List of Top Biotechnology Techniques

Principles Of Biotechnology

Biotechnology has grown and changed a lot over the years. The old way of doing it was to use microbes to make products that people could use. Now, the new way is to use genes to make vaccines. So, the European Federation of Biotechnology (EFB) has come up with a new definition of biotechnology that takes into account both the old and new ways of thinking about it. It says this:

“Biotechnology is the use of natural science, organisms, cells, cell parts, and molecular analogues to make products and provide services.” Modern biotechnology was made possible by two main techniques, which are:
Genetic engineering is a set of techniques that change the chemistry of genetic material in a way that changes the phenotype of the host when the changed genetic material is put into it.

When making things like vaccines, antibiotics, etc., it is important to keep the environment clean so that only the desired microbe can grow in large numbers.

Important Techniques of Biotechnology

The 3 important techniques of biotechnology are:

Recombinant DNA Technology (Genetic Engineering)

Plant Tissue Culture and
Transgenic (Genetically Modified Organisms).

Biotechnology technique – Recombinant DNA Technology

Recombinant DNA Technology is the method of making artificial DNA by combining different types of genetic material (DNA) from different sources. Genetic engineering is a common name for the science of recombining DNA.

In 1968, a Swiss microbiologist named Werner Arber found restriction enzymes. This was the start of recombinant DNA technology.
It’s not as easy as it sounds to put the gene you want into the genome of the host. It starts with choosing the gene you want to put into the host, then choosing the best vector for putting the gene into the host and making recombinant DNA.
Because of this, the recombined DNA needs to be put into the host. And finally, it has to be kept in the host and passed on to the next generation.

Tools Of Recombinant DNA Technology

Enzymes

Some enzymes, like restriction enzymes, help to cut, others, like polymerases and ligases, help to make.
In recombinant DNA technology, the restriction enzymes are a big part of figuring out where the gene you want to put into the vector genome will go. Endonucleases and Exonucleases are the two kinds.
Endonucleases cut into the DNA strand, while exonucleases remove the nucleotides from the ends of the strands.

The restriction endonucleases cut the DNA at certain places based on their sequence, which is usually a palindrome.
They look at how long the DNA is and cut it at a place called the “restriction site.” This makes the sequence have dead ends.
The same restriction enzymes cut both the desired genes and the vectors to get the complementary sticky notes. This makes it easy for the ligases to attach the desired gene to the vector.

The vectors

The vectors help move and connect the gene that is wanted.
These are a very important part of the tools used in recombinant DNA technology because they are the final carriers of the desired gene into the host organism.
Plasmids and bacteriophages are the most common vectors used in recombinant DNA technology because they can make a lot of copies of themselves.

The vectors are made up of an origin of replication, which is a sequence of nucleotides from which the replication starts, a selectable marker, which is made up of genes that show resistance to certain antibiotics like ampicillin, and cloning sites, which are the places where restriction enzymes can recognise where desired DNAs are inserted.

Host organism

Organism into which recombinant DNA has been introduced.
The host is the most important part of recombinant DNA technology. With the help of enzymes, it takes in the vector that has been engineered with the desired DNA.

These recombinant DNAs can be put into the host in a number of ways, such as by microinjection, biolistics or gene gun, alternating cooling and heating, using calcium ions, etc.

Process of Recombinant DNA Technology

Recombinant DNA technology involves a number of steps that must be done in a certain order to make the product that is wanted.

Step-1. Isolation of Genetic Material.

In the first step of Recombinant DNA technology, the desired DNA is separated from other macromolecules and made into a pure form.

Step-2.Cutting the gene at the recognition sites.

The restriction enzymes are a big part of figuring out where the gene you want to put into the vector genome will go. “”Restriction enzyme digestions” are the names for these kinds of reactions.

Step-3. Amplifying the gene copies through Polymerase chain reaction (PCR).

It is a way to make thousands or millions of copies of a single copy of DNA after restriction enzymes have been used to cut the gene of interest.

Step-4. Ligation of DNA Molecules.

In this part of the Ligation process, a cut piece of DNA and the vector are joined together with the help of an enzyme called DNA ligase.

Step-5. Insertion of Recombinant DNA Into Host.

In this step, the recombined DNA is put into a host cell that will accept it. Transformation is the name for this process.
Once the recombinant DNA is put into the host cell, it grows and makes the protein under the best conditions.
There are different ways to do this, as explained in Tools of recombinant DNA technology.

The recombinant gene is passed on to the next generation by the cells or organisms that were successfully changed.

E. The recombined DNA is put into coli, Bacillus subtilis, and yeast. Plasmids shouldn’t be in the bacteria that will be used as hosts. There are three ways that recombined DNA can be put into the host.

Transformation: It is the process by which a cell takes a piece of DNA from the outside world and adds it to its own chromosomal DNA.

Transduction: It is when a bacteriophage moves DNA from one living thing to another.
Vector less Gene Transfer: There are ways to move genes around that do not involve vectors. It could be done with a gene gun and tiny needles.

Step-6: Screening of the Transformed Cells, (i.e., Selection of clones with recombinant DNA):

The next step is to find out which cells have the recombinant DNA molecule with the gene of interest.

You can choose the clones you want based on whether they have the vector or the inserted gene. Some plasmid vectors, for example, make cells resistant to an antibiotic.
Another way is to find out which cells bind RNA that is complementary to the gene of interest or make proteins that are coded by that gene. Clones with recombinant DNA will stay the same for at least a few hundred generations.

Hazards of Genetic Engineering

There are several risks that come with recombinant DNA technology.

(1) Spread of New Diseases

Through the recombinant DNA technique, new dangerous forms of microorganisms can be made either by accident or on purpose.
If these microorganisms get out of the research lab through the drain, lab glassware, lab staff, etc., they could cause new diseases to spread and start. This could be a big problem. HIV (the AIDS virus) is thought to be one of these.

(2) Effect on Evolution

Nature has made it hard for prokaryotes and eukaryotes to trade DNA with each other.

Recombinant DNA technology makes it possible for DNA from these two kinds of organisms to be swapped. This stops evolution from happening naturally.

(3) Biological Warfare

There is always the chance that some countries with bad intentions could use genetic engineering for biological warfare.
Microorganisms that spread disease can be used against the enemy in this kind of war. This will only end in trouble.

So, genetic engineering also has some bad things about it. Many of the things that can be done with genetic engineering can also be done with less dangerous methods.

DNA Cloning

A clone is a group of separate things or cells that all come from the same progenitor.
Clones are genetically identical because the cell just copies itself to make more of the same cell. Scientists can make multiple copies of a single piece of DNA, called a gene, which can be used to make copies that are exactly the same. This is called a DNA clone.

Cloning is done by putting small pieces of DNA into a small molecule of DNA.
This molecule is made so that it can copy itself inside a living cell, like a bacterium. The tiny molecule that can make copies of itself is called the DNA vector carrier.
Most of the time, yeast cells, viruses, and Plasmids are used as vectors. Plasmids are molecules of DNA that are round and come from bacteria. They are not a part of the cell’s main DNA.

It has genes that give the host cell good qualities, like the ability to reproduce and to resist drugs.
They can be easily changed because they are small enough and can carry extra DNA that is woven into them.

Cloning can be studied under following Topics

1. Cell cloning

Cell cloning is the process of growing a cell in a lab so that it can make copies of itself. They have the same genes, the same bodies, and the same shapes.

Usually, only a totipotent can make its own clone. Plant cells are totipotent, but animal cells are not totipotent. They can grow many things (capable of getting transformed into any kind of cells).
Because of this, it is much easier to clone plant cells than animal cells.

2. Gene cloning

It means that a lot of copies of a DNA fragment are made. It can be made easily by using the rules of genetic engineering.

3. Microbial cloning

Microorganisms like bacteria and PPLO (Mycoplasma) reproduce without a male or female partner.
In a very short amount of time, it is easy to make copies of these organisms in a culture medium. In vitro, a single microbial cell that has had its genes changed can now make thousands of copies of itself.

4. Plant cloning

Plants are easier to clone because their cells can do many different things.

Meristematic cells, which are often found at the tips of a plant’s roots and shoots, help the plant make new plants and plantlets quickly.
Many plantlets can be made if these cells are taken out and grown in a clean environment.
Plant cloning is a great way to make crops that are resistant to GMF (Genetically Modified Food) as well as drought and pests.

5. Animal Cloning

“Dolly,” the world’s first cloned mammal, was made from a non-gametic cell of a sheep that had fully grown and changed.
It was born at the Roslin Institute in Edinburgh on February 13, 1996. (UK). Dr. Ian Wilmut and his colleagues came up with the idea. Polly and Molly were two sheep that scientists in Scotland were able to clone.
“Dolly” questions one of the most important ideas in developmental biology. Wilmut and his colleagues took cells from a six-year-old sheep’s udder.

Another adult sheep’s unfertilized egg was taken out. The egg’s centre was taken out. A non-dividing nucleus from an udder cell was taken out and put into an egg that had been stripped of its nucleus.
In the culture medium, the egg began to split, and an embryo formed.
Then, the young embryo was put into the uterus of a third sheep (called surrogate mother).

The surrogate mother, which was a fertile female sheep, gave birth to a healthy lamb that they named “Dolly.”
Since “Dolly” was cloned successfully, the idea of cloning humans has become more likely.
Many geneticists around the world are working on making clones of people.

But the governments of many countries have made it illegal because it causes social and moral problems.

Applications Of Gene Cloning

Here are some ways that gene cloning can be used:

Gene cloning is an important part of the field of medicine. It is used to make antibiotics, vitamins, and hormones.

The field of agriculture is one place where gene cloning is used. Cyanobacteria are responsible for fixing nitrogen, and desired genes can be used to make crops grow better and improve health. This method cuts down on the use of fertilisers, which makes it possible to grow food without chemicals.
It can be used in science to find and identify clones with a certain gene that can be changed by growing them in a controlled environment.
It is used in gene therapy, in which a healthy gene is put in place of a bad one. This idea can be used to treat health problems like leukaemia and sickle cell anaemia.

Biotechnology technique – Plant Tissue Culture

Plants are the only source of food, shelter, and clothing for people.
Because people need plants so much, they have come up with different ways to keep them from being destroyed by natural disasters or by humans.
Scientists came up with a way to keep plants from dying and grow a whole plant from a single cell or a small part of a plant while they were working on this. This method, called “tissue culture,” turned out to be a great thing for people.

Theoretically, any plant cell, except those without a nucleus or those surrounded by a rigid secondary wall, has the potential to grow back into the plant it came from.
This kind of cell is called “t totipotent.” The organism is made up of the way the organs are arranged in space. Tissues are made up of groups of similar cells, and organs are made up of groups of tissue systems (i.e., full plant).
Plants can be grown from organ explants (root and shoot tips, buds, leaf primordia, developing embryos, bud scales, etc.), tissue explants (pith, cortex, epidermis, phloem, nucellus), cells (parenchyma, collenchyma, uni- or binucleate pollen-grains), and protoplasts in vitro, which means in a lab in a controlled environment.

What is Totipotency?

Totipotency (from the Latin words “totus,” which means “whole,” and “potential,” which means “power”) is the ability or capacity of mature living cells to grow into a new organism under controlled conditions when they are taken out of the plant body.
Haberlandt, a German botanist, came up with the idea of totipotent cells in plants for the first time in 1902.
He said that every living cell in a plant can make a new plant because it comes from a fertilised egg and contains information about the plant’s genes.

He tried to make leaf cells grow on their own, but he was not successful.
Later in 1950 EE. Steward was able to show that cells can become any kind of cell. Cellular totipotency is a property of plant cells because a differentiated plant cell still has the ability to grow into a whole plant, while an animal cell loses the ability to grow back after it has grown into a specific type of cell.

Steward Experiment

Steward and the others all took 2mg. Carrot (Daucus carrota) phloem tissue was cut into thin slices and grew in a nutrient solution with coconut water.

Explants are the parts of a plant that are taken to grow new cells (Ex. Phloem tissue of Carrot root).
Some hours were given for shaking the liquid nutrient medium that had the explants in it. The cultured cells then keep dividing until they form a mass of tissue that hasn’t changed yet. This is called a callus.
Some of these cell clusters began to change into the first part of the root. When it moves to a medium that is halfway solid, it grows a root system and a new plant.

Then, these plants move to a pot or soil, where they grow into flowing plants.
Steward’s experiment shows that even fully differentiated (mature) plant cells can dedifferentiate, divide, come back together, and then redifferentiate to make a new plant.
Cells from a plant’s root, stem, leaf, vegetative bud, floral bud, anther, and embryo can all develop into different types of cells.

Applications of Totipotency

In tissue culture, the totipotency of plant cells has been used in the following ways:

It helps plants grow quickly and keep their good qualities.
Making more of rare plants.

To break the seed’s sleep.
Helpful plants spread quickly.
Develop haploid plant.

Produce virus free & disease resistant plant.
Help with fusion of protoplasts and somatic hybridization.
Plant crop varieties that produce a lot of food.

Help in embryo rescue.

Various Types of Plant Tissue Culture

Plant tissue culture comprises cell culture, protoplast culture, organ culture, meristem culture etc.
Organ culture includes any part of a plant that has its own identity, such as anthers, ovules, embryos, and buds.

In tissue culture, the part of the plant that is used to grow new cells is called an explant. It could be a protoplast, a cell, a tissue, or an organ.
Callus (pleural callus) is a group of regrown cells that aren’t in any particular order in a culture medium. Suspension culture is the process of suspending free callus cells in a liquid medium.
The ability of a plant cell to grow into a whole plant or to grow back into itself is called totipotency. This shows that each cell is able to grow into a whole plant.

The cell and tissue cultures cause the whole plant to grow back.
Somatic embryos develop in some plants, like carrots and sandalwood, but in wheat, rice, barley, and tobacco, both the root and the shoot develop from the calli.
Clone is the name for all of a somatic cell’s offspring that are made through vegetative reproduction. A clone’s single member is called a ramet.

Biotechnology technique – Transgenic (Genetically Modified Organisms)

Transgenics or Genetically Modified Organisms are terms for organisms that have been changed genetically (GMO).
In other words, a genotype made through genetic engineering is called transgenic (pleural transgenics). It could be a plant, an animal, or a microbe like a virus, fungus, or bacteria.
So, transgenic plants, also called Genetically Modified Plants (GM Plants), are plants that are made using genetic engineering.

Techniques like tissue culture and genetic engineering are used to make transgenic plants.
Genetic transformation is the process of putting a transgene into an organism and making it work.
Transgenes are foreign genes or genes that have been changed from the same species that are used to make transgenic plants or genotypes.

Transgenes can come from the same species (in a changed form), related wild species, species that are not related, or microbes (bacteria, fungi and viruses).

Advantages of Transgenic Breeding

Transgenic breeding is the use of biotechnology (tissue culture and genetic engineering) to improve the genes of crop plants, domestic animals, and useful microorganisms so that they can be used economically by people.
Transgenic plant breeding is the use of transgenes to improve the genes of crop plants.

Transgenic breeding is now used to improve the genes of different field crops in very specific ways. In the coming years, transgenic breeding is likely to play a big role in improving the genes of field crops.
Here are a few short points about the main benefits of transgenic crop breeding:

1. Rapid Method of Crop Improvement

Transgenic breeding is a fast way to make crops better.

With this method, stable transgenic plants can be made in 3–4 years, while it takes 12–15 years to make a new variety of plant using traditional breeding methods like pedigree, bulk, and back cross.
T0 progeny is the name for the first generation of transgenic plants that grow from seeds.
Transgenic plants that come from T0 plants are called T1 plants, those that come from T1 plants’ embryos are called T2 plants, and so on. Transgene showed a Mendelian pattern of segregation in barley and sunflower, which is a 3:1 ratio.

2. Overcome Crossing Barriers

Through transgenic breeding, genes can be moved between species or even between organisms that are not related to each other.
It permits gene transfer even between plants and animals. For example, a gene that makes fish resistant to freezing has been moved to tomatoes grown in farms.
In the same way, the ovalbumin gene from chicken has been put into alfalfa to make the protein better. There are many more ways that genes can move from plants to animals.

3. Evolution of New Genotypes

Because transgenic breeding lets genes move from one plant species to another, it can sometimes lead to the evolution of completely new plant species.
So, it will change the way that natural evolution works.

4. Application

Transgenic breeding can be used to improve the genes of both self-pollinating and cross-pollinating crop plants.

Transgenes can be used to improve both species that grow from seeds and those that grow from existing plants.

5. Effectiveness

Transgenic breeding has only been shown to work for improving the genes of monogenic traits.
So far, it has not been used to improve the genes of polygenic characters. It has been found to be very useful in making plants resistant to different diseases, insects, and herbicides.

Limitations of Transgenic Plant Breeding

There are some problems with transgenic breeding. The main problems with it are:

inconsistent performance,
Pleiotropic effect (meaning that a single gene has more than one effect) of transgene,
Effect of the transgene’s position,
expensive way to improve crops,
Transgenic plants don’t happen very often,
Risk that weed species that cause problems will change, etc.
Also, genetic engineering can’t be used to change polygenic traits.

Criticism Against Genetically Modified Crops

1. Unintended harm to other organisms

In a lab study that was published in the journal “Nature,” it was found that pollen from BT corn killed a lot of monarch butterfly caterpillars.
Monarch caterpillars eat milkweed plants, not corn, but pollen from BT corn could kill the caterpillars if it gets on milkweed plants in neighbouring fields and is carried by the wind.
Even though the “Nature” study wasn’t done in a real field, the results seemed to back up this point of view.

2. Reduced effectiveness of pesticides

Just like some mosquito populations have become immune to the pesticide DDT, which is now banned, many people worry that insects will become immune to BT or other crops that have been genetically modified to make their own pesticides.

3. Gene transfer to non-target species

Another worry is that weeds and crop plants that have been changed to be resistant to herbicides will get together and breed, giving the weeds the herbicide-resistant genes from the crops.
Then, these “super weeds” would also be resistant to herbicides. Other genes that were brought in may spread to non-modified crops that are grown next to GM crops.

Genetically Modified Food

Genetically modified food, or GM food, is made from crops that have been changed so that their genes are different.

GM food is different from food made from crops grown using traditional breeding methods in the following ways:

It has the protein made by the transgene in question, like Cry protein in the case of varieties that are resistant to insects.
It has the enzyme made by the antibiotic-resistant gene that was used in genetic engineering to move genes from one place to another.
It has the gene for antibiotic resistance in it.

Disadvantages of GM Food

The following things can go wrong because of GM food:

1. Allergies

The transgenic food could make you sick or give you allergies. Since the enzyme made by the gene that makes you resistant to antibiotics is a foreign protein, it can cause allergies.

2. Effect on Bacteria of Alimentary canal

The antibiotic-resistant gene in GM food can be taken up by the bacteria in a person’s digestive system. These bacteria might stop responding to the antibiotic, which would make them hard to control.

3. Economic concerns

Getting a genetically modified food on the market takes a long time and costs a lot of money, so agri-biotech companies want to make sure they get a good return on their investment.

What is a Transgenic Animal?

Transgenic animals are those that have genes from places other than their own.

Production of Transgenic Animals:

Recombinant DNA technology is used to put the foreign genes into the genome of the animal. Among the things that go into making transgenic animals are:

Finding, naming, and separating the gene you want,
Choose the right carrier (usually a virus) or send it directly,
Putting the gene you want together with the vector,
When a transferred vector is put into cells, tissues, an embryo, or an adult,
Integration of a foreign gene and its expression in a transgenic animal or tissue.

Why are Transgenic Animals Produced?

Its goal is;

Cows, sheep, and goats make a lot of important drugs and proteins that are used in medicine,
To use pigs to grow extra organs (like the heart and pancreas) for people (xenotransplantation),
By looking at different parts of cloning and how biochemicals affect mice,
Making human cell lines so that genetic diseases like Alzheimer’s, haemophilia, thalassemia, etc. can be cured.
Change broken parts with new ones grown from the patient’s own cells,
To make clones of people if ethics allow it.

References

Mustafa, M. G., Khan, M. G. M., Nguyen, D., & Iqbal, S. (2018). Techniques in Biotechnology. Omics Technologies and Bio-Engineering, 233–249. doi:10.1016/b978-0-12-815870-8.00013-9
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