REQUIREMENTS OF BIOTECHNOLOGY

1. Enzymes

2. Vector

3. Host

4. Techniques

I. ENZYMES

 Enzymes are proteins that participate in cellular metabolic processes with the

ability to enhance the rate of reaction between bio-molecules.

 Enzymes are proteins that act as catalysts within living cells. Catalysts increase

the rate at which chemical reactions occur without being consumed or

permanently altered themselves. A chemical reaction is a process that converts

one or more substances (known as reagents, reactants, or substrates) to another

type of substance (the product). As a catalyst, an enzyme can facilitate the same

chemical reaction over and over again.

 Reagents- used to test for the presence of another substance by chemical reaction

 Reactants- a substance that changes when it is combined with another substance

by chemical reaction

 Substrate- a substance acted upon

 Enzymes can have molecular weights ranging from about 10,000 to more than 1

million. Molecular weight is the sum of the atomic weights of a molecule's atoms.

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  STRUCTURE AND FUNCTIONS

On the surface of each enzyme is a special cleft called the active site. It provides a

place where reagents can 'meet' and interact. Much like a lock and its key, an enzyme's

active site will only accommodate certain reagents, and only one type of chemical

reaction can be catalyzed by a given enzyme.

In order to function, many enzymes require the help of cofactors or coenzymes.

Cofactors are often metal ions, such as zinc, copper, iron, or magnesium. Magnesium,

one of the most common cofactors, activates hundreds of enzymes, including those that

manufacture DNA and many that help metabolize carbohydrates.

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  CLASSIFYING ENZYMES

There are the 3 major classifications of enzymes:

 Oxidoreductases

-is an enzyme that catalyzes the transfer of electrons from one molecule, the

reductant, also called the electron donor, to another, the oxidant, also called the

electron acceptor

 transferases

is any one of a class of enzymes that enact the transfer of specific functional

groups (e.g. a methyl or glycosylgroup) from one molecule (called the donor) to

another (called the acceptor).

 Hydrolases

is an enzyme that catalyzes the hydrolysis of a chemical bond. (Hydrolysis

usually means the cleavage of chemical bonds by the addition of water. When a

carbohydrate is broken into its component sugar molecules by hydrolysis, this is

termed saccharification)

6 TYPES OF ENZYME CATALYST

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 Oxidation and reduction. Enzymes that carry out these reactions are called

oxidoreductases. For example, alcohol dehydrogenase converts primary alcohols

to aldehydes.

 Group transfer reactions. These enzymes, called transferases, move functional

groups from one molecule to another

 Hydrolysis. These enzymes, termed hydrolases, break single bonds by adding

the elements of water

 Formation or removal of a double bond with group transfer. The functional

groups transferred by these lyase enzymes include amino groups, water, and

ammonia.

 Isomerization of functional groups. In many biochemical reactions, the position

of a functional group is changed within a molecule, but the molecule itself

contains the same number and kind of atoms that it did in the beginning. In other

words, the substrate and product of the reaction are isomers.

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  Single bond formation by eliminating the elements of water. Hydrolases break

bonds by adding the elements of water; ligases carry out the converse reaction,

removing the elements of water from two functional groups to form a single bond.

Synthetases are a subclass of ligases that use the hydrolysis of ATP to drive this

formation

WHY DO WE NEED ENZYMES?

One of the immutable laws of nature is that energy is required to initiate a chemical

reaction. After all, if you want to change something, you have to put some work into it.

For every chemical reaction, the energy of activation--a specific amount of energy--must

be applied to the reagents before the reaction will proceed. The energy of activation is

like a hill between the reagents and the product, and the reagents must be pushed over

this hill before the reaction can continue.

Alcohol dehydrogenase is an oxidoreductase enzyme that converts alcohols to aldehydes

or ketones. This enzyme makes alcohol less toxic as it breaks it down.

It also plays a key role in the fermentation process.

VECTORS

 In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially

carry foreign genetic material into another cell, where it can be replicated and/or

expressed.

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  Vectors are usually viruses which are also helpful in genetic engineering. Virus is

an infectious organism, so when a new gene is inserted in it, it transfers that gene

into the host cell during causing infection. Scientists mostly block the function of

virus when they insert the foreign gene; this way virus will only be able to

replicate only the gene of interest and will insert it into the host cell.

Example of vectors:

Plasmid

A plasmid is a small, circular, double-stranded DNA molecule that is distinct from a

cell's chromosomal DNA. Plasmids naturally exist in bacterial cells, and they also occur

in some eukaryotes. Often, the genes carried in plasmids provide bacteria with genetic

advantages, such as antibiotic resistance lambda phages .

Lambda phages is a bacterial virus, or bacteriophage, that infects the bacterial species

Escherichia coli (E. coli). *A bacteriophage, also known informally as a phage, is a virus

that infects and replicates within a bacterium.

Cosmid

A cosmid is a type of hybrid plasmid that contains a Lambda phage cos sequence.

Cosmids (cos sites + plasmid = cosmids) DNA sequences are originally from the lambda

phage. They are often used as a cloning vector in genetic engineering

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. -A vector containing foreign DNA is termed recombinant DNA. Example of R-DNA:

The human insulin gene was recombined with bacterial DNA so that we can easily and

safely generate large amounts of insulin. The insulin gene was placed in bacterial plasmid

DNA in such a way that the bacteria that are given this plasmid will make the insulin

protein, even though it is of no use to the bacteria. We then grow massive amounts of

bacteria and isolate and purify the insulin from the bacteria

Artficial Chromosomes

 Bacterial Artificial Chromosomes. Based on bacterial mini-F plasmids.

 Yeast Artificial Chromosomes. This is an artificial chromosome that contains

telomeres (disposable buffers at the ends of chromosomes which are cut off

during cell division) with origins of replication, a yeast centromere (part of a

chromosome that links sister chromatids or a dyad), and a selectable marker

for identification in yeast cells.

 Human Artificial Chromosome. This type of vector ispotentially useful for

gene delivery into human cells, and a tool for expression studies and

determining human chromosome function. It can carry very large DNA

fragment

Viral vectors are tools commonly used by molecular biologists to deliver genetic

material into cells. This process can be performed inside a living organism (in vivo)

or in cell culture(in vitro)

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 HOST

A host is the organism that is modified in a genetic engineering experiment.

TECHNIQUES

GENE SYNTHESIS

The key to genetic engineering is finding the desired gene to be incorporated into the

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host to obtain the desired property. It is possible to chemically synthesize a gene in the

lab by laboriously joining nucleotides together in the correct order.

Recombinant DNA

Following steps are involved in recombinant DNA technique;

1) Gene of interest is isolated from the DNA molecule using the restriction enzymes.

2) After isolation, gene is inserted into a vector and is cloned to make multiple copies

of gene of interest.

3) When the cloning is done, the gene is incorporated into the plasmid.

4) Now the gene or DNA along with the plasmid is called as recombinant DNA

Electro and chemical poration

In this method, pores are created in the membrane of the cell and genes can be

transferred easily. Special chemicals are used to make pores in the cell surface.

Sometimes cells are exposed to weak electric current, it also makes pores in the

surface of the cells and genes can easily pass through these pores.

Bioballistics method

In this method, small silver particles are used to insert the genetic material into the

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 recipient cell. These silvers are coated with the genetic material and when released in

the cell, genetic material incorporates with the genes of the host cell. In one projectile

method, shot gun is used to insert the silvers into the host cell.

Microinjection

In this method, foreign gene is integrated into the cell by just injecting it into the

recipient cell. When large cell of plants and animals are concerned, then a fine glass

needle is used. The injected genes automatically enter into the nucleus where they

incorporate with the host cell’s genetic material and replicate.

SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis)

is commonly used in the lab for the separation of proteins based on their molecular

weight. It’s one of those techniques that is commonly used but not frequently fully

understood. Gel electrophoresis Gel electrophoresis is a basic technique used to separate

DNA, RNA or proteins. It is a common starting point for many biotechnology

experiments and is often paired with the blotting techniques.

This technique relies on electricity to separate out molecules in an agarose gel, a thick
jello-like substance. DNA, RNA and proteins are all electrically charged, so when an
electric current is applied to the gel, these molecules will naturally move toward the
opposite pole. Because the gel is difficult to travel through, the molecules will travel at

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different speeds depending on their size. Smaller molecules will be able to move faster
and will reach the far end of the gel, while larger molecules will be slowed down and
remain near the beginning.

The end result of gel electrophoresis is a gel with the molecules spread out from one end
to the other. If they have been coloured, the molecules appear as short bands to the
naked eye. The gel can be used in many different ways. Certain regions of the genome
will result in a pattern that is unique for every person when run on a gel, which can be
used for DNA fingerprinting. Gels are also used to detect the presence or absence of
specific DNA or RNA molecules or proteins, when they are combined with the blotting
techniques. The blotting techniques The Southern, Northern and Western blots are used
to detect DNA, messenger RNA (mRNA) and protein, respectively. Blotting refers to
the actual technique, where molecules that have been separated on a gel are transferred
or blotted onto a type of paper called nitrocellulose. The naming of the different blots
originated with the DNA blot, developed by Edward Southern, and the Northern and
Western blots followed.

 Southern blot Before the blot itself can be done, DNA that has been cut up with

restriction enzymes is separated by gel electrophoresis .For the blotting step, the

gel is placed on a sponge which is sitting in a buffer solution. The nitrocellulose

paper, where the DNA will be transferred to, is placed on top of the gel and then

covered with paper towels and a weight. The transfer of the DNA from the gel to

the paper happens by capillary action as the buffer moves toward the dry paper

towels. After several hours, the transfer is complete and the paper will have the

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 DNA fragments on it in the same pattern as they were in the gel. The paper can

then be incubated with a probe that is specific to a DNA fragment of interest. The

probe is radioactively labelled and once the incubation is complete, it can be

detected by autoradiography. Controls must be used to ensure that the

electrophoresis and the blot were successful. A comparison of the band patterns

by autoradiography shows the presence or absence of the DNA of interest

 Northern blot A Northern blot is done in the same way as a Southern blot, but it

uses mRNA instead of DNA.

 Western blot A Western blot also follows the same method as a Southern blot,

but is used to detect proteins instead of DNA. After the proteins are blotted onto

the paper, antibodies are used to detect their presence. The primary antibody binds

to the protein on the paper and the secondary antibody binds to the primary

antibody. The secondary antibody is either tagged with a colour or is attached to

an enzyme that can produce a colour in order to detect where it is on the paper.

Enzyme-Linked Immunosorbent Assay (ELISA) ELISA

is a technique used to detect the presence of specific antibodies or antigens in a sample.

It is a very simple test that can analyse a large number of samples at once, which makes
it a very important diagnostic technique. The ELISA method takes advantage of the
natural property of antigens and antibodies to bond together. A plastic dish with many
wells in it is coated with an antibody for a particular antigen. Then a different sample is

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added to each well - for example, blood samples from different people. Several wells
will contain positive and negative control samples. If the antigens in the blood match the
antibody in the well, they will bind. Those that do not bind will be washed off. A second
antibody is then added to the wells, which will only attach to the antigens. This second
antibody is attached to an enzyme ("enzyme-linked") that will produce a colour when a
solution is added to it. The entire dish can then be read by a scanner that looks for the
presence of the enzyme's colour. If a colour is present, it means that sample contained
the antigen of interest. If there is no colour, there was no antigen to bind to the first
antibody. The control samples are used to make sure the procedure was successful – the
positive control should be coloured and the negative control should not.

Polymerase Chain Reaction (PCR) PCR

is a technique used to make thousands of copies of a DNA strand in only minutes, using
an enzyme called DNA polymerase. PCR plays an important role in research, diagnosis
and forensics. To use PCR to amplify a DNA strand, the DNA sequences at both ends of
the strand must first be known. Scientists can make complementary DNA of these
regions, which are known as primers. The primers tell the DNA polymerase where to
start copying the DNA and then when to stop. In addition to the primers, a copy of the
DNA strand that needs to be copied, nucleotides and DNA polymerase are mixed in a
small tube and put in a machine that can closely control the temperature. Specific
changes in temperature are essential to the PCR process.

The starting temperature is 96°C, which denatures, or separates the two strands of the
DNA. The next step is called annealing, where the primers attach to the DNA strands.
This happens at 68°C. Once the primers have annealed, DNA polymerase will extend
them by adding nucleotides according to the DNA template at 72°C. Each cycle of these
three steps takes less than two minutes and it can be repeated multiple times to produce

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thousands of copies of the original DNA strand. PCR can be used for many purposes,
including genetic fingerprinting in forensics, paternity testing, mutation detection for
disease and cloning genes for research. Microinjection:- Microinjection is a technique of
genetic engineering which uses glass micropipette to inject a substance into the human
or animal cells. This is the only technique which does not need ant plasmids or vectors
for undergoing the process. During this process, usually genetic material of an organism
with a new gene is used to inject into the other organism's cells. As cells are very large,
whether animal cells or plant cells, that is why a small micropipette is used for this
purpose. When the genes of interest are inserted into the new cells, they find their
corresponding genes and combine with them to show certain features or characteristics.

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