A New Use for the Biotechnology Explorer GMO Investigator Kit
Summary
The purpose of the lab was to evaluate the efficiency of a new classroom kit for BioRad called
the Biotechnology Explorer GMO Investigator Kit. Genetically Modified Organisms (GMOs)
are organisms that have had foreign genes implanted in them with the goal of creating
“healthier” and “stronger” organisms. The initial objective of this experiment was to determine
if food labeled GMO free was actually GMO free. This portion of the lab did not yield much
success, but was primarily used to test the functionality of the BioRad kit itself. The heart of the
experiment was determining whether or not the BioRad kit could detect the concentration of
GMOs present in the given food sample, not just yield positive and negative results. By
performing the appropriate experiments it was found that there is a correlation between
concentrations of genetically modified genes that the kit is known to test for and the brightness
of the bands that appear after electrophoresis. More tests will need to be performed before a
precise quantitative correlation can be established.
Introduction
Genetically Modified Organisms (GMO)
Scientists can now isolate a gene from a particular organism giving them the ability to study its
impact on biomedical networks and organismal phenotypes. For the most part the function of
most genes is unknown at this point. The government is currently placing regulations on GMOs
for biosafety reason (Strauss 2003). Genetically modified crops are sold and exported to many
different countries around the world and in the United States about sixty percent of all the
processed foods in supermarkets have some form of a genetically modified (GM) ingredient
(Strauss 2003). The most common GM foods are soy, corn, canola, and some other vegetables
are genetically modified as well.
The first crops that were genetically modified expressed traits that were encoded by exogenous
and also were pest resistant. These different traits were found to be independent of each other,
which means that they can transfer the genes between phylogenically different organisms.
Instead of having to breed traits into different organisms, they now have a more efficient and
precise way to achieve the desired result (Strauss 2003). Examples of traits that been genetically
engineered are changes to plant reproduction, stature, and lipid and lignocellulose chemistry.
These genes are examples of genomics-guided transgenes (GGT).
Current Research and Field Testing
People are studying the GM crops to try to ensure that they are as nutritious or more in
comparison to their counterparts. There are some genes that would make the crop healthier for
humans to consume. For example, adding a gene that limits the amount of a toxic pesticide can
potentially be beneficial by reducing human exposure to the toxic pesticide, but at the same time,
chemicals like these might hurt the insect population, making their pollen harmful or sterile. At
this point nothing as been proven to resolve either of these concerns because there has not been
sufficient data collected.
The most important research done currently is field research because scientists are not sure how
a GGT will react in or on a certain organism and the only way to find out is by performing an
actual experiment. They have found that changing some genes in order to encourage stresstolerance in agronomic environments may not help the “fitness” of a plant and may actually hurt
it (Strauss 2003). Field research also needs to be conducted in order to identify valuable GGTs.
Many countries do not have the luxury to complete as many field tests as in the US and China;
European countries have limited most testing to recombinant field trials. In the US most field
trials are permitted as long as certain standards are met (Strauss 2003). The problem with field
tests is the possibility of vandalism and interference with the experiment. A costly test that
expends resources could be entirely spoiled and the actual experiment could hurt the near by
crops and organisms if not contained properly.
The current testing done by manufacturers is not that productive. The manufacturers are
required to extensively examine the crops before they sell them in order to try to prove that they
are not allergenic nor toxic. But only the manufacturers are doing the testing. There are
effectively no independent studies performed on GM crops, which means that the manufacturers
might not be as stringent as they should (Hopkin 2001).
Testing the GM crops can also be a problem because researchers typically cannot produce
enough of the modified protein to actually test it. The manufacturers attempt to solve this
problem by having bacteria produce the protein they wish to test. When doing this, the protein
often created by bacteria is not the same as the protein created by the crop rendering the test
useless (Hopkin 2001). One current test performed administers an amount of GM crops larger
than any human would consume to animals to determine the affects of the particular GM gene.
People are uncomfortable and will always be uncomfortable with advances in scientific research.
Most people are currently uncomfortable with field tests because the thought is that the test will
contaminate the ground and therefore contaminate other organisms and valuable resources.
Gaining public approval may be as important as biological advances at this point because
researchers will only be able to do a limited amount of research until GE crops are accepted.
Pros and Cons of Genetic Engineering
Robert B. Horsch is the vice president of product and technology cooperation at Monsanto
Company, an organization that plays a large role in the development and research of genetically
modified crops. Horsch believes that research in this area is necessary in order to solve many of
the food problems around the world. The primary benefit of GM crops is the decreased use of
pesticides, a trend already established among farmers (Nemecek 2001). The land also can
produce significantly more crop than it could initially. Greater yield on existing acres could also
lessen the requirement for more land dedicated to farming. Excess land not ideally suitable for
farming could be returned to wild land, which would also aid the environment.
Margaret Mellon, the director of the agricultural and biotechnology program at the Union of
Concerned Scientists in Washington, D.C., makes it clear that she is not opposed to
biotechnology. The therapeutic benefits in drugs outweigh any risks and there are no longer any
alternatives for the creation of some drugs. Mellon opposes GM crops because she does not
believe that currently there are any real benefits of GM crops (Nemecek 2001). The resistance
genes that target pests will only work for a short amount of time. After a while the pests and
viruses will evolve and scientists will need to target a new gene. Most scientists agree that gene
flow will occur which will make all crops exposed to the GMOs. Exposure has already created
herbicide-resistant weeds. There also have only been a handful of clinical studies, not enough to
actually decide whether the GM foods are healthy enough to pursue further clinical trials.
GM crops are completely unnatural, and therefore are riskier. Currently, the worst part about
GMOs is the fact that people will not know more about them until multiple clinical studies are
performed. It is theoretically possible that a resistance gene in the GM crop may “jump” from
the crop while in an individual’s stomach to bacteria in the consumer’s stomach, creating an
antibiotic resistance among disease causing bacteria (Hopkin 2001). Many people worried that
the change in genes and introduction of the new genes, which might be toxic, might make the
plant toxic or create allergic reactions that the unmodified crop would not. Adding a few genes,
controlled insertion, is much safer than introducing thousands of genes at the same time, which is
the method used when crops are crossbred the standard way.
The Experiment
The FDA’s regulations state that food can be labeled GMO free if the concentration of GMOs is
less than 5% of the total food product. One instrument used to gauge this is a BioRad product
titled: The Biotechnology Explorer GMO Investigator Kit. The kit targets the 35S promoter of
the cauliflower Mosaic virus (CaMV 35S) and the terminator of the nopaline sythase (NOS) gene
of Agrobacterium Tumerfaciens. These are the two most common GMO sequences that are
found in most all the food products that are permitted to be distributed in North America and
Europe. If either one of these sequences is found in a sample the kit is able to identify it.
Results
Testing The Kit
Corn food products were collected and samples of each were taken. BioRad provided a master
mix to use to determine whether or not there were GMOs present in the different corn products.
After polymerase chain reactions (PCR), the
products were electrophoresed against a 100 base
pair ladder to verify whether or not there were
GMOs present in the food sample. We used
Hawaiian papaya as a positive control because it is
comprised of 100% GMOs and BioRad provided a
negative control. The results after running the gel
illustrated positive results.
Figure 1: Testing the functionality of
the BioRad Kit
This gel was run to test to see if the kit
could actually identify the GMOs
present in the food samples. All of
these food products are comprised
primarily of corn and were all labeled
organic if not GMO free. This is what
was in each well:
1. 100 bp mw ladder 2. 60 bp ladder 3.
GMM free 4. PMM free 5. GMM
Papaya 6. PMM Papaya 7. GMM
White Corn Tortilla Chips 8. PMM
White Corn Tortilla Chips 9. GMM
Corn Soup 10. PMM Corn Soup 11.
GMM Corn Kernels 12. PMM Corn
Kernels
In all of the experiments two different master mixes
were used. Plant Master Mix (PMM) was created in
accordance with the given protocol from BioRad and
its purpose was to amplify all plant DNA segments
that exist in the sample. This way if there was no
DNA in a given well it can be determined that there
should be no band in the corresponding GMM well
either because it never existed or was destroyed.
GMO Master Mix (GMM) was also created in
accordance with the BioRad protocol. It targeted
and then amplified the 35S promoter of the
cauliflower Mosaic virus (CaMV 35S) and the
terminator of the nopaline sythase (NOS) gene of
Agrobacterium tumerfaciens. These are the two
most common GMO sequences that are found in most all the food products that are permitted to
be distributed in North America and Europe.
All of the PMM products ran to about 300 base pairs on the gel. (See Figure 1) This means that
all of the samples successfully contain DNA and the DNA segments were correctly targeted and
amplified. In the other wells the results varied. In lanes three, five and seven there appear to be
bands at abut 200 base pairs that signify that there are traces of the target GMO segments in the
DNA sample. There should not be any band in lane 3 because this is the negative control. In
this lane there is the GMO free sample that was provided by BioRad and GMM. The GMM was
only supposed to target the GMO segments that the GMO free sample was not supposed to
contain. This suggests that there was possible contamination in the water used to dilute the food
samples initially. All of the lanes containing GMM also exhibit bands of less than 100 base pairs
as well. These are all of the remainder DNA that was amplified but does not contain the other
DNA. This is known because when looking at the GMM Papaya sample in the gel there is no
second band because the entire food sample is 100 % GMO so there would be no remainder
DNA that would run farther. This also prompts the question if the concentration of GMO can be
detected because the GMM Papaya band is brighter than the rest of the GMM bands and the
more GMOs the sample contains the less of a second band present in the well.
Identifying Contamination and Tests for Concentration Detection
Two smaller experiments were carried out at
the same time on the two sides of the 100
base pair ladder as seen in figure 2. After
the first experiment was performed, the
negative control appeared suspicious due to
the slight positive results. In order to find
the source of contamination the controls
were separated and run individually with
autoclaved water in attempt to remove the
slight contamination from the water to
prevent the exhibition of bands that signify
the content of GMO segments. The second
of the two experiments was to determine
whether or not the BioRad kit could
illustrate a quantitative response regarding
Figure 2: Identifying Contamination and
Tests for Concentration Detection
The possible contamination of the provided
controls for the experiment prompted the
study of just the master mixes to determine
any contamination. The right lanes were run
as a small experiment to determine how the
kit would react to different concentrations of
GMOs and how they would exhibit the
results.
the level of concentration in a food sample.
Papaya was chosen to be the food sample
because it is entirely comprised of GMOs
1. PMM- 2. PMM 3. PMM + H2O 4. GMM5. GMM- 6. GMM + H2O 5. GMM 7. empty
8. 100 bp ladder 9. empty 10. 10% Papaya
GMM 11. 50% Papaya GMM 12. 100 %
Papaya GMM
and autoclaved water was used to dilute the papaya.
There were bands in all of the loaded wells, which means that the water was still contaminated.
The hope was that autoclaving the water would remove the band from the lane containing GMM
and water, which it did not. For the next experiment autoclaved water was to be used. The
results from this gel were consistent with the results from experiment 1; the same samples were
exhibiting similar and expected characteristics.
The faint band that appeared in the PMM + H2O was due to contamination because the master
mix should not have any present in the mix. This small band can be attributed to possible
contamination once again which means that the autoclaved water may still not be clean enough.
BioRad agreed to send “superclean water” to see if it could solve to problem.
The other side of the gel showed a possible correlation between the concentrations of GMO
samples and band length. The 100% papaya GMM sample had the shortest band length of about
500 base pairs long. The 50% papaya GMM sample had a length of about 250 base pairs and the
10% papaya GMM had a length of less than 100 base pairs long. The results seem a bit
confusing because the purpose of the GMM was solely to amplify existing GM genes. Because
of the number of cycles during PCR this should not have been a very noticeable difference. But
because there was a clear variation further tests were run to further explore the possibility of
there being a correlation between concentration of GMO and band length.
Expanded Concentration Testing
The purpose of the experiment was to examine the results of testing different concentrations of
GMO. Mixing different concentrations of papaya, which is known to be 100 % GMO with
different amounts of water, varied the concentrations. A full spectrum of concentrations was
examined ranging from 0% papaya all the way up to 100% papaya. A difference in the bands
was expected, but nothing much that informative appeared in our gel. Like the sample from the
first experiment, which contained GMM and papaya, the band length of these samples appeared
to be about 200 with a little variance. This meant that nothing could be deduced from this
experiment alone and the same experiment would have to be run again.
On the PMM side of the gel, the right set of wells, the 50% papaya PMM and the 0% papaya
PMM did not appear on the gel at all, although the
100% papaya PMM and the 10% papaya PMM did
show up and appeared to be the same length. It is
puzzling that the 50% papaya PMM sample did not
appear on the gel, but it is a positive result to see
the 0% papaya PMM sample not appear because it
means that there is no longer contamination in the
water samples.
The ladder on this side of the gel also appears to
be very weak, which means that it is impossible to
determine the lengths of the PMM samples
Figure 3: Expanded Concentration
Testing
This experiment was performed to
determine if the slight results in the past
experiment held true for a larger
concentration gradient. A difference in
the bands was expected, but all of the
papaya GMM samples look like they
yielded about the same results. On the
bottom half of the gel a select number of
PMM samples were run. Neither the 50%
papaya PMM sample nor the 0% papaya
PMM sample appeared.
because the ladder becomes blurry.
This gel was run ineffectively. By running the
samples in the split manner is was impossible to
accurately compare the GMM bands with the
PMM bands. Also, by running two separate rows,
the voltage at which they were run had to be
significantly decreased in order to run them for the
same amount of time. This means that all of the
bands were about equal to each other and almost
ran into the second set of wells. They were also
Upper: 1. 0% Papaya GMM 2. 5 %
Papaya GMM 3. 10 % Papaya GMM 4.
Empty 5. No result 6. 20% Papaya GMM
7. 30% Papaya GMM 8. 50% Papaya
GMM 9. 100% Papaya GMM 10. Empty
11. 100 bp ladder
Lower: 1. 0% Papaya PMM 2. empty 3.
10% Papaya PMM 4. empty 5. empty 6.
50% Papaya PMM 7. 100% Papaya PMM
8. empty 9. 100bp Ladder
very fuzzy which made it hard to determine which
band on the ladder they corresponded to.
To actually attain results from this experiment, the
gel needed to be re-run preferably in a single
welled gel in attempt to let the bands separate from each other and not run into the second set of
wells.
Retesting for the possible Concentration Correlation
An interesting pattern appeared in the GMM portion of this re-run gel. Because the data was
virtually useless from the previous gel, some of the concentration variant samples were re-run
producing interesting results. It
appeared that there is a relationship
between the brightness of the band and
the percent concentration of papaya in
each sample. (See figure 4) The
darkest of the bands was found in the
100% papaya GMM sample. To
determine what the actual brightness
signifies quantitatively more tests must
be performed.
Like in experiment 3 only the 100%
papaya PMM sample and the 10%
papaya PMM sample appeared.
Nothing again happened in the 0% and
50% lanes. There must have been
something contaminating the 50%
papaya PMM sample preventing them
from amplifying during the PCR stage
in the previous experiment. There were
no results in the 0% papaya PMM
sample again, which means that the
Figure 4: Retesting for the possible
Concentration Correlation
The expectation in this gel was to see variance in
the bands of the papaya GMM samples. As it
turned out, the brightness of the band appears to
correlate to the percentage of GMO in the
sample where the higher the concentration of
GMO the brighter the band. The few PMM
samples that were run illustrated the same results
as in the previous experiment and neither the
50% papaya PMM or the 0% papaya PMM
samples appeared
1. 100bp Ladder 2. empty 3. 100% Papaya
GMM 4. 50% Papaya GMM 5. 20% Papaya
GMM 6. 10% Papaya GMM 7. 5% Papaya
GMM 8. 0%Papaya GMM 9. 100% Papaya
PMM 10. 50% Papaya PMM 11. 10% Papaya
PMM 12. 0% Papaya PMM
contamination in the original samples
was from the water because there should be no band because there is no DNA present.
Discussion
The Biotechnology Explorer GMO Investigator Kit targets two separate GMO sequences that are
found in about 85 % of all GMO crops and are two of the GMOs that are allowed to be
distributed in North America and Europe. Initial tests of the kit showed that the kit is able to
identify whether or not there is GM DNA segments in the food product. The staple of this
experiment was corn and papaya. Corn was used because it frequently contains GMOs and
many corn products are labeled organic and GMO free. Hawaiian papaya was used because all
papaya was destroyed in Hawaii and now all the papaya produced there is 100% GMO.
The primary obstacle faced in the initial parts of the experiment was working though issues of
contamination. Negative controls were appearing to have positive results. As hypothesized, this
was due to contamination in the water. To try to remove all contamination the hood was cleaned
with hydrogen peroxide as well as all of the micropipettes. From this point on all classroom
spaces, materials, and instruments were sterilized before use. BioRad also shipped “superclean”
water that was to be used for the remainder of the experiment. The water solved the
contamination problem.
Part of the purpose of the lab overall was to also determine all flaws in the BioRad
Biotechnology Explorer GMO Investigator Kit. Confusion arose in the protocol. Because the
protocol was duplicated, one version for teachers, and a completely different version for
students, it was hard to tell what should be done when and some of the steps overlapped each
other. One specific example can be found in lesson 1 where the protocol is very unclear about
the contents of the “test” tubes for food samples when adding the appropriate amounts of the
master mixes. It was also found that the provided non-GMO food was hard to grind and make
from it useable solution.
The second phase of the experiment was to look deeper into the possible uses of the kit. The aim
of the following experiment was to determine if the kit could detect concentration and display
some sort of quantitative feedback regarding the variance in concentration. The experiment
yielded positive results and it was determined that there is a correlation between the brightness of
the band and the concentration of the respective food sample.
The next experiment would continue to test the correlation between the brightness of each band
and concentration of the sample itself. If possible a little more should be loaded into the wells to
try to permit the bands to become more apparent. Also a larger concentration gradient should be
tested to determine whether or not this holds true for all concentrations.
Materials and Methods
Experiment 1: Testing the functionality of the BioRad Kit
Used the Lesson 1: Extraction of DNA from food samples protocol to prepare the food samples.
Approximately 1.52g were measured out and used per food sample: frozen corn kernels (1.52g),
creamy corn soup (1.53g), white corn tortilla chips (1.52g), kettle corn tortilla chips (1.52g),
papaya (1.55g), and provided non-GMO sample (1.54g). Next the master mixes, PMM and
GMM, were created in accordance with the protocol and then left in the refrigerator at 4°C until
later use. Then the samples were prepared for PCR using the Lesson 2 protocol. When all tubes
are prepared place in the thermal cycler the heating process was as follows: 94°C for 2 minutes,
[94°C for 1 minute, 59°C for 1 minute, 72°C for 2 minutes] x 40 cycles, 72°C for 10 minutes and
then hold at 4°C until later use. We loaded 15 _L of each of the prepared sample into a 3 %
agarose gel with 12 wells. We used a 100 base pare ladder and ran the gel at 50 _. See results in
figure 1.
Experiment 2: Identifying Contamination and Tests for Concentration Detection
Two different experiments were conducted. For determining the source of contamination, the
following mixtures were created: “PMM mix alone”, “GMM mix alone”, “PMM mix + 20µL
H20”, “GMM mix + 20µL H20”, “GMM + Papaya100”, “GMM + Papaya50”, “GMM +
Papaya10”, “GMM + negative DNA”, “PMM + negative DNA”. For determining the effects of
varying the papaya concentrations, the following procedure was used: Papaya mix from
Experiment 1 was diluted into concentrations of 100%, 50%, and 10%. GMM mix was added to
all samples and autoclaved water was used. The above solutions were placed in the thermal
cycler and run as follows: 94˚C for 2 minutes, [94˚C for 1 minute, 59˚C for 1 minute, 72˚C for 2
minutes] x 40 cycles, 72˚C for 10 minutes and then held at 4˚C until later use. 10 µL of each of
the prepared samples was loaded into a 3 % agarose gel with 12 wells. A 100 base pare ladder
was also loaded. Electrophoresis was run at 50 _ for an hour and a half followed immediately by
staining with 10% syber green. See results in Figure 2.
**Between experiments two and three we received “superclean water” from BioRad as well as
filter tips. There appeared to be errors due to contamination, though to be from the water, in the
earlier experiments and in attempt to solve the problem we sterilized our workspace and took
apart the micropipettes to sterilize them. From this point on we only used the “superclean” water
given to us by BioRad.
Experiment 3: Expanded Concentration Testing
Used the same papaya mix that was initially used in experiment 1. Diluted the papaya into the
following concentrations of 100%, 50%, 40%, 30%, 20%, 10% papaya GMM solution. Then the
following PMM forms were made in the same manner: 100%, 50%, 10%, 0%. Solutions were
prepared in accordance with the different samples listed above. Following they were placed in
the thermal cylcer at the following temperatures: 94°C for 2 minutes, [94°C for 1 minute, 59°C
for 1 minute, 72°C for 2 minutes] x 40 cycles, 728C for 10 minutes and then held at 48C until
they were placed in the refrigerator. 10 µL of each sample were loaded into the 3 % agarose gel
at 50 _ with a 100 base pair marker. After the gels finished running they were stained in a 10%
sybergreen solution. See results in Figure 3.
Experiment 4: Retesting for the possible Concentration Correlation
Used the same papaya mix that was initially used in experiment 1 which was made according to
the Lesson 1 protocol. The dilutions of the papaya were prepared the same was as in experiment
3 and the rest of the set up for each sample according to the protocol in lesson 2. PCR tubes
were then placed in the thermal cycler which was run as follows: 94˚C for 2 minutes, [94˚C for 1
minute, 59˚C for 1 minute, 72˚C for 2 minutes] x 40 cycles, 72˚C for 10 minutes and then held at
4˚C until later use. Loaded 12 µL of each of the prepared sample into a 3 % agarose gel with two
rows of 12 wells. Used a 100 base par ladder in each row. Ran electrophoresis at 40 _ for two
hours. Stained in 10% syber green, covered in tin foil and refrigerated. See results in figure 4.
Acknowledgements
Dr. Julie Strong, Melissa Woodrow, Biotechnology Explorer Program, BioRad Laboratories.
References
Federoff, N.K., (2003). Prehistoric GM Corn. Science 302, 1158.
Hopkin, Karen (2001). The Risks on The Table. Scientific American April 2001, 60-61.
Kling, Jim (1999). Future Feast. Scientific American 1999, 88-91.
Nemecek, Sasha (2001). Does The World Need GM Foods. Scientific America 2001, 62-65.
Strauss, S.H. (2003). Genomics, Genetic Engineering, and Domestication of Crops. Science 300,
61-62.