGTGGTCCCAAAGATGGAC-3' (Forward Primer)
• 5'-ATATAGAGGAAGGGTCTTGCGAAGG-3' (Reverse Primer)
• 5'-GGGATCCACTTCATGCTTTCGTCC-3' (Forward Primer)
• 5'-GGGAACCACATCACCACGGTACAT-3' (Reverse Primer)

• PCR Characteristics
• Denaturing step  94 C 30
• Annealing step   60 C 30
• Extending step   72 C 30
• 34X..
• 35S ----162 base pairs
• Tubulin-187 base pairs

36
RG DP BS AS EC AS KR FD
CB AM c
TL ASca TJ TF SC DH DH DH
20001600 1000 500 20001600 1000
500
Results May 2010 15 Samples were successfully
amplified with only 4 products testing for GMO
FD Wheat (but we had not wheat products?? S
C TF DH corn pops
37
No Results.PCRs did not work
JO MS RH
AC AC BIR OSC ATM DD TP
C
38
PCR for Corn Transposon/Tubulin
M JP JS CC CG TL SM M ML SH
DR
39
GMO PCR Results.Biorad/Carolina Kits
M SL AB NB CW CT RH LB RB WM
RP TS AH HB EB CV AL AK M M LH
NF BF TW MH SB ZP MM S35 Promoter
CHIPS
Cracker Veg.
pepperoni Tor. Chip
pretzel PC

500
Tubulin Control PSII Control ?
PC
40
Results

• 14 Food items were tested for genetic
  modification using the S35 promoter from CMV as a
  marker
• Tubulin or Photosystem II used for a negative
  control
• 9 samples had PCR results
• 5 samples showed results for the S35 promoter

• Problem cracker and tortilla chip PCR product
  wrong size. Chips and pretzel dont have
  controls

41
Conclusions

• Tortilla chips, pretzels, veggie pepperoni and
  club crackers appear to contain food from GMOs
• Wrong size products, no tubulin make conclusion
  regarding pretzel and cracker suspect
• Surprizewheat products have generally not been
  reported to contain this genetic modification.
  Perhaps they also contain corn or soy products.
  Further testing would be necessary to confirm.
• Need to optimize the procedure.

42
Acknowledgement

• Funding for this project was made available by a
  generous grant from the Florida Farm Bureau.