BIOTECHNOLOGYCHAPTER – 11 :

PRINCIPLES AND PROCESSES

• Bio technology : • Bio technology deals with the techniques of using live

organisms or enzymes to produce products and processes useful to humans.

• The term Bio technology was coined by Karl Ereky in 1919.

• Principles of Biotechnology:• Two imp techniques that used in modern biotechnology

are, – Genetic engineering: Techniques to modification of chemistry

of genetic material (DNA and RNA) and to introduce them into host organisms to change the phenotypic character of the host.

– Maintenance of sterile: (microbial contamination-free): It is the processes to enable growth of only the desired microbe/eukaryotic cell in large quantities.

Genetic Engineering

• The three basic steps involved in Genetic engineering are,1. Identification of DNA with desirable genes.2. Introduction of indentified DNA in to the host.3. Maintenance of introduced DNA in the host and transfer

of that DNA in to its progeny.

• The techniques of genetic engineering include creation of recombinant DNA.

• r-DNA is used in gene cloning and gene transfer. It helps to introduce desirable genes into the target organism.

• It also helps to avoid undesired genes getting multiplied along with the desired genes.

• The tools of recombinant DNA technology . 1. Restriction enzymes.2. Polymerase enzymes.3. DNA - Desired gene.4. Ligases.5. Vectors.6. Host organism.7. Bioreactors.

• Hamilton smith discovered and isolated HIND II REN from Haemophilius influenzae in 1968. He received the Nobel Prize in Physiology or Medicine in 1978.

• Restriction enzymes:• REN are enzymes naturally occurring in bacteria as defensive enzyme. It

cuts and destroys bacteriophage DNA that infects bacterial cell. • It recognize specific palindromic sequences to cut in DNA. Different types of

REN are identified and isolated for different palindromic sequence.• The RENs are called molecular knives or molecular scissors as they cut DNA.

• Palindromic sequences: Palindromic sequences are ‘invert repeats’ which have the same nucleotide sequences when read in 5‘to 3' on both the strands.

• (As MALAYALAM.)

• The naming of restriction enzyme:1. The first letter of the name comes from the genus of bacterium from

which they are isolated. .2. Second letter from the species of the bacterium.3. The third letter indicate the strain of the organism.4. The roman numbers following the name indicate the order of

discovery.• example: EcoRI comes from Escherichia coli RY 13

• RENs are Eco RI, Hind III, Sma I, Hae III, etc.,.. • The fragments of DNA produced are called restriction fragments. The

fragments of DNA may have ‘blunt ends’ or ‘staggered ends’• (sticky ends). • • Endonuclease : They breaks DNA double helix at any point except the

ends. They produce internal cuts called nick's or cleavage.• • Exonuclease : They breaks or cuts the 5' or 3' ends of DNA molecule

i.e. they remove nucleotides from terminal ends of DNA.

• Desired gene: The functional or normal gene of our interest taken from donor cell.

It is also known as foreign gene or trans gene.

•Polymerase enzymes: 1. DNA Polymerase I.2. DNA Polymerase II3. DNA Polymerase III.

DNA ligase.• The enzyme that joins the two sticky ends

of DNA is called DNA ligase. It is used as molecular sitichers in genetic engineering.

• DNA ligase was discovered by H G Khorna. • Dr. Hargobind Khorana was born on 9th

January 1922 at Raipur, Punjab (now in Pakistan).

• Died November 9, 2011 (aged 89)Concord, Massachusetts, U.S.

• In 1968, He was awarded the Nobel Prize in Physiology or Medicine for the interpretation of the genetic code and its function in protein synthesis

1922 - 2011

• Vector: The carrier DNA that act as vehicle to carry desired gene to the host cell is called vector.

• The imp vectors used in Genetic engineering are,1. Plasmid.2. Phages.3. Plant virus.4. Animal virus.5. Cosmids.6. Artificial chromosomes.• Plasmid: The extra chromosomal self replicating

small circular DNA present in bacteria is called plasmid.

• Cloning vectors:• The plasmid and bacteriophages are used as cloning vectrors.

Bcoz it has the ability to replicate within bacterial cells independentlly.

• Features of cloning vector:• vector should contain three features for gene cloning. They are • 1) Ori site. 2) Cloning sites..3) selectable markers.

• Ori site: - Origin of replication• The sequence of DNA where the replication starts is called

ori site.• It helps the desired DNA linked with vector also replicates.

• Cloning sites:• The vector contains the recognition site or palindromic for the

commonly used REN to insert desired gene.

• Host cell: The cell to which desired new gene is introduced is called host cell. Any living cell can be used as host cell.

• Commonly E.coli bacterial cell is used as host cell in genetic engineering. Because,

1. It is a simple prokaryotic cell.2. It is a non-pathogenic bacteria.3. It can be cultured easily in laboratory condition.4. It has very short life span.5. It contains self replicating plasmid.6. The plasmid of E.coli can be easily handled as

vector.

• The steps involved in recombinant DNA terchnology.

1. Isolation of desired DNA .2. fragmentation of DNA by restriction

endonucleases.3. Isolation of a desired DNA fragment.4. Ligation of DNA fragment in to vector.5. Transferring the recombinant DNA in to host.6. culturing the host cell and extraction of desired

product.

Extraction of DNA or isolation of gene: 1. The cells of organism that contains desired

gene are collected. 2. The DNA of these cells is extracted by using

refrigerated centrifuge technology.3. The contents of the cell is treated with

enzymes like chitinase, lysozymes, cellulase , ribonuclease and proteases.

4. Finally chilled ethnol is added to get purified DNA.

5. The isolation of gene is done by shoot gun method. In this specific REN is used to cut and isolate desired gene. The isolated gene contains two sticky ends.

6. The desired gene is isolated by using gel electrophoresis.

• Gel Electrophoresis.1. The fragments obtained after cutting with restriction enzymes are

separated by using gel electrophoresis.2. Electric field is applied to the electrophoresis matrix (commonly

agarose gel).3. The negatively charged DNA fragments move towards the anode.4. Fragments are separate according to their size due to the sieving

properties of agarose gel. Smaller the fragment move faster than larger one.

5. Staining dye ethidium bromide followed by exposure to UV radiations are used to visualise the DNA fragments.

6. DNA fragments are visible as bright orange coloured bands in the agarose matrix.

7. These bands are cut from the agarose gel and extracted from the gel piece (elution).

8. DNA fragments are purified and used in constructing recombinant DNAs.

• Multiplication of gene: The isolated desired gene is multiplied into millions of copies using polymerase chain reaction.

• Ligation of desired gene in to vector. • A suitable plasmid is selected. It is treated with the

same restriction enzyme to break and open the plasmid at specific sites with sticky ends.

• Now the desired gene and the plasmid are mixed and enzyme DNA Ligase is added .

• The desired gene gets incorporated in to the plasmid .

• Transferring of recombinant plasmid in to the host cell. (transformation)

• The recombinant plasmid is transfered in to the host cell by cold calcium chloride method or by micro-injection.

• The bacterial cell ( host cell) and r-DNA are made to suspend in cold (5-6 0C ) calcium chloride solution. After some interval of time, the temp of solution is suddenly raised to 42 0C and again cooled.

• The increase in temperature increases the pore size of bacterial membrane. Through this pore r-DNA enters the bacterial cell .

• Micro-injection:• Method of introducing r-DNA directly by injecting in

to the host cell using fine needle is called microinjection.

• Biolastics or gene gun:• Method of introducing recombinant DNA in to host

cell by bombarding high velocity microparticles of gold or tungsten coated with DNA.

• Culturing the host cell and extraction of desired product:

• The r-DNA is made to express under appropriate conditions.

• The host cells are cultrured in a vessel called Bioreactor.

• The desired protein is extracted and purified by using different separation techniques called down stream processing.

• Bio -reactor :• Bio -reactor is an apparatus for culturing organisms like algae, fungi,

bacteria, animal cell or plant cells under controlled conditions.• It is used in industrial processes to produce pharmaceuticals, vaccines,

or antibodies. • In bio -reactor raw materials are biologically converted in to specific

products.• The most commonly used bioreactors are stirred tank reactor.• It is cylindrical with curved base to facilitate the mixing of the reactor

contents. The stirrer facilitates even mixing. Air is bubbled through the reactor.

• The reactor has, 1. an agitator or stirrer system, 2. an oxygen delivery system 3. a foam control system.4. a temperature control system.5. pH control system and6. sampling ports.

• Downstream processing: • Separation, purification of the products obtained

from recombinant DNA technique is called downstream processing (DSP).

• The product has to be formulated with suitable preservatives .

• Clinical trials and strict quality control testing is also conducted before marketing of the product.

• Vectors used for cloning genes in plants.• Ti plasmid: Tumour inducing Ti plasmid of

`Agrobactrium tumifaciens , used as cloning vector. It is nonpathiogenic to the plants. It can be used to deliver genes of our interest in to variety of plants.

• Vectors used for cloning genes in animals.• Retroviruses. The trimmed non pathogenic

retrovirus are used to deliver desirable genes into animal cells.