1 report for biotech fair 2008

8/11/2019 1 Report for Biotech Fair 2008

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Appendix H SP1

Biotech Science Fair 2008Secondary School Competition

Level: Upper SecondaryCategory: Life SciencesProject Title: Scientific Investigation on Genetically Modified Food

in SingaporeTeam Members: Deanna Lee Jia Yi (Team Leader)Liu Huimin Christine Ranita Yogeeswaran

Caryl Wong Hui Lin Yap TeikLynnSponsor Teacher(s): Mrs Ranee Mohan School:

Singapore Chinese Girls School

Contents 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


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Abstract

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.

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Declaration of Degree of Guidance from External Mentors /

EstablishmentsWe 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.

Introduction

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.

Theoretical Background

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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.

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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]

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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,

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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

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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

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Investigative ApproachIn 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.

Resources

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.

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Methodology

Extraction of DNA from Food Sample500l of InstaGene Matrix was pipetted intoeach sterile 1.5l microtube.The water bath was set to 95C.1g of each foodsample and controls were weighed out and put into the mortar.5mL of distilledwater 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 ofeach grinded sample was pipetted into a labelled sterile microtube containing500l of Instagene Matrix.Microtube containing the mixture of InstaGene Matrixand 50l of ground slurry were flicked, placed on a foam float and incubated inwaterbath at 95C for 5 minutes.

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

Setting up of PCR ReactionsStored tubes were removed from the freezer todefrost. PCR Mastermix and Plant (Green) primers were prepared. 300l of PCRMastermix to 6l of plant primers. The PCR mixture containing the plant primerswas pipetted into the individual PCR tubes for the plants. The process was thenrepeated for the GMO primers.20l of sample was pipetted from the DNA extractinto each of the 2 PCR tubes, one containing Plant primers and the other, GMOprimers.The PCR tubes were placed in the PCR thermocycler, with the specificationslisted below:

Table 1.1: Specification for PCR

Step Function Temperature/C Duration/min No. ofcycles

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

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10l of 5X Orange G Loading Dye was added into each PCR sample tube. It wasmixed 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 GelStaining Trays.The gels were stained with 100X Fast Blast DNA Stain for 5minutes.The gels were de-stained by using tap water multiple times, until thebands could clearly be seen.

Safety MeasuresWe 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.

FindingsAfter 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

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positive and negative control was achieved. Our results recorded in Table 1.2

showed that all the tested food samples have been genetically modified.

Analysis of Results

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

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sample is not a GM food. The results of our controls and test samples were

recorded in Table 1.2

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13. US IcebergBlue ChipCabbage

Present PresentGMO

Positive

14. Australian Wong

Bok

Present PresentGMO

Positive

15. China Broccoli Present PresentGMO

Positive

16. Positive GMOFood Control

Present PresentGMO

Positive

17. Negative GMOFood Control

Present Not Present GMONegative

* 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

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

Relevance to Practical Applications

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

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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.

Conclusion

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.

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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

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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.

References

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 - GeneticModification 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 yourfood 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 thealleviation of life-threatening micronutrient deficiencies in developingcountries;Golden Rice Project Homepage; [March 10, 2008]AvailableURL:http://www.goldenrice.org

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http://www.agriview.com/articles/2008/03/06/crop_news/crops01.txthttp://www.gmac.gov.sg/Index_FAQs_Genetically_Modified_Foods.htmlhttp://www.goldenrice.org/http://www.goldenrice.org/http://www.gmac.gov.sg/Index_FAQs_Genetically_Modified_Foods.htmlhttp://www.agriview.com/articles/2008/03/06/crop_news/crops01.txt


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5. Genetically Modified Food (GMF); MAF Information Services;PastoralHouse 25 The Terrace PO Box 2526 Wellington, NEW ZEALAND; [March6, 2008]

Available URL:http://www.maf.govt.nz/mafnet/schools/activities/gmfbio.htm

6. Jonathan Leake, Science Editor; Rapeseed Frankencrops in CanadaBreed 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 theenvironment - 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 safesay supporters; BBC News - Sci/Tech - GM foods safe say supporters; 7December, 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, Pages1047-1088; Safety Assessment, Detection and Traceability, and Societal

Aspects of Genetically Modified Foods European Network on SafetyAssessment 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, theHealth of Our Environment, and the Harmony of Our Relationship withNature; WHY THE VENTURE TO GENETICALLY ENGINEER OURFOOD OFFENDS SCIENCE, RELIGION, AND THE BILL OF RIGHTS - ASummary Overview; [March 6, 2008]

Available URL: http://www.biointegrity.org/Overview.html

13. Irish Doctors Environmental Association [IDEA]; Genetically modified foodand health - a cause for concern?; [March 10, 2008]

Available URL: http://www.ideaireland.org/gmfoodhealth.htm

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http://www.maf.govt.nz/mafnet/schools/activities/gmfbio.htmhttp://www.organicconsumers.org/patent/superweeds081301.cfmhttp://www.greenpeace.org/internationalhttp://www.foei.org/en/campaigns/gmohttp://news.bbc.co.uk/1/hi/sci/tech/1694077.stmhttp://www.agbioworld.org/biotech-nfo/articles/interviews/bearer.htmlhttp://www.agbioworld.org/biotech-nfo/articles/interviews/bearer.htmlhttp://www.biointegrity.org/Overview.htmlhttp://www.ideaireland.org/gmfoodhealth.htmhttp://www.ideaireland.org/gmfoodhealth.htmhttp://www.biointegrity.org/Overview.htmlhttp://www.agbioworld.org/biotech-nfo/articles/interviews/bearer.htmlhttp://www.agbioworld.org/biotech-nfo/articles/interviews/bearer.htmlhttp://news.bbc.co.uk/1/hi/sci/tech/1694077.stmhttp://www.foei.org/en/campaigns/gmohttp://www.greenpeace.org/internationalhttp://www.organicconsumers.org/patent/superweeds081301.cfmhttp://www.maf.govt.nz/mafnet/schools/activities/gmfbio.htm


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14. M.J. McKenzie 7 V. Mett 7 P.E. Jameson; Modified ELISA for thedetection 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, Iowa50131; An Immunoaffinity Immobilized Enzyme Assay for NeomycinPhosphotransferase II in Crude Cell Extracts; ANALYTICALBIOCHEMISTRY 194,64-68 (1991)

16. Esther Cabanes-Bastos, Anthony G. Day and Conrad P. LichtensteinCentre for Biotechnology, Imperial College of Science, Technology andMedicine, London SW7 2AZ (U.K.); Short Communications: A sensitiveand simple assay for neomycin phosphotransferase II activity in transgenictissue; 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 NeomycinPhosphotransferase Activityin Cell Extracts; ANALYTICAL BIOCHEMISTRY 193,316-318 (19%)

18. STEVEN G. PLATT AND NINGSUN YANG Agracetus, 8520 UniversityGreen, Middleton, Wisconsin 53562; Dot Assay for NeomycinPhosphotransferase Activity in Crude Cell Extracts; ANALYTICALBIOCHEMISTRY 162,52%535 (1987)

19. Glen J. Rogan, Joel E. Ream, Sharon A. Berberich, and Roy L. FuchsPlant Science Technology, Monsanto Agricultural Company, 700Chesterfield Village Parkway, St. Louis, Missouri 63198; Enzyme-LinkedImmunosorbent Assay for Quantitation of Neomycin PhosphotransferaseI1 in Genetically Modified Cotton Tissue Extracts; J. Agric. ~oodch em.1092, 40, 1453-1458 1453

Acknowledgement

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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.

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