Erythropoietin (EPO)

Production
ERYTHROPOIETIN (EPO) PRODUCTION
Application in the field of:
MEDICINE

I. INTRODUCTION
Erythropoietin is a hormone that your kidneys naturally make to stimulate the
production of red blood cells. It is produced primarily by specialized cells in the kidneys
in response to low oxygen levels in the blood. Genetic engineering techniques are used to
clone and express the gene encoding erythropoietin in host organisms. The EPO gene can
be inserted into expression vectors, which are then introduced into host cells, such as
bacteria (e.g., Escherichia coli), yeast, or mammalian cell lines (e.g., Chinese hamster
ovary cells), to produce large quantities of recombinant EPO.

II. PURPOSE/OBJECTIVES
The primary objective is to produce recombinant EPO in large quantities. By
introducing the EPO gene into host cells, the cellular machinery can transcribe and
translate the gene, leading to the synthesis of EPO protein. This recombinant EPO can
then be harvested and purified for therapeutic use. Overall, inserting the EPO gene into
expression vectors and introducing them into mammalian cell lines like CHO cells allows
for the efficient, scalable, and quality-controlled production of recombinant EPO,
fulfilling the objective of providing a safe and effective therapeutic product for treating
anemia and related conditions.

III. STEPS
Isolation of the EPO Gene:
 The EPO gene is isolated from a natural source, typically human genomic DNA or
a cDNA library derived from mRNA.
Design of Expression Vector:
 An expression vector is selected or designed to accommodate the EPO gene.
Insertion of EPO Gene into Expression Vector:
 The EPO gene is inserted into the expression vector using molecular cloning
techniques.
 Restriction enzymes are used to cut both the vector and the EPO gene at specific
sites, creating compatible ends for ligation.
 DNA ligase is then used to join the EPO gene fragment with the vector, forming
recombinant DNA.
Transformation of Host Cells:
 The recombinant expression vector containing the EPO gene is introduced into
host cells capable of expressing and secreting EPO.
 Mammalian cell lines, such as Chinese hamster ovary (CHO) cells, are
commonly used due to their ability to perform proper post-translational
modifications essential for EPO activity.
Selection and Screening:
  Transformed host cells are subjected to selection pressure to identify cells that
have successfully integrated the recombinant DNA.

Expression and Production:

 Selected host cells containing the recombinant EPO gene are cultured under
optimized conditions to induce gene expression and protein synthesis.
 EPO protein is synthesized within the host cells and secreted into the culture
medium.
 Bioreactor systems are often used to scale up production and achieve high
yields of recombinant EPO.
Purification and Characterization:
 The culture medium containing secreted EPO is harvested and subjected to purification
processes to isolate the recombinant protein from other cellular components.

IV. PICTURES

REFERENCES:
https://my.clevelandclinic.org/health/articles/14573-erythropoietin
https://go.drugbank.com/drugs/DB00016
https://www.yourhormones.info/hormones/erythropoietin/
https://www.medicalnewstoday.com/articles/erythropoietin
ARCTIC APPLES
Application in the field of:
AGRICULTURE

I. INTRODUCTION
Arctic® apples are a groundbreaking innovation in agriculture, representing a new
era of genetically modified fruits. Developed by Okanagan Specialty Fruits Inc.,
Arctic® apples are engineered to exhibit reduced enzymatic browning when sliced or
bruised, offering consumers a longer-lasting and visually appealing fruit experience.
Enzymatic browning is a natural process that occurs in fruits and vegetables when
certain enzymes react with oxygen upon exposure to air, leading to the formation of
brown pigments. This process often affects the appearance, texture, and flavor of sliced
or bruised produce, contributing to food waste and consumer dissatisfaction. chemical
additives or preservatives. The Arctic apple is the juiciest newcomer to produce aisles.
It has the special ability to resist browning after being cut, which protects its flavor and
nutritional value.

II. PURPOSE/OBJECTIVES
Arctic® apples have been genetically modified to suppress the expression of the
polyphenol oxidase (PPO) enzyme, which plays a key role in enzymatic browning. By
reducing PPO activity, Arctic® apples are able to maintain their natural color and
freshness for an extended period after being cut or bruised, without the need for
chemical additives or preservatives. The creation of Arctic® apples is driven by the
desire to improve the quality, freshness, and marketability of apples while reducing
food waste and advancing agricultural innovation. These objectives align with broader
goals of sustainability, consumer satisfaction, and technological progress in the food
industry.

III. STEPS
Genetic material is stored in our DNA and divided into functional units called
genes. The genes are “read” by copying the DNA sequence into a related molecule
called RNA. The RNA copy functions as a blueprint that instructs the cell how to build
the product for that gene, which is called a protein. The production of PPO enzymes,
therefore, can be blocked by simply removing their RNA blueprints. To do so,
researchers used a tool from molecular biology called RNA interference (RNAi). RNAi
is a natural biological process that recognizes and destroys specific RNA structures.
Biologists can use RNAi to lower PPO levels by introducing RNA sequences that cause
the degradation of PPO RNA. Using this technique, researchers developed an anti-PPO
gene that makes anti-PPO RNA, which destroys the PPO RNA before it can be used to
make PPO enzymes.

Once scientists created the anti-PPO gene, they needed to safely introduce it into the
apple genome. To make a variety called the Arctic Golden, researchers began with
Golden Delicious apple buds and inserted an engineered piece of genetic material called
a transgene that contained the anti-PPO gene. After confirming that the plant received
the transgene, the saplings were then allowed to grow into mature trees, one of which
produced the apple that is now known as the Arctic Golden.
 IV. PICTURES

REFERENCES:
https://en.wikipedia.org/wiki/Arctic_Apples
https://arcticapples.com/
https://www.nongmoproject.org/blog/gmo-apples-an-apple-a-day-may-not-brown-right-
away/
https://www.scientificamerican.com/article/genetically-modified-browning-resistant-apple-
reaches-u-s-stores/
https://sitn.hms.harvard.edu/flash/2018/arctic-apples-fresh-new-take-genetic-engineering/

BIOLUMINESCENT PLANTS
Application in the field of:
ENVIRONMENT

I. INTRODUCTION
Bioluminescent plants represent a fascinating intersection of genetic engineering and
natural phenomena. These plants are genetically modified to produce light through a
process called bioluminescence, similar to that found in fireflies, certain marine
organisms, and some fungi. This bioluminescence imparts a captivating glow to the
plants, illuminating them in low-light conditions and creating a striking visual display.
The resulting genetically modified plants emit a soft, natural-looking light that can be
visible to the naked eye, creating an enchanting ambiance in gardens, landscapes, and
indoor settings. Bioluminescent plants have garnered significant interest for their
aesthetic appeal, educational value, and potential applications in sustainable lighting
solutions.

II. PURPOSE/OBJECTIVES
The development of bioluminescent plants contributes to our understanding of
plant biology, genetic engineering, and the mechanisms underlying bioluminescence in
nature. Studying the genes and pathways involved in plant bioluminescence can yield
insights into fundamental biological processes and inform future research endeavors.
Bioluminescent plants offer a sustainable and eco-friendly alternative to conventional
lighting sources. By harnessing the natural light-producing capabilities of
bioluminescent organisms, these plants reduce energy consumption, minimize light
pollution, and contribute to environmental sustainability.

III. STEPS
1. Gene Selection: The first step is to select genes responsible for bioluminescence from
bioluminescent organisms such as fireflies, marine bacteria, or fungi. The most
commonly used gene for bioluminescence is luciferase, which catalyzes the light-
emitting reaction.
2. Vector Construction: Once the bioluminescent gene(s) are selected, they are inserted into
a plant transformation vector. This vector typically contains regulatory elements such as
promoters and terminators to control the expression of the bioluminescent gene in plant
cells.
3. Plant Transformation: The constructed vector is introduced into plant cells using a
suitable transformation method. This can include Agrobacterium-mediated
transformation, biolistic particle bombardment, or protoplast transformation. The goal is
to deliver the vector into the plant cells' genome.
4. Regeneration: Transformed plant cells are cultured under selective conditions to
encourage their growth and development into whole plants. This often involves the use of
tissue culture techniques to regenerate shoots or callus tissue from transformed cells.
5. Selection and Screening: Transformed plant tissues are screened for the presence of the
bioluminescent gene(s) using molecular techniques such as PCR or Southern blotting.
Additionally, plants showing bioluminescence under specific conditions may be selected
for further analysis.
 6. Characterization: Transgenic plants that successfully express the bioluminescent gene(s)
are further characterized to assess the brightness, duration, and stability of the
bioluminescent phenotype. This may involve quantitative measurements of light emission
using specialized equipment.
7. Propagation and Maintenance: Transgenic plants with desirable bioluminescent traits are
propagated through vegetative or sexual reproduction methods to generate a stable
population of bioluminescent plants. These plants are then maintained under controlled
conditions for further study or commercial use.

V. PICTURES

REFERENCES:
https://environment.co/experiments-in-bioluminescence-can-trees-glow-in-the-dark/
https://www.smithsonianmag.com/innovation/creating-a-new-kind-of-night-light-glow-in-
the-dark-trees-9600277/
https://blog.suny.edu/18414/future-cities-lit-by-beautiful-bioluminescent-trees/
https://sitn.hms.harvard.edu/flash/2020/glowinthedarkplants/