Step 1: DNA Extraction

Find an organism with a specific trait that you want to add to another organism and
extract its DNA.
Step 2: Gene Cloning
Clone the gene that controls the specific trait you are interested in by separating it from
the rest of the genes within the DNA that was extracted.
Step 3: Gene Design
Design the gene to produce the desired enzyme (protein) in a specific way in a different
organism (the crop plant).
Step 4: Transformation
Insert the gene into the cells of a crop plant. This can be done using a gene gun,
agrobacterium, microfibers, or electroporation.
Step 5: Backcross Breeding
Use (traditional) backcross breeding to produce a high yielding crop that exhibits the
characteristics of an elite crop line and the desired trait from the transgene. The
resultant crop plant is called a GMO, in other terms genetically modified organism.

1. Denaturation (separation of two strands of DNA by temperatures of around 94 to 98 degrees

Celsius) in pcr everything in controlled by temperature.

2. Annealing (binding of DNA primer to the separated strands. I believe it Occurs at 50 to 65 degrees
Celsius, which is lower than the optimal temperature of the DNA polymerases)

3. Elongation (elongation of the strands using the DNA primer with heat-stable DNA polymerases,
most frequently Taq (Thermus aquaticus) or Pfu (Pyrococcus furiosus) polymerases. And it occurs
around over 70 degrees Celsius)
The process of genetic engineering
Genetic engineering is widely used in biological research. Mouse models are engineered for
biomedical studies, bacteria are engineered to produce medications such as insulin, and crops are
engineered for agriculture. All of these products of genetic engineering were created using the
same basic steps: identifying a trait of interest, isolating that genetic trait, inserting that trait into
the genome of a desired organism, and then growing the engineered organism (Figure 1). These
steps are explained in detail below, using examples from Monsanto as the details of their
technologies are publicly available. Other major companies such as Syngenta, BASF, Dow,
Bayer, and Du Pont use similar methods, as outlined in brief on their respective websites [3, 4, 5,
6, 7].
Step 1: Identify a trait of interest
In order to identify a desirable new trait scientists most often look to nature. Successful
discovery of a new genetic trait of interest is often a combination of critical thinking and luck.
For example, if researchers are searching for a trait that would allow a crop to survive in a
specific environment, they would look for organisms that naturally are able to survive in that
specific environment. Or if researchers are aiming to improve the nutritional content of a crop,
they would screen a list of plants that they hypothesize produce a nutrient of interest.

An example of a trait currently in GMOs that was identified through this combination of luck
and critical thinking is tolerance to the herbicide Roundup (see this article). Monsanto created
Roundup Ready plants after finding bacteria growing near a Roundup factory that contained a
gene that allowed them to survive in the presence of the herbicide [8]. Although it is not on the
market in the United States, Syngenta has designed Golden Rice with an increased amount of
pro-vitamin A, which the human body may turn into the vitamin A (see this article). Researchers
at Syngenta identified the gene sequence that produces pro-vitamin A and compiled a list of
plants to screen with that sequence [9]. With a little luck, there was a plant in nature, maize, that
contained a gene that would make Golden Rice produce pro-vitamin A at a level that could meet
the nutritional needs of vitamin A deficient communities.

Step 2: Isolate the genetic trait of interest

Comparative analysis is used to decode what part of an organisms genetic makeup contains the
trait of interest. The genomes of plants with the trait are compared to genomes in the same
species without the trait, with the goal of identifying genes present only in the former [8]. The
genomes of different species with the same trait may also be compared in order to identify a
gene, as was the case while developing Golden Rice [9]. If there is no database of genetic
information for comparison, scientists will purposefully delete, or knock out, parts of the
genome of interest until the desired trait is lost, thereby identifying the genes that lead to the
trait.

In order to expedite this process, Monsanto has developed and patented a method known as seed
chipping [8]. Through this method Monsanto shaves off parts of seeds for high-throughput
genetic sequencing while leaving the rest of the seeds viable for planting. This creates a genetic
database for plants before they are even grown, where a barcode system is used to match plants
to their genotypes. Researchers may then use this database to identify new traits of interest as
well as to optimize the desirable traits in a crop by selecting for the best genotypes based on
plant phenotypes.

Step 3: Insert the desired genetic trait into a new genome

Altering the genome of plant seeds is difficult due to their rigid structure. Many biotech
companies use gene guns that shoot metal particles coated with DNA into plant tissue with a .
22-caliber charge [8]. Monsanto no longer uses gene guns, but instead takes advantage of
bacteria, called Agrobacterium tumefaciens, that naturally invade seeds and alter plants by
inserting pieces of their own DNA into a plants genome.

In biotechnology research it is common to genetically engineer bacteria to produce a desired

protein. This is done by using enzymes to cut and paste a DNA strand of interest into a plasmid,
which is a small, circular molecule of DNA [10]. Bacteria are then shocked using heat or
electricity so that the cells accept the engineered plasmid. By modifying A. tumefaciens, which is
easier to modify than plant seeds themselves, researchers may use the bacterias naturally
invasive behavior as a Trojan horse for inserting desirable traits into a crops genome.
Step 4: Growing the GMO
After a genetic trait has been successfully inserted into an organisms genome, the modified
organism must then be able to grow and replicate with its newly engineered genome. First, the
genotype of the organisms must be checked so that researchers are only propagating organisms
in which the genome was modified correctly.

Biotech companies invest large sums into keeping these plants alive and reproducing once they
have been successfully created. The companies use special climate-controlled growth chambers,
and biologists often check on the plants by hand to make sure that they are growing as expected
[8].

During this process biotech companies will use automated machines, such as Monsantos GenV
planter, in order to track plants and calculate optimal seeding and growth conditions to create the
best possible yields. GMO seeds often come with instructions on spacing and nutrition that result
from these studies.

Future directions for the creation of GMOs

Humans ability to modify crops for improved yields and nutrients in a given environment is a
keystone of agriculture. The technological advancement from selective breeding to genetic
engineering has opened up a large realm of possibilities for the future of our food. As techniques
for genetic engineering, such as new RNAi- and nuclease-based technologies that allow for
direct modification of the genome (see this article and this article), steadily improve, our ability
to create new GMOs will also grow [11]. As our scientific capabilities expand it is essential that
we discuss the ethics and ideals surrounding GMOs so that we may effectively and safely use
this technology in a way that is acceptable to the public.
 Table 1.
Summary of the FDAs Inventory of Completed Biotechnology Consultations on Genetically
Engineered Foods as of June 30th, 2015. Crops listed in order of relative abundance of
genetically engineered crop consultations (corn having the most consultations). This information
is available to the public:
http://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=Biocon

Chelsea Powell is a PhD student in the Chemical Biology Program at Harvard University.

This article is part of the August 2015 Special Edition, Genetically Modified Organisms and Our
Food.

References
1. Questions & Answers on Food from Genetically Engineered Plants. U.S. Food and Drug
Administration. U.S. Food and Drug Administration, 22 June 2015.
http://www.fda.gov/Food/FoodScienceResearch/Biotechnology/ucm346030.htm
2. Cossins, Daniel. Will We Ever Eat Genetically Modified Meat? BBC. BBC, 9 Mar. 2015.
http://www.bbc.com/future/story/20150309-will-we-ever-eat-gm-meat
3. http://www.syngenta.com/global/corporate/en/products-and-innovation/research-
development/rdapproach/Pages/research-areas.aspx
4. https://www.basf.com/en/company/research/our-focus/plant-biotechnology.html
5. http://www.dowagro.com/innovation/
6. http://www.cropscience.bayer.com/en/Products-and-Innovation/Research-and-Innovation.aspx
7. http://www.dupont.com/industries/agriculture.html
8. Boyle, Rebecca. How To Genetically Modify a Seed, Step By Step. Popular Science.
Popular Science, 24 Jan. 2011. http://www.popsci.com/science/article/2011-01/life-cycle-
genetically-modified-seed
9. Paine, Jacqueline A., Catherine A. Shipton, Sunandha Chaggar, Rhian M. Howells, Mike J.
Kennedy, Gareth Vernon, Susan Y. Wright, Edward Hinchliffe, Jessica L. Adams, Aron L.
Silverstone, and Rachel Drake. Improving the Nutritional Value of Golden Rice through
Increased Pro-vitamin A Content. Nature Biotechnology 23.4 (2005): 482-87.
http://www.ncbi.nlm.nih.gov/pubmed/15793573
10. Genetic Engineering. BBC. BBC, 2015. Web.
http://www.bbc.co.uk/education/guides/zg2bkqt/revision/2
11. Hsu, Patrick D., Eric S. Lander, and Feng Zhang. Development and Applications of
CRISPR-Cas9 for Genome Engineering. Cell 157.6 (2014): 1262-278.
http://www.sciencedirect.com/science/article/pii/S0092867414006047
12. Biotechnology Consultations on Food from GE Plant Varieties. Biotechnology
Consultations on Food from GE Plant Varieties. FDA, 30 June 2015.
http://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=Biocon

http://sitn.hms.harvard.edu/flash/2015/how-to-make-a-gmo/ time : 11.07 tgl

4/4/2017