Biotechnology: Principles and Processes Class XII Chapter 11.pptx

BIOTECHNOLOGY: Principles and
Processes
CHAPTER - 11

Introduction
Biotechnology can be defined as the use of
microorganisms, plants or animal cells or their
components to produce products and processes
useful to humans.
According to the EFB (European Federation of
Biotechnology), biotechnology is the integration of
natural science and organisms, cells, parts there of
and molecular analogues for products and
services.
The term ‘Biotechnology’ was coined by Karl Ereky
in 1919.

We will learn about
Concept of Genetic engineering
TOOLS OF RECOMBINANT DNA TECHNOLOGY
Restriction Enzymes
Cloning Vectors
Competent Host (For Transformation with Recombinant DNA)
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
Isolation of the Genetic Material (DNA)
Cutting of DNA at Specific Locations
Amplification of Gene of Interest using PCR
Insertion of Recombinant DNA into the Host Cell/Organism
Obtaining the Foreign Gene Product
Downstream Processing
Microinjection
Electroporation
Biolistic gene gun
Sticky end
Gene therapy
Recombinant protein
Transformation
Selectable marker
Electrophoresis
Insertional activation
Biotechnology
Genetic engineering
Transgenic
Gene cloning
Recombinant DNA
Restriction Enzymes- Molecular scissors
Palindromes
Recognition sites/Restriction sites
Plasmid
Polymerase Chain Reaction

BIOTECHNOLOGY
Traditional Biotechnology:
Includes the processes that are based on natural capabilities
of microorganisms. Curd, vinegar, ghee, wine and beer, idli,
dosa, cheese paneer, etc are made using traditional
biotechnology.
Modern biotechnology:
New and highly useful crop varieties, animal breeds are
created. Recombinant proteins are produced using
techniques of modern biotechnology.

PRINCIPLESOFBIOTECHNOLOGY
Genetic engineering : Techniques to alter the chemistry of genetic
material (DNA and RNA), to introduce these into host organisms and
thus change the phenotype of the host organism.
Maintenance of sterile (microbial contamination-free) ambience in
chemical engineering processes to enable growth of only the desired
microbe/eukaryotic cell in large quantities for the manufacture of
biotechnological products like antibiotics, vaccines, enzymes, etc.

Sterile Conditions
Autoclaving- of apparatus, glassware, media by high pressure treatment
Sterilization- of chemicals through filters
Laminar air flow- working space containing sterile air

Why do we need modern Biotechnology?
PRODUCING HYBRIDS
TRADITIONAL BREEDING
OR RECOMBINANT DNA
TECHNOLOGY?

Identification of DNA with desirable genes
Introduction of identified DNA into the host
Maintenance of introduced DNA in the host
and transfer of host DNA to it’s progeny
Wet lab/ dry lab
(Bioinformatics)
Gene transfer to
vector and then the
r-vector to organism
Recombinant DNA
must have ORI
Three basic steps in genetically modifying an organism

Construction of First Artificial Recombinant DNA
(i) It was achieved by linking a gene encoding antibiotic resistance with a native plasmid (an
autonomously replicating circular extrachromosomal DNA) of Salmonella typhimurium.
(ii) Stanley Cohen and Herbert Boyer accomplished this in 1972.
(iii) They isolated the antibiotic resistance gene by cutting out a piece of DNA from a
plasmid.
(iv) The cutting of DNA at specific locations was carried out by molecular scissors, i.e.
restriction enzymes.
(v) The cut piece of DNA was then linked to the plasmid DNA with the enzyme DNA ligase
(molecular glue).
(vi) The plasmid acts as vectors to transfer the piece of DNA attached to it.
(vii) When this DNA is transferred into E. coli, it could replicate using the new host’s DNA
polymerase enzyme and make multiple copies.
(viii) This ability to multiply copies of antibiotic resistance gene in E. coli was called cloning
of antibiotic resistance gene in E. coli.
Stanley Cohen of Stanford and Herbert Boyer of UCSF applied for a patent on recombinant DNA technology in 1974; it was granted in 1980.

First rDNA from “different organisms”
In 1972, Paul Berg succeeded in inserting DNA from a
bacterium into the SV40 virus' DNA.
He thereby created the first DNA molecule made of
parts from different organisms.
Nobel Prize in Chemistry- 1980
(shared the Nobel Prize in Chemistry with Walter
Gilbert and Frederick Sanger)
The Berg letter:
https://wellcomecollection.org/works/ue8p83nr/items?canvas=4

Worksheet 1 20.1.2022
Name two core techniques that enabled birth of modern biotechnology
Name the scientists who accomplished formation of first artificial rDNA?
Where do you see formation of a natural rDNA?
What is the advantage of using genetic engineering over traditional hybridization methods?
In a chromosome there is a specific DNA sequence called_______________, which is
responsible for initiating replication.
To clone a particular gene, it must be linked to a DNA molecule that has a __________ .
Define cloning.
Define plasmid
Three basic steps in genetically modifying an organism —

TOOLS OF RECOMBINANT DNA TECHNOLOGY
Vectors (DNA)
Gene of interest (DNA)
Restriction endonucleases
DNA Ligases
DNA Polymerases
(Alkaline) Phosphatases
Reverse Transcriptase
Host (Cell/organism)

Restriction enzymes
or ‘molecular
scissors’ are used for
cutting DNA.

Nucleases and its types
Restriction enzymes belong to a
class of enzymes called
nucleases.
Nucleases are of two types:
Exonucleases They remove
nucleotides from the ends.
Endonucleases They cut at
specific positions within the
DNA.

Restriction endonuclease- History
(i) Two enzymes from E. coli that were responsible for restricting the growth of
bacteriophage were isolated in 1963, one of them added methyl group to DNA and the other
cut DNA into segments. The later was called restriction endonuclease.
(ii) The first isolated was Hind II in 1968. They found that it always cut DNA molecules at a
particular point by recognising a specific sequence of six base pairs known as recognition
sequence.
(iii) Discovery of Restriction Endonuclease: Arber, Nathans and Smith (Nobel Prize 1978).
(iv) Besides Hind II, more than 900 restriction enzymes have been isolated now, from over
230 strains of bacteria, each of which recognises different recognition sequences.
1972- first rDNA

Restriction Endonuclease: Nomenclature
(a) The first letter is derived from the genus name and the next two letters from
the species name of the prokaryotic cell from which enzymes are extracted.
(b) The Roman numbers after name show the order in which the enzymes were
isolated from the bacterial strain.
For example, Eco RI comes from Escherichia coli RY13 and Eco RII comes from E.
coli R 245, etc.
For example HindIII comes from Haemophilus influenzae.

Source book: Principles of Gene Manipulation and Genomics, Primrose and Twyman

Restriction Enzymes: How do they act?
Each restriction endonuclease recognises a specific
palindromic nucleotide sequences in the DNA.
This site is called the Recognition or restriction site.
Palindrome in DNA is a. sequence of base pairs that reads
same on the two strands when orientation of reading is kept
same.
For example, the following sequences reads the same on the
two strands in 5′ -> 3′ direction as well as 3′ -> 5′ direction.
5′ — GAATTC — 3′
3′ — CTTAAG — 5′
Eco RI

RE which produce sticky ends are useful in RDT

Mechanism of Action of Restriction Enzymes
(a) Restriction enzymes recognize restriction site, bind and cut each of the two strands of DNA at specefic points.
(b) This leaves single stranded portions at the ends.
(c) There are overhanging stretches called sticky ends on each strands.
These are named so, because they form hydrogen bonds with their complementary cut counterparts.
(d) The stickiness of the ends facilitates the action of the enzyme DNA ligase.
(e) These sticky ends are complementary to each other when cut by same restriction enzyme, therefore can be joined
together (end-to-end) using DNA ligases.
Restriction endonucleases and DNA Ligases are used in genetic engineering to form recombinant molecules of DNA,
which are composed of DNA from different sources/genomes.

RECOMBINANT DNA MOLECULE
Recombinant DNA Molecule
Gene of interest

How do we
confirm that the
DNA has been
cut? Or joined..
GEL ELECTROPHORESIS

Separation and Isolation of DNA Fragments
(i) The cutting of DNA by restriction endonucleases results in the fragments of DNA.
(ii) The technique, which separates DNA fragments based on their size is called gel
electrophoresis.
(iii) DNA fragments are negatively charged molecules. They can be separated by forcing
them to move towards the anode under an electric field through a medium/matrix.
(iv) The most common medium used is agarose, a natural polymer extracted from sea
weeds. Hence is also referred to agarose gel electrophoresis.
(v) The DNA fragments separate (resolve) according to their size through sieving effect
provided by the agarose gel.
(vi) The smaller the fragment size, the farther it moves.

How to visualize the DNA on gel?
(vi) The separated DNA fragments can be visualised only after staining the DNA
with a compound known as ethidium bromide followed by exposure to UV
radiation.
(vii) The DNA fragments can be seen as bright orange coloured bands.
Isolation/ Purification of DNA fragments from the gel:
These separated bands are cut out from the agarose gel and extracted from the
gel piece. This is called elution.
The purified DNA fragments can be used in constructing recombinant DNA by
joining them with cloning vectors.

Worksheet 2 22.1.2022
Mention 5 tools of rDNA technology
Name the enzyme: Hemophilus influenzae, strain d, 3rd enzyme
Source of EcoRI -
Differentiate between endonuclease and exonuclease -
What is a palindromic nucleotide sequences -
What is ‘recombinant’ molecule of DNA -
Gel image: Which band represents smaller DNA fragment. Give basis for your inference.
Why were ‘restriction’ enzymes named so ?
How many cuts do you need in a circular plasmid to make it linear?

There are two sites of EcoRI on a plasmid. After
digesting it with the enzyme, only one band is
seen on the gel? Why?

Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.
Cloning
vectors
PLASMIDS AND BACTERIOPHAGES
HAVE THE ABILITY TO REPLICATE
WITHIN BACTERIAL CELLS
INDEPENDENT OF THE CONTROL
OF CHROMOSOMAL DNA.

Vectors used in RDT
(a) Plasmids Autonomously replicating circular extra-chromosomal DNA.
(b) Bacteriophages Viruses infecting bacteria.
(c) Cosmids Hybrid vectors derived from plasmids which contain cos site of X phage.
(d) BAC: Bacterial Artificial Chromosome
(e) YAC: Yeast Artificial Chromosome
(f) MAC: Mammalian Artificial Chromosome
Cloning vectors are the DNA molecules that can carry a foreign DNA segment into the host cell.
The vectors used in recombinant DNA technology can be:

PLASMIDS
1. Autonomously replicating ds circular extra-chromosomal DNA molecules
2. They are found naturally in some bacteria and yeast. They can be made artificially in lab
too.
3. Copy number per cell: They can be high or low copy number. Some plasmids may have
only one or two copies per cell whereas others may have 15-100 copies per cell. Their
numbers can go even higher.
4. The plasmids are by far the most widely used, versatile, easily manipulated vectors.
5. E.g. pBR322, pUC 19 (artificially created)

Features that are required to facilitate cloning into a Vector/Plasmid.
Characteristics of a Vector/ Plasmid
(a) Origin of replication (Ori)
(b) Selectable marker
(c) Cloning sites
Vectors for cloning genes in plants and animals.

Origin of replication (ori)
1. This is a sequence from where replication
starts and any piece of DNA when linked to
this sequence can be made to replicate
within the host cells.
2. This sequence is also responsible for
controlling the copy number of the linked
DNA.
3. If a genetic engineer wants to recover many
copies of the target DNA(insert) it should be
cloned in a vector whose origin support high
copy number.

(ii) Selectable marker
The vector requires a selectable marker, which helps in
identifying and eliminating non transformants and
selectively permitting the growth of the transformants.
(Transformation is a procedure through which a piece
of DNA is introduced in a host bacterium)
Normally, the genes encoding resistance to antibiotics
such as ampicillin, chloramphenicol, tetracycline or
kanamycin, etc., are considered useful selectable
markers for E. coli.
The normal E. coli cells do not carry resistance against
any of these antibiotics. (Only transformed cells do)

(iii) Multiple cloning site (MCS)
DNA region within a Plasmid that contains multiple
unique recognition sites for the commonly used
restriction enzymes.
For each enzyme, the vector needs to have very
few, preferably single, recognition site.
The vector can be cut at these sites so as to enable
ligation of the foreign gene (gene of interest).
The ligation of alien DNA is carried out at a
restriction site present in one of the two antibiotic
resistance genes

Are these sites randomly present on the vector?
IF not, where are these specifically present?
How do we cut the DNA at these sites?
How do we insert the DNA?
How do we check the DNA is inserted or not ?

Inserting gene within a AbR Gene! How does it work?
Ligate a foreign DNA at the BamH I site of tetracycline resistance gene in the vector pBR322.
The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA
Recombinants will still have the ampicillin resistance.
The transformants growing on ampicillin containing medium are then transferred on a medium containing tetracycline.
The recombinants will grow in ampicillin containing
medium but not on that containing tetracycline.
Non-recombinants will grow on the medium
containing both the antibiotics.
Tet X
Amp ✓
Tet ✓
Amp ✓
What about non transformants?
Tet- No
Amp- No

But the process of selecting recombinants by
simultaneous analysis of two plates…
It’s a cumbersome process!
Let us look for an alternative…. (one step process)
β-galactosidase
IPTG
(Inducer)
Blue coloured
product
Colonies appear blue
LacZ gene
X-Gal
LacZ gene
X-Gal is a Substrate (like Lactose)

Using LacZ gene – (instead of selectable marker gene)
DNA is inserted
within the coding
sequence of
β-galactosidase.
This results
into
inactivation of
the enzyme
Principle: Differentiate recombinants from non-recombinants on the basis of their ability to produce colour in
the presence of a chromogenic substrate.
INSERTIONAL INACTIVATION
Blue
colonies
White
colonies
Non
recombinant
Recombinant
✓
X
Blue White Screening -Insertional Inactivation
DNA was not inserted and plasmid self ligated

Vectors for
cloning genes
in plants and
animals

Ti (Tumor inducing) plasmid in Plants
In plants, the Tumour inducing (Ti) plasmid of Agrobacterium tumefaciens is used as a cloning
vector.
Agrobacterium tumefaciens is a pathogen of several dicot plants.
It delivers a piece of DNA known as T-DNA in the Ti plasmid which transforms normal plant
cells into tumour cells to produce chemicals required by pathogens.
How we use it as vector:
◦ Remove T-DNA region- This makes the vector disarmed
◦ Insert the gene of interest in disarmed Ti plasmid
◦ Unaware Agrobacterium, transfers our gene to the plant
genome instead.

Vectors for animals
Retrovirus : PATHOGEN
Retroviruses can transform normal cells into cancerous cells
Useful vector for delivering gene of interest to humans
Retroviruses have been disarmed and are used to deliver
desirable genes into animal cells

Competent Host
(For Transformation with
Recombinant DNA)

Why do we need a ‘competent’ host?
Competent host organism (for transformation with recombinant DNA) is
required because DNA being a hydrophilic molecule, cannot pass through cell
membranes, Hence, the bacteria should be made competent to accept the
DNA molecules.
(i) Competency is the ability of a cell to take up foreign DNA.
(ii) Methods to make a cell competent are as follows-
◦Chemical method- Heat shock method
◦Physical methods- Gene gun, microinjection, electroporation
◦Disarmed pathogen vectors: when allowed to infect the cell,
transfer the recombinant DNA(desired gene) into the host.

Chemical method of TRANSFORMATION:
Heat shock method
Chemical method In this method, the cell is treated with a
specific concentration of | a divalent cation such as calcium to
increase pore size in cell wall.
The cells are then incubated with recombinant DNA on ice,
followed by placing them briefly at 42°C and then putting it
back on ice. This is called heat shock treatment.
This enables the bacteria to take up the plasmid (recombinant)
DNA.

Physical method of gene transfer
In this method, a recombinant DNA is
directly injected into the nucleus of an
animal cell by microinjection method.
In plants, cells are bombarded with high
velocity microparticles of gold or
tungsten coated with DNA called as
biolistics or gene gun method.
Gene
gun
Microinjecton

PROCESSES OF RECOMBINANT DNA TECHNOLOGY
Isolation of Genetic Material
Cutting of DNA at specefic location
Amplification of Gene of Interest using cloning in
vector/PCR
Insertion of Recombinant DNA into host
cell/organism
Obtaining foreign gene product
Important

ISOLATIONOF GENETIC MATERIAL
Break the cell
open- Lysis of
cell by
treatment of-
1.bacterial cells
with lysozyme
2. plant tissue
with cellulase
3. Fungal cells
with chitinase.
Release of DNA
along with other
macromolecules
such as RNA,
proteins,
polysaccharides
and also lipids.
RNA can be
removed by
treatment with
ribonuclease
Proteins can be
removed by
treatment with
protease.
Addition
of chilled
ethanol
Purified DNA
ultimately
precipitates
out
Seen as collection of fine
threads in the suspension

http://nobel.scas.bcit.ca/
https://www.sciencelearn.org.nz/

CUTTINGOF DNA AT SPECEFICLOCATION
Restriction enzyme digestions
Under optimal conditions, purified DNA is cut by restriction enzyme.
This step is done to vector as well
Agarose gel electrophoresis is used to check the digested DNA.
Cut out gene of interest is then incubated with the cut out vector DNA along with DNA Ligase
rDNA is prepared

NPT II
RB LB
NOS Pro SlMBD2
RIP1
Promoter
NOS
ter
pCAMBIA2301 Rip-1-amiRNA SlMBD-Nos
XbaI
SacI
NOS
ter
21bp S 21bp AS
mir164 Loop
Example of a prepared rDNA

Confirmation of insert (SlMBDs) by digestion of back
transformed constructs isolated from Agrobacterium.
pCambia 2301 digested with XbaI and SacI
1.9 kb
750 bp
pCambia 2301
© Bhoomika, 2017

Amplificationof Geneof Interest usingPCR
PCR stands for Polymerase Chain Reaction.
It was developed by Kary Mulis for which he was warded Nobel Prize in 1993
With this technique, we can get multiple copies of the DNA or gene of interest in vitro by using set of
primers and enzyme DNA polymerase.
PCR components:
1. A DNA template: which contains the region to be amplified.
2. A set of Primers (Forward and Reverse) :small chemically synthesised oligonucleotides that are
complementary to the regions of DNA
3. dNTPs : deoxyribonucleotide TriPhosphates
4. A heat stable DNA Polymerase- Taq Polymerase : isolated from a bacterium, Thermus aquaticus,
which remains active during the high temperature induced denaturation of double stranded DNA

PCR : Each cycle
has three steps
(A) Denaturation
(B) Primer annealing
(C) Extension of primers
~30
cycles

Applications of PCR
1. Diagnosis of Pathogens
2. Diagnosis of specefic mutations
3. DNA Fingerprinting
4. Detection of specefic microorganism
5. In prenatal diagnosis

PCR result of 15 samples- out of which 4 are positive

Insertionof RecombinantDNA intohost cell/organism – andscreening
Recipient cells is made ‘competent’ to receive, take up DNA present in its surrounding
The recombinant DNA bearing gene for resistance to an antibiotic is transferred into
E.coli cells
Host cell become transformed into ampicillin-resistance cells
Only transformants will grow on Ampicillin containing medium
Screening of transformants with selectable marker gene- Antibiotic resistance genes
Insertion
screening

This is -
AMPLIFICATON OF GENE INVIVO AMPLIFICATON OF GENE INVITRO
PCR

Gene of interest
Recombinant DNA
Host Cell containing recombinant Vector
PCR/Genome
Vector
Transfer to plant
Transgenic plant
Expression of foreign
gene in host cells
Optimized condition for Induction
RE RE
Stored in E. coli for
further outsourcing

Obtainingforeigngene product
After having cloned the gene of interest and having optimised the conditions to induce the expression of the
target protein, the protein can be produced on a large scale.
The cells harbouring cloned genes of interest may be grown on a small scale in the laboratory. The cultures
may be used for extracting the desired protein and then purifying it by using different separation techniques.
But, small volume cultures cannot yield appreciable quantities of products.
To produce in large quantities of recombinant protein, bioreactors are required, where large volumes (100-
1000 litres) of culture can be processed.
BIOREACTORS
Bioreactors re large vessels in which raw materials are biologically converted into specific products, individual
enzymes, etc., using microbial plant, animal or human cells.
A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth
conditions (temperature, pH, substrate, salts, vitamins, oxygen).
Small scale
Large scale: Industrial Biotechnology

(a) Simple stirred-tank bioreactor (b) Sparged stirred-tank bioreactor through which
sterile air bubbles are sparged
Bioreactor: Stirred type, the most common
1. A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents.
2. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor.
3. Alternatively air can be bubbled through the reactor.
4. 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.

DownstreamProcessing
After completion of the biosynthetic stage, downstream processing is required.
Downstream Processing involves processes that make the product obtained ready for marketing.
This process includes separation and purification.
Suitable preservatives are added to it and send for clinical trial in case of drugs before releasing to market for public use
Strict quality control testing for each product is also required.
Steps of downstream processing
(for understanding purpose)

Image Sources: http://theupturnedmicroscope.com/

1. What is a recombinant protein?
2. Name the components a bioreactor must possess to achieve the desired product?
3. What is a continuous culture system? What are its advantages?
4. Explain the role of enzymes in the extraction of DNA from Rhizopus in its purest form.
5. Since DNA is a hydrophillic moelcule, it cannot pass through cell membranes. Name and explain the technique
with which the DNA is forced into (ii) a bacterial cell (ii) a plant cell (iii) an animal cell.
6. Describe two methods of selection of recombinants (bacteria).
7. A selectable marker is used in the section of recombinants on the basis of their ability to produce colou*r in
presence of chromogenic substrate.
(a) Mention the name of mechanism involved.
(b) Which enzyme is involved in production of colour?
(c) How is it advantageous over using antibiotic resistant gene as a selectable marker?

1. How is copy number of the plasmid vector related to yield of recombinant protein?
2. Restriction enzymes should not have more than one site of action in the cloning site of a vector. Comment.
3. A plasmid without a selectable marker was chosen as vector for cloning a gene. How does this affect the
experiment?
4. A mixture of fragmented DNA was electrophoresed in an agarose gel. After staining the gel with ethidium
bromide, no DNA bands were observed. What could be the reason?
5. Describe the role of Agrobacterium tumefaciens in transforming a plant cell.
6. What would happen when one grows a recombinant bacterium in a bioreactor but forget to add antibiotic to
the medium in which the recombinant is growing?
7. Make a chart (with diagrammatic representation) showing a restriction enzyme, the substrate DNA on which
it acts, the site at which it cuts DNA and the product it produces.
8. A plasmid DNA and a linear DNA (both are of the same size) have one site for a restriction endonuclease.
When cut and separated on agarose gel electrophoresis, plasmid shows one DNA band while linear DNA
shows two fragments. Explain.

Describe the various steps involved in
Recombinant DNA technology with the help of
a well labeled. Diagram? 5

What after infection with Agrobacterium
mediated transfer of gene?
Tissue culture ----> Transgenic

Biotechnology: Principles and Processes Class XII Chapter 11.pptx

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