BIOTECHNOLOGY- principles and

processes
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 host DNA in vector

Transformation
of host cell
Basic steps involved in process

Isolating Isolating
genomic genomic DNA
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 desired
of host cell gene.
Biotechnology led to production of many products and
provides many services for human welfare.
Tools od 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

strain, ‘I’ – order in which the enzyme were isolated from that
strain of bacteria
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

not produce any colour- identify recombinant colony
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
DNA- Amplification & involve ‘thermostable DNA
polymerase’- Thermus aquaticans- to facilitate replication even
at high temperature (denaturation)
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

& considered as Selectable marker
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