Lecture-5.1

Genetic Engineering

Genetic engineering is a set of technologies used to change the genetic makeup of cells,

including the transfer of genes within and across species boundaries to add one or more new

traits that are not naturally already found in that organism so as to produce improved or novel

organisms. Genetic engineering is the deliberate, controlled manipulation of the genes in an

organism with the intent of making that organism better in some way.  Genetic engineering

involves changing an organism’s DNA to give it some new useful traits/characteristics.

Tools used in Genetic Engineering:

Restriction enzymes also known as “molecular scissors” have a site specific cleavage property

i.e. hey can recognize and cu DNA on a specific site which is known as restriction site. Different

restriction enzymes cut DNA in different ways. Each enzyme has a different restriction site

Some Restriction enzymes cut straight across and leave “blunt ends” and some make staggered

cuts and leave “sticky ends”.

Cloning Vector:

Vector is a DNA molecule that carries foreign DNA into a host cell, replicates inside a bacterial

cell and produces many copies of itself and the foreign DNA. Plasmid is a type of cloning vector.

Plasmids are loops of DNA in bacteria. These are extra chromosomal circular DNA molecule

which is capable of self replication i.e. it can divide on its own.

Using vector as a carrier:

The vector (plasmid) and DNA to be cloned (gene of interest) are digestion/cut with same

restriction enzymes to generate complementary ends.

The foreign DNA is ligated (joined) into the vector with the enzyme DNA ligase

The plasmid carrying the gene of interest is introduced the host cells (bacterial cells) by

transformation

Genetic Engineering Process steps:

1) Isolation of Gene of Interest: The first step is to find and isolate the gene of interest (GOI) or

target gene which will be inserted into the host. The desired DNA is cleaved from the donating

chromosome by the action of restriction enzymes, which recognize and cut specific nucleotide

segments.

2) Insertion of gene of Interest into the vector: The target gene is inserted into a vector,

usually a plasmid. Plasmids are an ideal vector because they replicate easily inside host bacteria

and readily accept and transfer new genes. Plasmid is cut with the same restriction enzyme and

the target gene is ligated/added/joined to the plasmid with the help of an enzyme called DNA

ligase.

3) Transformation: The plasmids carrying the desired traits are introduced into host cells via a

process called transformation. When the host cell reproduces the plasmids also reproduce,

making multiple clones of their DNA.

4) Amplification: The bacterial cells are allowed to increase in number. As the cells divide the

plasmid containing the target gene also divides. Many copies of the target gee are produced

which results in multiple copies of the protein of interest.

4) Recovery of desired product: The desired protein product is then isolated from bacterial

cells and processed further so that it can be used.

Genetic Engineering Example: Production of Insulin:

Insulin is a protein that helps the body to regulate the level of sugar. The pancreas of a person

who has diabetes is not able to make enough insulin. Until the 1980s, a person with diabetes had

to take artificial insulin that was extracted from the body of a pig. But there were problems

associated with using nonhuman insulin. Now human insulin can be made in a laboratory. The

production of human insulin resulted from genetic engineering.

Bt Crops

What is Bt?

Bacillus thuringiensis (Bt) is a spore forming (flash animation) bacterium that produces crystals

protein (cry proteins), which are toxic to many species of insects.

How does Bt work?

Bt has to be eaten to cause mortality. The Bt toxin dissolve in the high pH insect gut and become

active. The toxins then attack the gut cells of the insect, punching holes in the lining. The Bt

spores spills out of the gut and germinate in the insect causing death within a couple days. Even

though the toxin does not kill the insect immediately, treated plant parts will not be damaged

because the insect stops feeding within hours. Bt spores do not spread to other insects or cause

disease outbreaks on their own.

1. Insect eats Bt crystals and spores.

2. The toxin binds to specific receptors in the gut and the insects stops eating.

3. The crystals cause the gut wall to break down, allowing spores and normal gut bacteria to

enter the body.

4. The insect dies as spores and gut bacteria proliferate in the body.

Bt action:

Bt action is very specific. Different strains of Bt are specific to different receptors in insect gut

wall. Bt toxicity depends on recognizing receptors, damage to the gut by the toxin occurs upon

binding to a receptor. Each insect species possesses different types of receptors that will match

only certain toxin proteins, like a lock to a key.

These crystal proteins are toxic to very specific species of insects yet harmless to humans and the

natural enemies of many crop pests (benenificial insects). The crystal proteins bind specifically

to certain receptors in the insect's intestine. Not all insects carry the same receptors allowing for

high species specificity. Humans and other vertabrates do not have these receptors in their

bodies, so the toxin is unable to affect us.

Most GM crops grown today have been developed to resist certain insect pests. There are GM

plants being developed today to produce specific vitamins, resist plant viruses and even produce

products for medical uses.

Benefits of genetic engineering:

In agriculture: Increased crop yields, reduced costs for food or drug production, reduced need

for pesticides, enhanced nutrient composition and food quality, resistance to pests and disease,

greater food security, and medical benefits to the world's growing population.

Advances have also been made in developing crops that mature faster and tolerate aluminum,

boron, salt, drought, frost, and other environmental stressors, allowing plants to grow in

conditions where they might not otherwise flourish

A number of animals have also been genetically engineered to increase yield and decrease

susceptibility to disease. For example, salmon have been engineered to grow larger and mature

faster and cattle have been enhanced to exhibit resistance to mad cow disease.

Lecture-5.2: Genetic Engineering-A Potential solution

Genetically Modified Organism (GMO) or Transgenic Organisms:

GMOs, or “genetically modified organisms,” are plants or animals that have been genetically

modified. Genetic modification involves the mutation, insertion, or deletion of genes.

Transgene is a segment of DNA containing a gene sequence that has been isolated from one

organism and is introduced into a different organism. A transgenic organism is one that carries a

foreign gene that has been deliberately inserted into its genome.

Bioremediation:

Bioremediation is the use of microbes to clean up contaminated soil and groundwater. Microbes

are very small organisms, such as bacteria, that live naturally in the environment. Bioremediation

stimulates the growth of certain microbes that use contaminants as a source of food and energy.

Contaminants treated using bioremediation include oil and other petroleum products, solvents,

and pesticides.

The use of genetic engineering to create organisms specifically designed for bioremediation has

great potential. The bacterium Deinococcus radiodurans (the most radioresistant organism

known) has been modified to consume and digest toluene and ionic mercury from highly

radioactive nuclear waste. Mycoremediation is a form of bioremediation in which fungi are used

to decontaminate the area.

Cynobacteria:

One of the most common air pollutants is carbon dioxide. Carbon dioxide is a greenhouse gas

and its main sources are from combustions such as vehicle and power generator. Since carbon

dioxide is a greenhouse gas, it traps heat and ultimately causes global warming. And global

warming then lead to a greater impact to the environment such as rises in sea water level and

even food shortage due to death of crops by high temperature.

Thus to reduce the impact caused by carbon dioxide, apart from increase the intake of carbon

dioxide by the plants, researchers from University of California, Los Angeles (UCLA) had also

genetically modified (GM) a cyanobacteria which is able to consume carbon dioxide via

photosynthesis under sunlight to produce isobutanol, a liquid fuel. With the ability to produce

fuel, it will be an incentive for energy infrastructures such as automobiles to use GM

cyanobacteria, since by using it will cut done on their cost for fossil fuel and thus able to earn

more profits. Apart from that by using the bacteria in the infrastructure, it will reduce the carbon

dioxide, minimizing pollution at its root source. Therefore, using GM cyanobacteria will be a

win- win situation for both energy infrastructure and the environment.

Oil-eating bacteria:

Even though petroleum products are the major source of energy for industry as well as

day today life, it also poses major concern over hydrocarbon release during its production. These

are released into soil, air and water which posses a great danger to the natural habitats. The oil

spills from marine water are treated using bioremediation methods.

An oil spill is an environmental hazard that is dangerous to many species of plants and

animals. One of the methods of cleaning up oil spills that has been investigated is the use of oil-

eating bacteria. These strains of soil bacteria naturally use oils in the environment as their food.

They also need some inorganic nutrients, oxygen and water in their environment in order to

survive. The oil-digesting abilities of soil bacteria are thought to vary depending on the amount

of oil found in the natural environment of different bacterial strains.

Pseudomonads are a family of bacteria that have the uncanny ability to break down and

assimilate large, complex organic compounds, such as camphor. Individual Pseudomonas strains

possess only a handful of the genes that enable it to break down the hydrocarbons in crude oil.

Dr. Ananda Mohan Chakrabarty, an Indian-born scientist figured that a strain that contained all

the genes might be able to handle a significant amount of oil, and so he inserted plasmids

containing the genes into a single strain of Psuedomonas Putida and cultivated it in his GE

laboratory. The result was a recombinant organism, a genetically modified pseudomonad

capable of breaking down (in theory, at least) large amounts of crude oil.

GloFish: The GloFish is a patented brand of genetically modified (GM) fluorescent zebrafish

with bright red, green, and orange fluorescent color. The original zebrafish from which the

GloFish was developed measures three centimeters long and has gold and dark blue stripes. In

1999, Dr. Zhiyuan Gong and his colleagues at the National University of Singapore were

working with a gene called green fluorescent protein (GFP), originally extracted from a jellyfish,

that naturally produced bright green bioluminescence. They inserted the gene into a zebrafish

embryo, allowing it to integrate into the zebrafish’s genome, which caused the fish to be brightly

fluorescent under both natural white light and ultraviolet light. Their goal was to develop a fish

that could detect pollution by selectively fluorescing in the presence of environmental toxins. It

is the first genetically modified animal to become publicly available as a pet.

See-Through Animals:

Dissecting animals for science has sparked controversies worldwide, even prompting some

companies to create computer simulations as cruelty-free alternatives. For high school students

everywhere, this revealing amphibian may be a cut above regular frogs. That’s because the see-

through frog does not require dissection to see its organs, blood vessels, and eggs.

Applications of Transgenic bacteria:

Transgenic bacteria can be used to produce human proteins.

Transgenic plants are common in agriculture.

Transgenic animals are used to study diseases and gene functions.

For rest of the examples: Refer to the Lecture’s Power Point Presentation

Lecture- 5.3

Cloning

The term cloning describes a number of different processes that can be used to produce

genetically identical copies of a biological entity. The copied material, which has the same

genetic makeup as the original, is referred to as a clone. Clones are organisms that are exact

genetic copies. Every single bit of their DNA is identical.

There are two different types of artificial cloning: reproductive cloning and therapeutic

cloning. Reproductive cloning produces copies of whole animals. Therapeutic cloning produces

embryonic stem cells for experiments aimed at creating tissues to replace injured or diseased

tissues.

Therapeutic cloning:

Therapeutic cloning refers to the removal of a nucleus, which contains the genetic

material, from virtually any cell of the body (a somatic cell) and its transfer by injection into an

enucleated egg cell (from which the nucleus has also been removed). The newly reconstituted

entity then starts dividing. After 4-5 days in culture, embryonic stem cells can then be removed

and used to create many embryonic stem cells in culture. These embryonic stem cell ‘lines’ are

genetically identical to the cell from which the DNA was originally removed. Therapeutic

cloning is also known as somatic cell nuclear transfer (SNCT).

Therapeutic cloning re-programs an adult nucleus to develop into any body part. Cells of

a particular tissue generally express a characteristic set of genes. When an adult cell’s nucleus is

transferred to an enucleated egg, the adult nucleus becomes re-programmed in the environment

of the egg. That is, genes that were not used before (switched off) become reactivated. Instead of

the adult nucleus causing the egg to behave like an adult cell, the egg causes the nucleus to go

backwards along a differentiation sequence, resulting in an embryonic type cell which can divide

into any body type.

Therapeutic cloning or Somatic cell nuclear transfer (SCNT) steps:

1. A somatic (i.e. body) cell is taken from a donor.

2. The DNA (46 chromosomes in humans) is removed from that cell.

3. A female gamete (an egg or ovum) is harvested from a second donor and enucleated, i.e.

its nucleus is removed. This removes the DNA (23 chromosomes in humans) contained in

the nucleus.

4. The DNA from step 2 is inserted into the enucleated egg; at this point in human cloning,

the egg would contain a full set of 46 chromosomes, all from a single donor. It would be

the functional equivalent of a human zygote (the single cell organism created at

conception by the fusion of sperm and ovum).

5. The resulting cell is allowed to mature for a few days, until it is a blastocyst (a pre-

implantation embryo of about 128 cells).

6. Stem cells are removed, thus destroying the blastocyst. These stem cells can develop

into any type of body cell when provided with suitable growth conditions.

Reproductive cloning:

Reproductive cloning is the process in which the newly formed embryo resulting from a

therapeutic cloning procedure, were transferred into the womb of a woman to develop into a new

organism. The scientific community overwhelmingly rejects the use of therapeutic cloning for

the purposes of human reproductive cloning.

Difference between reproductive and therapeutic cloning:

Reproductive cloning involves creating an animal that is genetically identical to a donor animal

through somatic cell (any normal body cell like skin cell) nuclear transfer. In reproductive

cloning, the newly created embryo is placed back into the uterine environment where it can

implant and develop. Dolly the sheep is perhaps the most well known example. In therapeutic

cloning, an embryo is created in a similar way, but the resulting "cloned" cells remain in a dish in

the lab; they are not implanted into a female's uterus

Therapeutic cloning does not involve the creation of a perfectly copied human being. It is

reproductive cloning that result in a copy of a specific human being. In therapeutic cloning, no

sperm fertilisation is involved nor is there implantation into the uterus to create a child.

Dolly the Sheep: Dolly was the first ever cloned animal. She was produced from a single

microscopic cell. Cloning techniques might be used widely now in some part of worlds for food

but dolly remains remarkable in being the first mammal to be cloned from an adult somatic cell,

using the process of nuclear transfer. She was cloned by Ian Wilmut, Keith Campbell and

colleagues at the Roslin Institute near Edinburgh in Scotland. She was born on 5 July 1996 and

she lived until the age of six. She died in 2003, living about half as long as a typical sheep. She

developed a lung disease common in older sheep.

Problems associated with Cloning:

Animals as Drug factories: Animals have long been used for the production of drugs. For

example, hundreds of thousands of pigs have been sacrificed over the years to provide insulin for

diabetics. Transgenesis (Transfer of genes from one organism to another) takes this process to

another level. Transgenes introduced into the nucleus of cow, sheep, and pig eggs become part of

the animal's own DNA. One such transgene causes a cow to secrete human proteins in its milk,

thus turning the cow into a living pharmaceutical factory. This process is also employed in

sheeps to produce alpha-1-antitrypsin, a drug used in the treatment of cystic fibrosis.

Scientists playing GOD: Advancements in the field of Genetic Engineering allow scientists to

create animals that are on one hand completely foreign to the earth and on the other, specifically

tailored to possess only the traits that humans desire in animals. They are tampering with nature

by mixing genes among species. By doing this they are not only disturbing the species

boundaries but also creating an imbalance in nature

Future of Cloning:

Human patients may be able to grow their own cloned organs one day. Some of the most

useful application of cloning technology will be to clone skin cells for grafting in burn

victims, or bone marrow cells for leukemia patients. The rejection danger is eliminated,

as well as the need for immunosuppressive drugs.

Cloning research may make organ transplantation a more successful process.

Also, cloning research will allow genetic manipulation to produce animals that are

disease resistant, just as research in agronomy has yielded similar disease resistance in

plant crops.

Another application of cloning technology is in the field of animal husbandry. Present

methods of breeding superior livestock involve the artificial insemination of an animal

with frozen semen and embryo transfer. The problem with artificial insemination is that it

only provides half of the desired elite genes. Cloning, however, would reproduce the

entire gene set of an elite individual.

In the future, cloning research may allow scientists to reprogram cells.

Clones of transgenic animals would produce herds of superior livestock.

Scientific research itself will benefit from cloning techniques. Animals such as monkeys

and mice could be cloned especially for research. Genetically identical laboratory animals

would reduce variability in experiments.