THE ASIAN SCHOOL

KINGDOM OF BAHRAIN
GRADE 12
BIOLOGY
CHAPTER-11 BIOTECHNOLOGY : PRINCIPLES

NOTES
Principles of Biotechnology
Two basic core techniques used in biotechnology are:
1. Genetic engineering
It is technique to produce recombinant DNA. It alters the chemistry of genetic material
(DNA or RNA). When introduced into the organism, the organism produces the desired
product.
2. Bio process engineering
Maintainance of sterile ambience or to have contamination free environment.
Genetic engineering
• It is popular term used for recombinant DNA technology.
• Recombinant DNA technology involves combining of DNA from two different
organisms and to generate rDNA.
Two basic steps in this technique are:
1. Cutting or isolating a segment of DNA or a desired gene with the help of an
enzyme( restriction enzyme)
2. 2. Joining this DNA or gene with the DNA of a different organism (by DNA ligase)
The new DNA formed contains fragment of foreign DNA is called rDNA.
The organism that contains an artificially inserted gene is known as transgenic
organism or genetically modified organism (GMO)
Artificial recombinant DNA molecule
• The construction of the first recombinant DNA emerged from the possibility of
linking a gene
• Stanley cohen and Herbert boyer accomplished this in 1972 by isolating the
anitibiotic resistance gene
• Plasmid
• Autonomously replicating circular extra chromosomal DNA.
• The gene encoding antibiotic resistance in the native plasmid of salmonella
typhimurium
• They isolated this gene by cutting out a piece of DNA from a plasmid which was
responsible for conferring antibiotic resistance.
• The cutting of DNA was done by molecular scissors “restriction enzymes”
• The cut piece was linked with the plasmid DNA.
• Plasmid DNA acts as vectors to deliver any alien piece of DNA into host.
• The linking of alien piece into plasmid is done with enzyme DNA ligase.
• This formed an in vitro circular autonomously replicating DNA known as
recombinant DNA.
• This DNA is transferred into Escherichia coli, a bacterium closely related to
Salmonella typhimurium.
• The plasmid is able to replicate using host’s DNA polymerase enzyme and make
multiple copies.
• This process of making copies is known as cloning of antibiotic resistance gene in
E.coli.
Tools of recombinant DNA (r DNA) technology
Basic tools are:
1. Enzymes
2. Cloning vectors
3. Host organism
History
• The first restriction endonuclease is HIND II was isolated by Smith, wilcox and
Kelley from Haemophilous influenzae bacterium.
• HIND II functions on specific DNA nucleotide sequence
• HIND II always cut DNA at particular point by recognizing a specific sequence of 6
base pairs
• This specific base sequence is known as recognition sequence
Example : EcoRI
• The first letter of the name comes from the genus
• The second two letters comes from the species of the prokaryotic cell from which it
was isolated.
• Here EcoRI comes from Escherichia coli RY 13
• The R is derived by the name of strain used
• Roman number indicate the order in which enzymes were isolated.
Enzymes :
• The restriction enzymes are called ‘molecular scissors’ and are responsible
for cutting DNA.
• They are present in bacteria to provide a type of defence mechanism called
the ‘restriction modification system’
Restriction endonuclease
• They are known as molecular scissors
• They belong to a larger class of enzymes called nucleases.
• They are of kinds:
1. Exonucleases: it removes nucleotides from the ends of the DNA.
2. Endonucleases: it removes at specific positions within the DNA.
• Each restriction endonuclease functions by inspecting the length of DNA
sequence.
• Once it will find the sequence it will cut each of the two strands in their sugar
phosphate backbone.
• Each restriction endonuclease recognizes a specific palindromic nucleotide
sequences in the DNA.
Diagrammatic representation of rDNA technology (study from textbook)
Separation and isolation of DNA fragments
• The fragments can be separated by a technique known as gel electrophoresis.
• DNA fragments are negatively charge molecules they will move towards anode
under an electric field through medium.
• The most commonly used matrix is agarose gel.
• Agarose is a natural polymer extracted from sea weeds.
 • The DNA fragments separate according to their size through sieving effect provided
by the agarose gel.
• So the smaller the fragments are farther they will moves.
• The DNA fragments were visualised only after staining the DNA with ethidium
bromide.
• With exposure to UV light.
• Orange coloured bands of DNA can be seen.
• The separated bands are cut out from agarose gel and extracted from the gel piece.
This step is known as elution
Cloning vectors or vehicle DNA
Vectors are used as carriers to deliver the desired foreign DNA into a host
cell.
A vector must possess the following features when acting as a cloning vehicle.
• Should easily be isolated from the organism.
• Should be small in size because the larger vector DNA molecules have the possibility
of breaking.
Example :
• Plasmids and bacteriophages have the ability to replicate within bacterial cells
independent of chromosomal DNA.
The following are the features that are required to facilitate cloning in a
vector;
(refer textbook for the features)
Vectors for cloning genes in plants and animals
Bacterial and viruses as natural vectors
• They insert their genes into eukaryotic cell and force them to do what they want
Example
• Bacterium Agrobacterium tumifaciens
Ø a pathogen in dicot plants can deliver a piece of DNA known as T-DNA.
Ø T-DNA can transform normal cell into a tumour.
Ø This tumour produce chemicals required for a bacterium.

• Retrovirus
Ø In animals transform normal cells into cancerous.
Using bacteria and virus as vectors
• They are used as vectors to deliver genes of interest to human beings.
1. Agrobacterium tumifaciens the tumour inducing plasmid (Ti) plamid is no more
pathogenic to plants
It delivers a piece of DNA known as T- DNA in the Ti plasmid which transforms
normal plant cells into tumor cells to produce chemivals against pathogens.
Ti plasmid cloning vector
• Similarly retrovirus are modified into vectors to deliver desirable genes of interest
in animals.
• Once the vector is ready it is transferred to suitable host like bacteria, plants and
animal where it multiplies.
Competant host
• DNA as hydrophilic molecule cannot pass through the cell membrane of the host.
• The bacterial cell first be made ‘competent’ to take up DNA.
• It can be done by various means:
Direct method/ chemical method
• In this transfer of r-DNA :
1. Electroporation
• In this technique bacterial cells are treated with specific concentration of divalent
cations such as calcium,
• Which increases the efficiency with which DNA enters the bacterium through pores
in its cell wall.
• Such cells are incubated with recombinant DNA on ice. Then they are placed briefly
at 420C (heat shock) and put them back on ice. This enable the bacteria to take up
recombinant DNA.
Physical methods
1. Micro-injection
• Recombinant DNA is directly injected into the nucleus of an animal cells.
2. Bolistic (Gene gun )
• Cells are bombarded with high velocity micro- particles of gold or tungsten coated
with DNA. This method is suitable for plants.
Disharmed pathogen vector
• When it infects the cell, transfer recombinant DNA into host.

Processes of Recombinant DNA technology

1. Isolation of genetic material DNA
2. Cutting of DNA at specific location
3. Amplification of gene of interest using PCR
4. Ligated with vector
5. Insertion of recombinant DNA into host cells
6. Obtaining the foreign gene product
7. Downstream processing
(Isolation of genetic material DNA: refer textbook)
(Cutting of DNA at specific locations: refer textbook)
Amplification of gene of interest using PCR
By two means we can make copies of desired gene:
a) By inserting it into a bacterium, and when the bacteria divide millions of cells
contains the copies of the fragment can be obtained.
b) Another quicker way and direct approach is to use DNA polymerase through the
PCR.
Polymerase chain reaction
• It was developed by Kary Mullis in 1983, won nobel prize in chemistry in 1993.
• Multiple copies of the gene (DNA) of interest are synthesized through PCR in-vitro
in following three steps.
1. Denaturation
• DNA fragment is mixed with the two sets of primers (small 20-30 chemically
synthesized oligonucleiotides complementary to the regions of DNA) and then this
mixture of primer and DNA fragment is heated to 980C.
• This temperature dissociates the double stranded DNA fragment into single strands.
2. Annealing
• Now allow this solution to cool at about 600C.
• The complementary primers gets attached to the base pairs
3. Extension (750C- 950C)
• Next a very heat stable variety of enzyme, DNA polymerase which is isolated from
bacterium, Thermus aquaticus (Taq polymerase)is added along with large supply of
four nucleotides Now using the primer, DNA polymerase copies the gene template
and add complementary nucleotides to the extending strand.
• By the end , both strands of the gene template are replicated and there are two
copies of the original DNA fragment.
• The steps 1, 2, 3 are repeated again and again to get more copies.
• More DNA polymerase not to be added as the same enzyme is used again and again.
• Being heat-resistant, this enzyme remains active even at high temperature.
(Insertion of recombinant DNA into the host cell: refer textbook)
(Obtaining the foreign gene product: refer textbook)
BIOREACTORS
• These are the vessels in which raw materials are biologically converted into specific
products, enzymes etc. using microbial plant, animal or human cells.
• Bioreactors provides optimal growth conditions (temperature, PH , substrates, salts,
vitamins, oxygen) for achieving desired product.
• Most commonly used bioreactors are of stirring type
(stirred – tank reactors)
• It is usually cylindrical or with curved base to facilitate the mixing of contents.
• The stirrer facilitates mixing and oxygen availability.
• Better temperature and pH control, foam control system to prevent foam and
shearing damage to cells due to agitation, system sterilization,
Sparged stirred tank
• Alternatively air can be bubbled through the reactor.
Parts of bioreactor
• An agitator system
• An oxygen delivery system
• A foam control system
• Temperature control system
• PH control system
• Sampling ports ( for periodic withdrawal of culture)

Downstream processing
All the processes to which a product is subjected to before being marketed as a
finished product are called downstream processing.
It includes:
• Separation of the product from the reactor
• Purification of the product
• Formulation of product with suitable preservatives
• Quality control testing and clinical trails in case of drugs.