BIOTECHNOLOGY :

PRINCIPLES AND
PROCESSES

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 BIOTECHNOLOGY : PRINCIPLES AND PROCESSES

Biotechnology deals with techniques of using live organisms or enzymes from organisms
to produce products and processes useful to humans.
European federation of biotechnology (EFB) definition : The integration of natural science
and organisms, cells, parts thereof, and molecular analogues for products and services.

Principles of biotechnology

Genetic engineering / Recombinant DNA technology Bioprocess engineering

– Recombinant DNA formation – Maintenance of sterile ambience
– Gene transfer (environment)
– Formation of GMO (Genetically modified – Culture in large quantities.
organism) – Quality testing etc.

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 BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
Steps of recombinant DNA technology

Identification of Transfer of DNA Maintenance of introduced

DNA with desirable into host DNA in the host and
gene Transfer to the progeny Recombinant
Foreign DNA DNA Molecule
Vector DNA
(plasmid)
Same restriction enzyme cutting both foreign Transformation
DNA and vector DNA at specific point E.coli
(Cloning Host)
Cells divide

Ligases join foreign DNA to plasmid

Diagrammatic representation of recombinant DNA technology

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BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
Tools of r-DNA technology Restriction endonuclease:
Restriction enzyme /
Restriction endonuclease Cuts DNA at specific sites
(Molecular scissors) known as recognition /
Vector restriction / cloning site.
Plasmid (extra chromosomal First discovered RE is Hind-II.
DNA of bacteria), More than 900 restriction
Retro virus - for animal cell enzymes that have been
Ti Plasmid (T-DNA) - for plant isolated from over 230 strains
cells. of bacteria each of which
Passenger
recognise different recognition
Gene
DNA of interest
sequences
cDNA, sDNA (synthetic gene)
Host cell
Animal cell, Plant cell,
Bacterial cell, Yeast
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 BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
Nomenclature of Restriction enzyme:

EcoRI
nd rd th
First letter indicates 2 and 3 letter 4 letter indicates Roman numerical
bacterial genus indicates species of strain of bacteria indicates order of
name bacteria isolation
Escherichia coli RY13 I

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 BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
Action of Restriction Enzyme Vector DNA Foreign DNA

· The enzyme cuts both DNA

strands at the same site
EcoRI

· EcoRI cuts the DNA between Sticky end

bases G and A only when the
sequence GAATTC is present in
the DNA Sticky end
DNA fragments join at sticky
ends

Recombinant DNA
Steps in formation of recombinant DNA by action of
restriction endonuclease enzyme - EcoRI
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 BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
Vector DNA : The DNA used as a carrier for transferring EcoRI Cla I Hind III
a fragment of foreign DNA into a suitable host. Pvu I
Features required to facilitate cloning into a vector Pst I BamH I
R R
amp tet
Origin of replication (Ori)
 This is a sequence from where replication starts. pBR322 Sal I
ori rop
 It is also responsible for controlling the copy
number of the linked DNA.
Pvu II
Selectable marker
 It is used for selection of transformant
and non transformant. E. coli cloning vector pBR322 showing
Eg-Antibiotic resistant gene, Enzyme restriction sites (Hind III, EcoR I, BamH I,
forming gene. Sal I, Pvu II, Pst I, Cla I), ori and antibiotic
Restriction sites / Cloning sites resistance genes (ampR and tetR). Rop
 The ligation of alien DNA is carried out codes for the proteins involved in the
at a restriction site present in one of the replication of the plasmid.
two selectable marker gene.
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 BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
Process of r-DNA technology
Wells
(A) Isolation of DNA:
DNA
(i) Lysing enzymes Bacterial cell - Lysozyme
Fungal cell - Chitinase Largest bands Smallest
Plant cell – Cellulase & Pectinase
Animal cell – Lipase & Protease
(ii) DNA purification
Ribonuclease – Removal of RNA.
Protease – Histones & other proteins.
(iii) DNA precipitation – Chilled ethanol A typical agarose gel electrophoresis showing
(iv) Spooling. migration of undigested (lane 1) and digested
(B) Fragmentation of DNA by restriction endonuclease set of DNA fragments (lane 2 to 4)
Incubation of purified DNA molecules
withelectropheresis
(C) Gel RE. (D) Staining with EtBr (ethidium bromide)
In agarose gel DNA is loaded at cathode or wells (E) Visualisation in ultra voilet light – Bright
Negatively charged fragments run orange colour bands
towards anode. (F) Elution – Separation of gene from gel.
DNA separate according to their size (G) Entry into vector.

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 BIOTECHNOLOGY : PRINCIPLES AND PROCESSES

A DNA was cleaved by using restriction endonuclease and restriction fragment (a, b and c) produced as a digestion
are shown below. Select the option that represents the correct gel electrophoresis separation of these fragments

(1) (2)

(3) (4)

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 BIOTECHNOLOGY : PRINCIPLES AND PROCESSES

If the alien DNA is ligated at the Pvu-I site of ampicillin resistance gene in the given vector
then gene which get inactivated due to insertion of alien DNA helps in the selection of :

(1) Transformants (2) Recombinants

(3) Non-transformants (4) Both (2) and (3)

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 BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
Region to be amplified
Amplification of gene of
5' 3'
3' 5' ds DNA interest by using PCR
Heat Thermal cycle
Denaturation 94°
5' 3' In vitro DNA replication
3 5' Primers Using DNA primer
5 3' ' Annealing 54°
3 ' Taq polymerase
5'
' DNA polymerase (Thermostable enzyme)
(Taq polymerase)
+ deoxynucleotides
5 3' Amplified
' 3 5' Extension 72°
3' ( 1 billion
'5 30 cycles times)
3 ' 5'
'
Polymerase chain reaction (PCR) : Each cycle has three steps:
(i) Denaturation; (ii) Primer annealing; and (iii) Extension of primers
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 BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
Gene transfer in Host cell
(A) Indirect method (Vector mediated)
(i) In Bacterial cell
Heat shock treatment
Treat with Keep cell at
Bacterial cell + r-DNA Ca+2 ion Keep Keep again Heterologous
42°C for
(Host) at 0°C at 0°C host
some time
(ii) In plant cell
Agrobacterium Chromosomal (B) Direct method
T-DNA DNAT-DNA
integrat
Micro injection Gene gun / Biolistic method
es Foreign gene is Foreign gene coated on
Tumor
Chromosome (crown gall) injected in host gold or tungsten particles
Ti plasmid Transformed bombarded at host
plant cell
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BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
Selection of transformant with recombinant DNA
(A) Selection by two antibiotic R R Foreign DNA
resistance genes – amp pBR32 tet Bam HI Same RE cutting both foreign &
Insertional inactivation 2 vector DNA at specific point
Vector DNA
Desired gene
ampR pBR32 DNA ligase
2
R
pBR32 tet amp pBR3 Desired gene
R
ampR
2 22
Self ligated Vector DNA Recombinant vector DNA
(Non-recombinant) Transformation Bacterial chromosome

Transformant with Transformant with Non-transformant

non-recombinant DNA recombinant DNA (without plasmid)
All cells growing on ampicillin Selection of
containing medium transformed cells Death

Transformant with Transformant with Transformant with

non-recombinant DNA recombinant DNA recombinant DNA
Selection of recombinants
Transformed cells growing on
tetracycline containing medium (Death)
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 BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
(B) Selection by one Lac z gene and one antibiotic resistance gene – Insertional inactivation
Transcription
& Translation -galactosidase X-gal Recombinant bacteria  white colonies
Lac z gene
enzyme medium Non recombinant bacteria  blue colonies

Obtaining the foreign gene product :

If any protein encoding gene is expressed in a heterologous host, it is called a recombinant protein.
Recombinant host cell is cultured in large tanks called bioreactors in optimum condition
(temperature, pH, substrate, salts, vitamins, oxygen)
Types of culture

Batch culture Continuous culture

Medium is added only once in In this used medium is drained out from one side while
bioreactor and then extracted fresh medium is added from other side to maintain the
after complete process. cells in their most active log / exponential phase.

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 BIOTECHNOLOGY : PRINCIPLES AND PROCESSES

Bioreactors in cultures :
• The most commonly used bioreactors are of stirred type.
• A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the
mixing of the reactor contents. The stirrer facilitates even mixing and oxygen
availability throughout the bioreactor. Alternatively air can be bubbled through
the reactor.
• The bioreactor has an agitator system, an oxygen delivery system and a foam
control system, a temperature control system, pH control system and sampling
ports so that small volumes of the culture can be withdrawn periodically.

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 BIOTECHNOLOGY : PRINCIPLES AND PROCESSES

Increased surface area

for oxygen transfer

Motor
Acid/Base
Foam braker Gas
for pH control
entrainment
Flat bladed
Steam for impeller
sterlisation Culture broth
Bubbles\
dramatically
increase the oxygen
Sterile Air transfer area
(a) Simple stirred – tank bioreactor (b) Sparged stirred – tank bioreactor

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 BIOTECHNOLOGY : PRINCIPLES AND PROCESSES

Downstream processing
(after completion of biosynthetic stage)

Separation and Addition of suitable Quality control Clinical trials

purification of preservative testing
product

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