Chapter-11

Biotechnology : Principle and Processes

Class 12

- SOURAV DAS
BIOLOGY FACULTY
EXCELADZE
Introduction to biotechnology

Definition:
• Biotechnology is the use of living systems and organisms to
develop or make useful products, or "any technological
application that uses biological systems, living organisms or
derivatives thereof, to make or modify products or processes
for specific use“
• European Federation of Biotechnology (EFB) has defined
biotechnology as “The integration of natural science and
organisms, cells, parts thereof, and molecular analogues
for products and services”.
Oldest form of biotechnology
 Application of
fermentation in
production of wine
and other alcoholic
beverages is also a
biotechnological
technique
Principles of biotechnology

• Two important technique which enable development of modern

biotechnology:

1. Alteration of chemistry of DNA & RNA to introduce into host

organism to change phenotype of host- Genetic
engineering

2. Maintenance of sterile ambience to enable growth of desired

microbe/ eukaryotic cell in large quantities for manufacture
of biotechnological products like vaccine, enzymes,
beverages, drugs etc.- Chemical engineering
• Asexual reproduction in organism preserves genetic
information
• Sexual reproduction (hybridization) leads to variation- includes
undesirable gene with desirable gene
• Genetic engineering- isolate & introduce only one or set of
desirable genes without introducing undesirable genes in
target organism
• Techniques of genetic engineering- creation of recombinant
DNA, use of gene cloning & gene transfer to host
• Recombinant DNA (rDNA)/ alien DNA- cannot multiply itself
until integrated in host genome
• When inherited in host DNA- ability to replicate due to
origin of
replication (host DNA)- initiates replication
• Alien DNA- linked with host DNA replicates & multiply itself
along with host DNA- Cloning
• Gene transfer to host require Vector
• Commonly used vector- Plasmid (small, circular, double
stranded, self replicating extra chromosomal material of bacteria)
• First recombinant DNA was constructed- Stanley Cohen &
Herbert Boyer (1972) by linking gene encoding for
antibiotic resistance with plasmid of Salmonella
typhimurium
• Isolation of desirable gene (antibiotic resistant)- cutting out
piece of DNA from a plasmid responsible of antibiotic
resistance which involve ‘molecular scissors’- restriction
enzymes
• Desirable gene/ alien DNA – linked with plasmid (vector) to
transfer into host organism
• Linking of DNA involves DNA ligase- acts on cut DNA molecules
& join their ends- new combination circular autonomously
replicating DNA created in vitro- Recombinant DNA
• rDNA transferred into Escherichia coli- DNA replicate using
host’s DNA polymerase- multiple copies of antibiotic resistant
gene- Cloning & E. coli will become antibiotic resistant

• Basic steps in genetically modifying organism:

1. Identification of DNA with desirable gene
2. Introduction of the identified DNA into host
3. Maintenance of introduced DNA in the host and transfer of the
DNA into its progeny
Steps to genetically modify organisms
Basic steps involved in process

Screening
Isolating Fragmenting DNA
Genomic DNA DNA fragments

Culturing the Introducing Insertion of

cells DNA in DNA in vector
host

Transformatio
n of host
cell
Basic steps involved in process

Isolating Isolating
genomic DNA
genomic
DNA from the donor.

Fragmenting
Fragmenting this DNA using
this DNA molecular
scissors.
Basic steps involved in process

Screening the
Screening
the
fragments for a
fragments “desired
gene”.

Inserting the
Insertion of
fragments with the
DNA in a desired gene in a
vector ‘cloning vector’.
Basic steps involved in process

Introducing the recombinant

Introducing vector into a competent host
in Host
cell

Culturing these cells to obtain

Culturing multiple copies or clones of
the cells desired DNA fragments

Using these copies to

transform suitable host cells
Transformation so as to express the
of host cell desired gene.
Biotechnology led to production of many products
and
provides many services f o r human welfare.
Tools of recombinant DNA technology

• Recombinant DNA technology is accomplished with tools:

1. Restriction Enzymes: Restriction endonuclease &

Restriction exonuclease

2. Polymerase enzymes

3. Ligase

4. Vectors

5. Host
Restriction enzymes

• Enzymes which is used to make cut in DNA in

recombinant DNA technology
• 1963 two enzymes were isolated from Escherichia coli
which restricts the growth of bacteriophage
• One enzyme added methyl group to DNA & other cut
DNA and was named ‘Restriction endonuclease’
• Function of Restriction endonuclease depend on
specific DNA nucleotide sequence
• First endonuclease isolated was Hind II; studies
proved that Hind II always cut DNA at particular point
by recognizing a specific sequence of six base pairs-
recognition sequence of enzyme
• 900 restriction enzymes isolated from 230 strains
of bacteria
• Restriction enzymes belongs to nuclease class of enzymes. Its
of
two types:

1. Restriction exonuclease – removes nucleotides from the end

of DNA

2. Restriction endonuclease – cut at specific positions within

DNA

• While naming restriction enzymes- first letter represent

genomic name & second two letter represent species name of
the prokaryotic cell from which the enzyme was isolated
• Eg.- EcoRI- isolated from Escherichia coli RY 13; R- name of
How restriction endonuclease act??
• R.E inspects DNA sequence to find its specific recognition site
to bind & cut specifically each of the two strand of the double
helix at their sugar phosphate backbone
• R.E recognizes specific palindromic nucleotide sequence
in DNA
• Palindrome in DNA- sequence of base pairs that reads the
same on two strands when orientation of reading is kept
the same, i.e., 5’ 3’ in both strands
• R. E cut the strand away from the center of palindrome sites
but between same two bases on the opposite strand leading to
formation of single stranded portion at the ends- Sticky ends
• Sticky end- hydrogen bond with their complementary
cut counterparts- DNA ligase
Application of restriction endonuclease
• Used in the field of genetic engineering to form recombinant
molecules of DNA- composition of DNA from different sources
• Same R.E (vector & source DNA)- same kind of sticky ends
which can be joined by DNA ligase
Diagrammatic representation of recombinant dna technology
Separation and isolation of dna fragments
• Action of R. E result fragments of DNA- separated by Gel
Electrophoresis
• Depends on negative nature of DNA
• External electrical field is applied to the medium- DNA move to
anode
• Medium/ matrix used is agarose gel- natural polymer,
extracted
from sea weed
• Medium- acts as sieve & separate DNA according to their
sizes; smaller fragments- farther
• Separated DNA- stained with Ethidium bromide & exposed to
UV radiations- bright orange coloured bands of DNA in gel
• Separated bands of DNA- cut out from agarose gel &
extracted from gel piece- Elution
• Purified DNA fragments- constructing recombinant DNA by
joining them with cloning vector
 Electrophoresis- separation of
DNA fragments based on size

Staining- Ethidium bromide

Exposure to UV radiation
Cutting out DNA fragments
Cloning vectors
• A cloning vector is a small piece of DNA, taken from a virus or
a plasmid of bacteria, that can be stably maintained in an
organism, and into which a foreign DNA fragment can be
inserted for cloning purposes
• Common vectors- Plasmids & Bacteriophage

• Salient features of cloning vectors:

1) Ability to replicate within bacterial cell independent
of chromosomal DNA
2) High copy number per cell; Plasmids- 15- 100
copies per cell
3) Alien DNA when integrated with vector can multiply
equal to
copy number of vector
4) Vectors can be engineered for easy linking of foreign DNA
& selection of recombinant from non- recombinants
Features to facilitate cloning into vectors

• Features required to facilitate cloning into

vectors:
1. Origin of replication (ori)
2. Selectable marker
3. Cloning sites
4. Vectors for cloning genes in plants & animals
1. Origin of replication (ori):
• Sequence on DNA where replication starts/ initiates
• Alien DNA when linked to it- replicate within host cell
• Regulates & control copy number of linked DNA/ alien
DNA/ target DNA
• If multiple copies of target DNA required, then target DNA be
cloned in a vector whose origin of replication supports high
number

2. Selectable marker:
• Identify & eliminate non- transformants from transformants &
selectively permit growth of transformants
• Transformation- procedure through which rRNA introduced
into host bacterium- change in phenotype
• Eg.- E. coil- resistance against ampicillin, chloramphenicol,
tetracycline or kanamycin- selectable markers which is
lacked in normal E. coli
3. Cloning sites
• Region in the vector where ligation of target DNA takes place
• Created by commonly used specific R. E at the recognition
sites/ restriction sites within a vector where ligation of
alien DNA takes place
• pBR322- genetically engineered plasmid; widely used E. coli
cloning vectors; the ampR gene- encodes for ampicillin resistance
protein, and the tetR gene- encodes for tetracycline resistance
protein; have specific restriction sites for different Restriction
endonucleases (BamH I, Sal I, Hind III, Cla I, EcoR I, Pvu I Pvu II,
Pst I)
• Presence of more than one recognition site within vector will
complicate gene cloning
• Ligation of alien DNA- restriction site present in one of two
antibiotic resistance genes
• Inactivate the gene- enable to choose transformants from non
transformants
• If alien DNA is ligated at Bam HI site in tetracycline resistance gene-
recombinant DNA loose its resistance against Tetracycline
(transformants/ recombinants)
• Transformants can be selected from non transformants- plating
bacteria on ampicillin containing medium- bacteria grows
• Transformants growing in ampicillin transferred to tetracycline
medium- transformants cannot grow & non recombinants grow- due
to gene gets inactivated- insertion
• Selection of recombinants due inactivation of antibiotics-
cumbersome process due to simultaneous plating on
two different antibiotics
• Alternative selectable marker developed- differentiates to
produce colour in the presence of chromogenic
substance
• Recombinant DNA sequence is inserted within coding
sequence of enzyme β- galactosidase- inactivation of
enzyme- insertional inactivation
• Presence of chromogenic substrate (β- galactosidase)- blue
coloured colonies (bacteria with plasmid with no insert)
• Presence of insert- inactivation of β- galactosidase- colonies do
4. Vectors for cloning genes in plants & animals:
• Bacteria & virus- transform eukaryotic cell by delivering genes
• Agrobacterium tumifaciens (pathogen of dicot plants)- deliver
a DNA piece- ‘T- DNA’ & transform normal cell to tumor which
produces chemicals required by the pathogen
• Retrovirus- transforms normal animal cell to cancerous cell
• An understanding of mechanism of delivering genes by
pathogens to their host can help to understand the tools of
pathogen & can be used to deliver genes of interest in
human
• Tumor inducing plasmid (Ti) of Agrobacterium tumifaciens-
modified into cloning vector- non pathogenic to plants &
have ability to deliver genes of interest to variety of plants
• Retrovirus- disarmed; delivers genes of interest in animals
• When gene of interest is ligated into a suitable vector-
transferred into suitable host (bacteria, plant or animal)- multiply
Plant vector- Ti plasmid
Animal vector
Competent host (for transformation with rDNA)
• rDNA should enter cell to transform cells
• But DNA cannot enter cell in a medium without treatment-
hydrophilic
• Host should be made competent to take up DNA (vector)

Different techniques to make host competent:

1.Chemical Treatment:
• Treating hosts (bacteria) with specific concentration of a
divalent cation (Ca)- increases efficiency & make DNA enter
through cell wall of host
• Heat shock (sudden change in temperature)- mixture of rDNA &
host cells
• Incubation of host & rDNA on ice & then placing them at 42ºC &
then back to ice
• Enable bacteria to take up rDNA
2. Microinjection:
• rDNA directly injected into nucleus of host cell
• Common for animal cell

3. Biolistics & gene gun:

• Host cell bombarded with high velocity micro- particles of gold
or tungsten
• Micro- particles are coated with recombinant DNA
• Used for plant cell

4. Disarming pathogen:
• Pathogenicity of vector is removed- ‘disarmed pathogens’
• Disarmed pathogen vector- allowed to infect host & transfer
r DNA
Techniques to introduce recombinant dna
Process of recombinant DNA technology

• Recombinant DNA technology involves following steps:

1. Isolation of genetic material DNA

2. Fragmentation of DNA by restriction endonuclease

3. Isolation of desired DNA fragments

4. Ligation of the DNA fragments into a vector

5. Transferring rDNA into host

6. Culturing host cells in a medium at large scale

7. Extraction of desired product

1. Isolation of Genetic material (DNA)
• Most of eukaryotic cells have DNA as genetic material cells
• To form rDNA, the DNA should be pure, i.e., free from all
biomolecules
• DNA- located inside nucleus enclosed by plasma
membrane, genes interwined with histone protein
• Cell have to break open to release DNA & other macro
molecules (RNA, proteins, polysaccharides & lipids)
• Cells are lysed by lytic enzymes: lysozyme (bacteria),
cellulase (plant cell), chitinase (fungus)
• Macromolecules- treated with specific enzymes to remove;
RNA- ribonuclease, Proteins- Protease,
• Purified DNA precipitates with addition of Ethanol- appear as
collection of fine threads in suspension
Steps for DNA extraction
2. Cutting of DNA at specific location
• Purified DNA (source DNA/ desirable gene DNA & vector DNA)
incubated with specified restriction enzymes for the process-
Digestion at optimal condition
• Progression of restriction enzyme digestion is checked- agarose
gel electrophoresis (source DNA & vector DNA)
• DNA- negatively charged so move towards positive
electrode (anode)
• Once ‘gene of interest’ & cut vector with space for gene of interest is
isolated, DNA ligase is added
• This step is a preparation of recombinant DNA
3. Amplification of Gene of Interest using PCR
• PCR (Polymerase Chain Reaction)- technology used to amplify
a single or a few copies of a piece of DNA to generate
thousands to millions of copies of a particular DNA sequence in
vitro.
• Components required- Primers (small chemically
synthesized oligonucleotides that are complementary to the
regions of DNA), Nucleotides & DNA polymerase- incubated
together
• Steps in PCR
1. Denaturation of double stranded DNA by heating- single
stranded DNA
2. Primer is added- binds to complementary region-
Annealing
3. DNA polymerase- polymerization with nucleotides in
medium;
genomic DNA act as template
4. Replication repeated many times- 1 billion copies of desirable
Polymerase chain reaction
4. Insertion of Recombinant DNA into Host cell/ organism
• Insertion of rDNA possible by making host ‘competent’

• Achieved by- Chemical treatment, microinjection, biolistic &

gene gun method or disarming pathogen
• Phenotype of organism alters when rDNA is transferred to host

• Eg.- rDNA with gene resistance against antibiotic Ampicillin

transferred to E. coli (host)- phenotype alters; resistant
against ampicillin- transformants
• When plated on agar plate with ampicillin- transformed will
grow
(change in phenotype) & non- transformed- die
• Presence of ampicillin resistance gene- select transformed
cells
5. Obtaining the Foreign gene product
• Alien DNA binds with cloning vector- rDNA, transferred to
suitable host (bacteria, plant or animal cell)
• rDNA- expresses itself (target protein) under appropriate
optimal conditions & after cloning of rDNA to particular number
of copies
• Target protein- produce in large scale
• In heterologous host if protein encoding gene is expressed-
recombinant protein
• Heterologous host- gene or gene fragment that does
not naturally present in host & the gene expresses itself
(recombinant protein)
• Cells which harbor cloned genes of interest- grown in
laboratory in small scale
• Culture of dividing host- used to extract desired protein &
then purified by different separation techniques
• In large scale cells can be cultured in continuous culture system
• Here the used medium is drained out form one side & fresh
medium added from other to maintain the cells-
physiologically most active log/ exponential phase
• This culture method produce a larger biomass leading to
higher yields of desired protein
• Biomass- biological material derived from living, or recently
living
organisms
• To produce large quantities of products- Bioreactors are
employed (100- 1000 liters)- large volumes of culture is
processed
• Bioreactors- vessels in which raw materials (recombinant
organism like microbial plant, animal or human cell & culture
medium with ambient condition)- biologically converted into
specific products (protein/ enzymes)
• Optimal growth is achieved with growth conditions-
temperature, pH, substrate, salts, vitamins & oxygen
Structure of Bioreactors:
• Commonly used- stirring type

• Cylindrical & with curved base to facilitate mixing contents

• Equipped with stirrer- even mixing & oxygen

availability throughout reactor
• Oxygen delivery system- bubble air through reactor

• Agitator- thorough mixing of contents

• Also have foam control system, temperature control system,

pH control system & sampling port
• Sampling port- withdraw small volumes of culture periodically
to check the progression
Bioreactors
6. Downstream Processing
• Downstream processing refers to the recovery and
purification of biosynthetic products, particularly
pharmaceuticals, from natural sources such as animal or
plant tissue or fermentation broth
• Products obtained from bioreactors- subjected to series of
process before marketing as a finished product
• Process include separation & purification of biological
products
• Products are formulated with suitable preservatives & then
should undergo thorough clinical trials like drugs
• Strict quality control testing is required for the final biological
products