Screening of Food Products Containing CP4 EPSPS Gene

Chelsea Hawkins, BS Student

Our Lady of the Lake College, Baton Rouge, LA

Methods

Background

DNA Extraction

GMO: Genetically Modified Organisms

Most common GMOs include: corn, soy, canola, sugar beets,
cotton, and papaya
Almost all GMOs are modified for herbicide/pesticide
tolerance
Glyphosate is modified with adjuvants that introduce genes
into the plant
Roundup is being studied as potentially more fatal than
glyphosate itself
There is evidence that glyphosate is an endocrine disrupting
chemical
Glyphosate linked to male infertility in low doses, more
specifically Roundup
Once the GM food is consumed, the effects associated with the

Add 5000L of water per

gram of food
Grind mixture until smooth
slurry forms
Add additional 5000L of
water per gram of food
Take 250L of slurry and add
to screw tube + 500L of
InstaGene Matrix buffer
Heat 5-6 minutes at 95
Centrifuge at 6000 rpm for 5
minutes
Take 50L of supernatant and
place 20L in two PCR tubes.
Rinse M&P with 10% bleach
and water
Repeat with each food.

specific gene introduced can disturb the biological processes

or the effects of the glyphosate can be more severe than the
gene itself
Health Effects: Birth Defects, Cardiovascular Disease, Cancer,
and Alteration to Endocrine, Neurological, and Reproductive
functions
EPSPS gene present in plants, but modified to CP4 EPSPS to
tolerate glyphosate
promoter, Cauliflower Mosaic Virus, and NOS terminator,

(CaMV35S)

PCR Preparations

Weigh 2.1g of agarose

Test
Test
GM
GM
NonNon Place 980mL of water in tower
(in L)
Food 1 Food 1
+ve
+ve
GM
GM
Fill tower to 1000mL with 5x
DNA
20
20
20
20
20
20
TAE buffer
Place agarose in flask
GMP
1.45 --------- 1.45 --------- 1.45 -------- Fill flask with 70mL of 1x TAE
PSP
--------- 1.45 --------- 1.45 --------- 1.45
buffer
MM
19.55
19.55
19.55
19.55
19.55
19.55
Heat mixture for 1 minute
Allow mixture to cool briefly,
Total
41
41
41
41
41
41
but not solidify
Test
Test
Test
Test
GM
GM
Non- Non- Pour mixture into gel tray
(in L)
Food 2 Food 2 Food 3 Food 3 +ve
+ve
GM
GM
Let sit 15 20 minutes
DNA
20
20
20
20
20
20
20
20
Remove comb from gel
GMP
4
-------4
-------4
-------4
-------- Place gel in electrophoresis
tray
PSP -------4
-------4
-------4
-------4
Fill tray with buffer
MM
21
16
21
16
21
16
21
16
Mix 10L of orange loading
Total
45
40
45
40
45
40
45
40
dye with each tube
Pipet 15-20L of MWR into
PCR Cycling Conditions
first well
Step
Function Temperature Duration
Cycles
Pipet 20L of each PCR
Denaturation
Denature
2 minutes
1
product into other wells
94C
Run at 100V for 30 minutes
Denature
1 minute
94C
Place gel in Fast Blast Stain
Amplification
Anneal
1 minute
40
59C
De-stain with warm water
Extend
2 minutes
72C
Use ChemiDoc to take image
Extension
Extend
10 minutes
1
72C
Hold
Hold
Indefinite
1
4C

(NOS)

Codes for 5-enolpyruvulshikimate-3-phosphate synthase enzyme

Can be found in Alfalfa, Argentine Canola, Cotton, Creeping Bentgrass, Maize,
Polish Canola, Potato, Soybean, Sugar Beet, and Wheat

Functions to decrease the binding affinity for glyphosate, which increases the
plants tolerance to the glyphosate

commonality to households. Corn meal is an item that is used for

more than making cornbread. Cornbread mix is commonly used to
make many meals. With the preference for organic foods growing,
the organic corn is also growing in popularity among households.
The knowledge of which foods are genetically modified will
continue to play a prominent role in research of health effects
associated with the consumption of these foods.
The impact of this research would reach far into all
communities. Infertility has always posed a problem regardless of
gender, race, or ethnicity. Here is something to think about: What
if something you were eating caused you to become infertile?
This research would address this issue. Perhaps glyphosate has
entered into your system and disrupted your reproductive
functions. Would there be a way to detect if this was the cause?
Most research articles focus solely on male infertility. This
experiment poses the question for future research that would
address both male and female infertility. There are reports that
establish a basis for disrupted reproductive function in animals
caused by pesticides. The validation of these concepts is
prominent and would be a step forward in preventing detrimental
glyphosate health effects.

Terminator

References

About the CP4 EPSPS Gene:

The foods in this experiment were chosen based upon their

Results

Nopaline Synthase

CP4 EPSPS

PCR

Test Food 1 (TF1): Cornbread Mix

Test Food 2 (TF2): Yellow Corn Meal
Test Food 3 (TF3): Frozen Organic Sweet Corn

A GM plant will show the presence of the CaMV35S

Promoter

DNA
Electrophoresis

Conclusion

Part of the shikimate pathway that is involved in the production of aromatic amino

1000bp
700bp
500bp

1000bp
700bp
500bp

200bp

200bp

100bp

100bp

acids and other aromatic compounds in plants

Shikimate Pathway is said not to be present in humans

It is mainly overlooked that this pathway is present in human gut bacteria,

meaning it is possible that it attributes to associated health effects

Legend:
GM: Genetically Modified
MWR: Molecular Weight Ruler
GMP: Genetically Modified Primer

Acknowledgements: I would like to express my gratitude to Dr. Ratna Gupta for guiding me throughout this experience. Her valuable feedback and constant encouragement throughout the
duration of this project have shaped my idea of laboratory research. This course, under the instruction of Dr. Gupta, was an extremely knowledgeable experience for me.

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1. Busbee,
D. C.
(2007).
Alteration
estrogen-regulated
expression
human cells induced
by the agricultural
horticultural
information
onand
these
lines.herbicide

glyphosate. Human & Experimental Toxicology, 747-752. Retrieved from

http://web.a.ebscohost.com.ezproxy.ololcollege.edu:2048/ehost/pdfviewer/p
dfviewer?sid=45af106b-d034-499d-a214-51636e76eb2e
%40sessionmgr4004&vid=1&hid=4204
2. Center for Environmental Risk Assessment. (2010). A Review of the
Environmental Safety of the CP4 EPSPS Protein. Washington, DC: ILSI
Research Foundation. Retrieved from http://ceragmc.org/docs/cera_publications/pub_01_2010.pdf
3. Feeney, D. F. (2014, April). How Roundup (AKA. Glyphosate) Affects
Your Health. Nutritional Perspectives: Journal of the Council on Nutrition,
33. Retrieved from
http://web.a.ebscohost.com.ezproxy.ololcollege.edu:2048/ehost/pdfviewer/p
dfviewer?sid=3315ae23-6fc8-4ff7-b11a45fd66cd5572%40sessionmgr4001&vid=1&hid=4204
4. ILSI Research Foundation. (2015, February 27). GM Crop Database.
Retrieved from Center for Environmental Risk Assessment: http://ceragmc.org/GmCropDatabaseEvent/MON89788