Appendix H SP1

Biotech Science Fair 2008

Secondary School Competition
Level: Upper Secondary
Category: Life Sciences
Project Title: Scientific Investigation on Genetically Modified Food
in Singapore
Team Members: Deanna Lee Jia Yi (Team Leader)
Liu Huimin Christine Ranita Yogeeswaran
Caryl Wong Hui Lin Yap Teik
LynnSponsor Teacher(s): Mrs Ranee Mohan School:
Singapore Chinese Girls School


Cont ent s Page
Item Page

1.

Abstract

3

2.

Declaration of Guidance from External Mentors / Establishments

4

3.

Introduction

4

4.

Theoretical Background

5

5.

Investigative Approach

11

6.

Resources

11

7.

Methodology

11

8.

Safety Measures

14

9.

Findings

14

10.

Analysis of Findings

14

11.

Relevance to Practical Applications

19

12.

Conclusion

19

13.

References

21

14.

Acknowledgements

24



Abst r ac t
The aim of this project is to establish a list of consumed foods that have been
genetically modified and are sold in Singapore. According to the legislations in
Singapore, it is not compulsory for wholesalers to label their imports if they were
genetically modified. There are also no regular tests established to check if
genetic engineering has been carried out on these imports. As a result, the
consumers are often unaware of the source of their platter. Through this study,
we hope to increase the public awareness on the presence of genetically
modified food among our daily foods.Background research was carried out on
different foods from different sources. Food samples were then collected to
investigate if they have been genetically modified. DNA was extracted from the
food samples and specific GM sequence was amplified using Polymerase Chain
Reaction (PCR). Agarose gel electrophoresis was carried out to separate the
bands of the PCR product. The gels were stained and analyzed for the presence
of a GM band. The results showed that all our samples have been genetically
modified.




2
Dec l ar at i on of Degr ee of Gui danc e f r om Ex t er nal Ment or s /
Est abl i shment sWe had help from Miss Christy Goh, a trainer with Genecet
Biotechnologies Pte Ltd. Her role was mainly to teach us the molecular
techniques and to help troubleshoot when we had technical problems.
Throughout the course of this project, Miss Goh explained to us the principles
behind the techniques that we used during experiments.

I nt r oduc t i on
This project was selected due to the ongoing debate about the need to label GM
foods. This is also in conjunction with the Biotech 2008 A World in Crisis
Biotechnology to the Rescue theme. Scientists claim that the production of
Genetically Modified Foods first started due to the food shortages around the
world. This food shortage has escalated in the recent years and is viewed as an
on coming crisis in the near future. This means that people will be coming into
contact with genetically modified food more often. With this in mind, we wanted to
study the extent of the presence of genetically modified organisms in the foods
we consume and at the same time increase public awareness so that they can
make informed decisions on whether or not to consume genetically modified
foods.


Theor et i c al Bac k gr ound
3
Genetically modified food is food that has been biologically engineered to enable
it to look more appealing, enhance its taste, increase yield or to increase its
resistance to pests. The International Service for the Acquisition of Agri-biotech
Applications reported in 2007, that a total of 52 countries have been granted
regulatory approvals for biotech crops to be imported for food and feed use, and
for release into the environment since 1996. A total of 615 approvals have been
granted for 124 events for 23 crops. [1] The Singapore Genetic Modification
Advisory Committee (GMAC) also states that there is no current legislature or
guidelines in Singapore that requires manufactured and imported genetically
modified food to be labeled [2]. Manufacturers are allowed to leave out or even
remove the labels that state the products have been genetically modified. This is
done out of fear that Singaporeans may not be willing to consume GM food if it
was labeled. Dr Wong Kwok Onn, Head of Survey and Safety Review Branch at
the Agri-Food and Veterinary Authority mentioned this in a recent Straits Times
article. [3]. Therefore consumers in Singapore are unable to identify the
difference between genetically modified and non-genetically modified products
amongst their daily essential food items.

Our group has carried out a survey to study the awareness of Singaporeans on
GMO food. From the results (Figure 1.1), we concluded that Singaporeans,
despite knowing what genetically modified foods are, are unable to differentiate
between genetically modified foods such as corn and soyabean apart from foods
that have undergone selective breeding such as Guapple.
4
Since scientists have yet to fully evaluate the effects GM food on our health and
our environment, consumers of GM food are unaware of the potential benefits or
dangers of GM food.
There is a possibility that GM Food is actually beneficial to mankind. The World
Health Organisation (WHO) had estimated that up to half a million children suffer
from Vitamin A deficiency and will eventually become blind. Scientists have
created Golden Rice which has enhanced nutritional value of Vitamin A. They
believe this would fix the Vitamin A Deficiency crisis in third world countries. [4]
Aside from health benefits, growing and producing GM food has also been made
to be more efficient. This is achieved through providing the plant with pest and
disease resistance or increased crop tolerance to a wider range of climates, or
making the food more attractive to the consumer. This would allow farmers to
worry less about their yields and profits. [5]Since there are no conclusive
research on the effects of the artificially altering genes of foods, concerned
members of the public are worried about their own health and the potential
damage to the environment such as the forming of superweeds. Researchers
have found that GM rapeseed can blow into and contaminate neighbouring farms,
and that different GM strains can interbreed, producing superweeds that are
resistant to a wide range of herbicides. A farmer from Manitoba had experienced
this when he had sprayed glycophosphate, which left the rapeseed unaffected
but killed the surrounding grass. Another situation of superweeds also caused a
wheat farm to be towered over with weeds growing two feet above the wheat
crops.[6]
5
Furthermore, the United Kingdom's Sunday Times [6] states that organic farmers
are most at risk, as their organic farms may be contaminated by the superweeds,
and this might lead to them no longer being able to claim organic statuses and
will thus lose their livelihood. Situations such as these have led to
demonstrations, public protests, debates and campaigns being held to object the
manufacture of GM food. Activists from Greenpeace [7] and Friends of the Earth
International (FOEI) [8] have been campaigning against GM food.However, there
are supporters of GM food, such as Cropgen, which states that GM technology is
independently tested and that their reports show that regulations are in place.
Their report offers a guide to the data collected on the four main GM crops
approved for consumption in the UK, as well as an overview of the regulatory
procedures in the UK and the European Union. The report claims that GM
technology provides an environmentally friendly system of farming and the
potential for many consumer benefits.[9] This claim is supported by another
paper which assesses the safety of foods derived from genetically modified crops.
The report touches on the development of the concept of substantial equivalence,
which is the comparison of the food derived from the GM crop with a traditional
counterpart that is generally accepted as safe based on the history of human
food use. This ensures that foods derived from GM crops are as safe and as
nutritious as the currently consumed plant derived foods. [10]Biotechnologists
have claim that GM is the same as traditional breeding that traditional farmers
have been doing for the past 100 years; mixing and matching and getting the
best trait from like species through a process of natural selection.[11] However,
6
the Alliance for Bio-Integrity shows through an overview on GM food, that during
natural selection, the transferred genes are conveyed in complete groups and in
a fixed sequence that harmonizes with the sequence of genes in the partner cell.
In contrast, bioengineers isolate a gene of interest from one type of organism and
splice it haphazardly into the DNA of a dissimilar species. [12]
Furthermore, the Alliance for Bio-Integrity have reported that genetic modification
process involves selecting the section of DNA that promotes gene expression in
a pathogenic virus, and fusing it with the gene of interest before it is transplanted
into another organism. As the transplanted gene is foreign to its surroundings
and cannot properly function without further modification, the behavior of the
transplanted gene may be altered, and may disrupt the coordination of processes
carried out within the modified organism and may even cause unknown, unstable
substances to be produced as a result. It has also been reported in an article
written by Irish Doctors Environmental Association, that approximately 40% of all
GM foods on sale contain DNA from a soil bacterium, which produces an
insecticide (Bt toxin). As a result, such plants are classified as insecticides in the
United States. A publication of clinical studies on the human health effects of GM
food has been conducted and this study highlighted the possibility of GM material
being potentially passed from the consumed food to the bacteria in the gut.
[13]The same article also reported that rats fed with GM maize have significant
alterations in blood cell numbers: higher white blood cell counts; and lower
reticulocyte counts, increased blood sugar and decreased kidney weight. Since
7
such changes in the bodies of rats were observed, there may be a possibility that
our human bodies may produce a delayed reaction in years to come.[13]
As the advantages and disadvantages of production of GM food have not been
proven yet, we would like to establish a list of food consumed in Singapore that
are genetically modified with the use of a GMO Investigator kit. This is to allow
consumers to have a choice in their food selection, if they should choose to avoid
genetically modified food products.







Figure 1.1 Survey results on which food sold in Singapore are thought to be
Genetically Modified.







8

Which food is thought to be Genetically Modified and sold in
Singapore
90
108
89
111
85
50
60
70
80
90
100
110
120
20
30
40







I nvest i gat i ve Appr oac hIn this investigative experiment, we aimed to
establish a list of GM food in Singapore. To do so, the experiment was carried
out with the Biotechnology Explorer GMO Investigator Kit that was purchased.
DNA was extracted from different food samples, and the kit required us to set up
polymerase chain reactions (PCR). This kit made use of duplex PCR whereby
two pairs of primers simultaneously amplify two target sequences in the DNA.
The Electrophoresis of PCR products was carried out, followed by the staining of
gels and analysis of our results to determine which foods have been genetically
modified.
Resour c es
We were very fortunate that our school already had equipment for molecular
laboratory work in the Lifescience laboratory. This included micropipettes, PCR
machine and electrophoresis chambers. We had to purchase the laboratory kit
(InstaGene Matrix) for DNA, PCR Mastermix, GMO primers and Plant (Green)
primers.

9
Met hodol ogy
Extraction of DNA from Food Sample500l of InstaGene Matrix was pipetted into
each sterile 1.5l microtube.The water bath was set to 95C.1g of each food
sample and controls were weighed out and put into the mortar.5mL of distilled
water was added to 1g of the test sample.Contents in mortar were grinded with
pestle until a smooth suspension was obtained. 5ml of distilled water was added,
again and grinded further, so as to obtain a smooth mixture for pipetting.50l of
each grinded sample was pipetted into a labelled sterile microtube containing
500l of Instagene Matrix.Microtube containing the mixture of InstaGene Matrix
and 50l of ground slurry were flicked, placed on a foam float and incubated in
waterbath at 95C for 5 minutes.

The contents in the tubes were spun down in a microcentrifuge at 13.4rpm for 5
minutes.
The tubes were stored in the freezer.

Setting up of PCR ReactionsStored tubes were removed from the freezer to
defrost. PCR Mastermix and Plant (Green) primers were prepared. 300l of PCR
Mastermix to 6l of plant primers. The PCR mixture containing the plant primers
was pipetted into the individual PCR tubes for the plants. The process was then
repeated for the GMO primers.20l of sample was pipetted from the DNA extract
into each of the 2 PCR tubes, one containing Plant primers and the other, GMO
primers.
The PCR tubes were placed in the PCR thermocycler, with the specifications
listed below:

Table 1.1: Specification for PCR
Step Function Temperature/C Duration/min No. of
cycles
Initial Denaturation Denature 94 2 1
PCR Amplification Denature
Anneal
Extend
94
59
72
1
1
2
40
Final Extension Extend 72 10 1
Hold Hold 4 Indefinite 1

Gel Electrophoresis of PCR Samples
10

10l of 5X Orange G Loading Dye was added into each PCR sample tube. It was
mixed well by pipetting up and down gently.

1X TAE Buffer was poured into the electrophoresis gel chambers.

20l of PCR sample was loaded into individual wells.

20l of PCR molecular weight ruler was loaded into the first well of every gel.

Positions of the samples in each well were recorded.

Gels were run at 100V for 30 minutes.


Staining of the Gels

Agarose Gels from the Electrophoresis Chambers were transferred into the Gel
Staining Trays.The gels were stained with 100X Fast Blast DNA Stain for 5
minutes.The gels were de-stained by using tap water multiple times, until the
bands could clearly be seen.
Saf et y Measur esWe wore gloves at all times and sterilized all our equipment
with ethnol before and after each use. We used appropriate tools for different
instances, for example, we used a pipette filler to transfer and measure liquids
which might be harmful. We had read the kit instructions thoroughly before
following them exactly as instructed. We did not return chemicals, once removed
from the bottles, back into the bottles unless instructed to do so by our teacher or
mentor.
Our experiments were very safe as we abided strictly to the instructions on the kit.
Furthermore, we had teacher mentors who guided us during each step and made
sure we stayed out of any danger.
Fi ndi ngsAfter the electrophoresis of the PCR products, the staining process of
the gels enabled the bands to be clearly seen. The optimum results of the GMO
11
positive and negative control was achieved. Our results recorded in Table 1.2
showed that all the tested food samples have been genetically modified.

Anal ysi s of Resul t s
As the validity of our results depend on the bands generated in the GMO positive
and negative controls, we had to check the results of the controls first:

1. To ensure that the PCR had worked, we checked for the presence of a
455 bp band in the GMO positive control + plant primers lane and the
presence of a 200 bp band in the GMO positive control + GMO primers
lane.
2. The presence of a 455 bp band in the GMO negative control + plant
primers lane indicated that the DNA was successfully extracted from the
certified non-GMO control.

3. The PCR reactions were confirmed to be uncontaminated as there was no
200 bp band from the negative control + GMO primers lane.

And from there on, we could deduce our results by checking for the presence of
a 455bp band that indicated the test food's DNA had been successfully extracted.
The presence of a 200bp band indicated that the test sample had been
genetically modified, while the absence of the 200 bp band indicated that the test
12
sample is not a GM food. The results of our controls and test samples were
recorded in Table 1.2
13

Table 1.2 Analysis of Electrophoresis Gel Run Results
Sample Plant band (455bp) GMO band (200bp)

Result
1. Thai Sweet Corn

Present Present

GMO
Positive
2. Malaysian Salted
Soyabeans
Absent Absent

N/A
3. Malaysian Sweet
Corn
Present Present

GMO
Positive
4. Japanese Cherry
Tomtaoes
Present Present

GMO
Positive
5. Thai Organic
Tomatoes
Present Present

GMO
Positive
6. Malaysian
Tomatoes
Present Present

GMO
Positive
7. Australian Carrot
Present Present

GMO
Positive
8. Thai Solo Papaya
Present Present

GMO
Positive
9. Hong Kong
Papaya
Present Present

GMO
Positive
10. Malaysian Papaya
Present Present

GMO
Positive
11. Malaysian
Honeydew

Present Present

GMO
Positive
12. Beijing Cabbage Present Present
GMO
Positive
14
13. US Iceberg
Blue Chip
Cabbage
Present Present
GMO
Positive
14. Australian Wong
Bok
Present Present
GMO
Positive
15. China Broccoli Present Present
GMO
Positive
16. Positive GMO
Food Control
Present Present

GMO
Positive
17. Negative GMO
Food Control
Present Not Present

GMO
Negative
* For Gel Pictures please refer to the Appendix attached to this report.

In addition to our list of genetically modified foods, we have come up with a world
map which pinpoints the countries from which the tested GM positive food
samples from our gel electrophoresis results were imported from. At a glance,
countries that export the highest number of tested GM positive crops can be
identified. This will be to the publics advantage. Those who wish to keep away
from genetically modified foods can then be wary of the food imported from
specific countries that tend to have GM foods in their imports.






Fig. 2 World Map of Countries that Export GM Foods
15

*A larger image of the world map is attached to the Appendix.

Rel evanc e t o Pr ac t i c al Appl i c at i ons
As an addition to our list of genetically modified food sold in Singapore, the group
has plans to develop a household kit that can be used easily, for the detection of
GM food. The list of GM Foods in Singapore that the group has compiled only
includes common foods consumed like tomatoes, papaya and corn. There are a
lot more varieties of plant food for us to test on. The actual process of testing
requires a detailed understanding of scientific concepts like molecular genetics
and skills involving the use of micropipettes and in depth analysis. Thus
households lacking an acquired level of scientific knowledge cannot use the
16
actual process of testing for GM Foods. They would also lack access to
laboratory equipment like PCR Machines which are costly.

Therefore, in order to develop this user-friendly household kit that will not require
the user to understand scientific concepts. We plan to come up with the kit by
making use of proteins and antibodies. Our research studies are still on-going to
develop such a kit.

Conc l usi on
Based on the analysis of our results, a large majority of corns, tomatoes and
papayas were genetically modified. As our food samples were largely imports
from other countries, our conclusion therefore fits our initial assumption that
some of Singapores imported foods are genetically modified.

There were some surprising discoveries, for example, food samples that were
clearly labeled organic, non-GMO or GMO Free gave positive results when
tested. Dr Wong mentioned in The Straits Time Article [3] that manufacturers do
not accurately label their foods because they are afraid that Singaporeans will be
unwilling to consume their products. This is the first probable cause for this
unexpected result.


17
As a follow up to our current list of genetically modified foods, the group had
planned to create a user-friendly household kit to allow users to test their foods
easily. However, due to budget and time constrains, we will only be able to carry
out the theoretical aspect of this research. We intend on using the kit to Identify
positive GM food by detecting for the presence of Neomycin Phosphotransferase
II (NPTII) protein. We use the NPTII protein because plant transformation usually
involves the use of marker genes which confer antibiotic resistance, thus is is
found in most GM foods. One of the most commonly utilized antibiotic resistance
markers is the NPTII gene, which induces resistance to the antibiotics kanamycin
and geniticin in transformed tissue. Such foods that contain the Neomycin
Phosphotransferase II (NPTII) protein are cucumber, tomatoes, kiwi fruits and
barley.

Currently, we are still researching on the different methods to detect NPTII in
GM foods. These methods include the Dot Assay and a modified ELISA
(Enzyme-Linked-Immuno Sorbent Assay) method.

The dot assay is based upon the ability of nitrocellulose membrane to eliminate a
positive interfence without a prior electrophoretic protein separation step, which
is extremely time-consuming. This dot assay has its advantages, as it provides a
convenient and rapid procedure for analyzing crude cell extracts for the presence
of NTPII. Also, it is comparable in sensitivity to the conventional electrophoretic
18
procedure. However, we, as students, may not be able to use this dot assay
method as it involves radioactivity, which may be hazardous and dangerous.

The modified ELISA method is modified in the sense that it has an Iblock. This
Iblock gives a consistently low background and eliminates washing procedures.
Some advantages of this modified ELISA method are the elimination of flase
positives due to non-specific protein kinase reactions (which may happen in the
dot assay), the ability to test a diverse range of species, it reduces the amount of
plant tissue needed, provides for rapid identification of the NPTII protein, and it is
safe and inexpensive.


Ref er enc es
1. Jane Fyksen, Crops Editor; Biotech Crops Expanding Worldwide; Agriview
- Biotech Crops Expanding Worldwide; [March 6, 2008]Available URL:
http://www.agriview.com/articles/2008/03/06/crop_news/crops01.txt

2. Genetically Modified Food - Frequently Asked Questions; GMAC - Genetic
Modification Advisory Committee, Singapore. [February 20, 2008]
Available URL:
http://www.gmac.gov.sg/Index_FAQs_Genetically_Modified_Foods.html

3. Jessica Lim; Genetically Modified Food; Do you know how much of your
food is Genetically Modified?; 14 March 2008; The Straits Times, Page
Home 1

4. Golden Rice is part of the solution - Biofortified rice as a contribution to the
alleviation of life-threatening micronutrient deficiencies in developing
countries;Golden Rice Project Homepage; [March 10, 2008]Available
URL:http://www.goldenrice.org

19
5. Genetically Modified Food (GMF); MAF Information Services;Pastoral
House 25 The Terrace PO Box 2526 Wellington, NEW ZEALAND; [March
6, 2008]
Available URL:
http://www.maf.govt.nz/mafnet/schools/activities/gmfbio.htm

6. Jonathan Leake, Science Editor; Rapeseed Frankencrops in Canada
Breed Superweeds; Sunday Times (UK) - August 12, 2001; [March 10,
2008]
Available URL:
http://www.organicconsumers.org/patent/superweeds081301.cfm

7. Say no to genetic engineering; Say no to genetic engineering -
Greenpeace International; [March 11, 2008]
Available URL: http://www.greenpeace.org/international

8. GMOs out of our food and the environment; gmos out of our food and the
environment - friends of the earth international 2008-02-28; [March 11,
2008]
Available URL: http://www.foei.org/en/campaigns/gmo

9. Alex Kirby; BBC News Online environment correspondent; GM foods safe
say supporters; BBC News - Sci/Tech - GM foods safe say supporters; 7
December, 2001; [March 11, 2008]
Available URL: http://news.bbc.co.uk/1/hi/sci/tech/1694077.stm

10. Food and Chemical Toxicology; Volume 42, Issue 7; July 2004, Pages
1047-1088; Safety Assessment, Detection and Traceability, and Societal
Aspects of Genetically Modified Foods European Network on Safety
Assessment of Genetically Modified Food Crops

11. Sonia Chopra; Biotechnologys Standard Bearer; September 7, 2000;
[March 6, 2008]Available URL: http://www.agbioworld.org/biotech-
nfo/articles/interviews/bearer.html

12. ALLIANCE FOR BIO-INTEGRITY - Preserving the Safety of Our Food, the
Health of Our Environment, and the Harmony of Our Relationship with
Nature; WHY THE VENTURE TO GENETICALLY ENGINEER OUR
FOOD OFFENDS SCIENCE, RELIGION, AND THE BILL OF RIGHTS - A
Summary Overview; [March 6, 2008]
Available URL: http://www.biointegrity.org/Overview.html

13. Irish Doctors Environmental Association [IDEA]; Genetically modified food
and health - a cause for concern?; [March 10, 2008]
Available URL: http://www.ideaireland.org/gmfoodhealth.htm

20
14. M.J. McKenzie 7 V. Mett 7 P.E. Jameson; Modified ELISA for the
detection of neomycin phosphotransferase II in transformed plant species;
Plant Cell Reports (2000) 19: 286289

15. LaRhee Henderson, A. Gururaj Rao, and John Howard Pioneer Hi-Bred
International, Inc., Department of Biotechnology Research, Johnston, Iowa
50131; An Immunoaffinity Immobilized Enzyme Assay for Neomycin
Phosphotransferase II in Crude Cell Extracts; ANALYTICAL
BIOCHEMISTRY 194,64-68 (1991)

16. Esther Cabanes-Bastos, Anthony G. Day and Conrad P. Lichtenstein
Centre for Biotechnology, Imperial College of Science, Technology and
Medicine, London SW7 2AZ (U.K.); Short Communications: A sensitive
and simple assay for neomycin phosphotransferase II activity in transgenic
tissue; Gene, 77 (1989) 169-176 Elsevier GEN 02943

17. N. Ramesh and William R. A. Osborne1 Departnent of Pediatrics,
University of Washington, Seattle, Washington 98195; Assay of Neomycin
Phosphotransferase Activity
in Cell Extracts; ANALYTICAL BIOCHEMISTRY 193,316-318 (19%)

18. STEVEN G. PLATT AND NINGSUN YANG Agracetus, 8520 University
Green, Middleton, Wisconsin 53562; Dot Assay for Neomycin
Phosphotransferase Activity in Crude Cell Extracts; ANALYTICAL
BIOCHEMISTRY 162,52%535 (1987)


19. Glen J. Rogan, Joel E. Ream, Sharon A. Berberich, and Roy L. Fuchs
Plant Science Technology, Monsanto Agricultural Company, 700
Chesterfield Village Parkway, St. Louis, Missouri 63198; Enzyme-Linked
Immunosorbent Assay for Quantitation of Neomycin Phosphotransferase
I1 in Genetically Modified Cotton Tissue Extracts; J. Agric. ~oodch em.
1092, 40, 1453-1458 1453





Ac k now l edgement
21
22
We would like to express our heartfelt gratitude to our school, Singapore Chinese
Girls' School for giving us this wonderful opportunity. We thank our Principal, Mrs
Low Ay Nar and all our Vice-Principals for their support. We thank Mrs
Yogeeswaran (HOD/Science) and Mrs Cha their keen support and involvement
in this project . We also thank our teacher in charge, Mrs Ranee Mohan and our
mentor, Miss Christy Goh for their guidance and encouragement.