UNIVERSITY INSTITUTE OF SCIENCES

Academic Unit V
Bachelor of Engineering
(Computer Science & Engineering)
Biology For Engineers
23SZT148

Recombinant
DNA Technology
DISCOVER . LEARN . EMPOWER
Recombinant DNA Technology

Stem Cell Biology

Tissue Engineering

2
 Course Outcome
CO
Title Level
Number
Identify the biological concepts from an Remember
CO1
engineering perspective.
Development of artificial systems mimicking Understand
CO2
human action.
Explain the basic of genetics that helps to identify
CO3 Understand
and formulate problems

Apply knowledge of measurement system,

biomedical recording system and biosensors to Understand
CO4
excel in areas such as entrepreneurship,
medicine, government, and education.
Will be covered in this lecture
Integrate biological principles for developing next
CO5 Understand
generation technologies https://byjus.com/biology/tools-recombinant-dna-technolog
y/
 SYLLABUS
Unit-3 Bio Medical Instrumentation Contact Hours: 15
Chapter 1 Electrical stimulus and biophysical activity: Patient, electrical
Electrical stimulus shock and hazards, leakage current, Electrical activity of heart (ECG),
and biophysical Electrical activity of brain (EEG), Electrorentinogram (ERG), Electro-
activity occologram (EOG), Electromyogram (EMG).

Chapter 2 Biomedical recording systems: Basic Recording system, General

Biomedical consideration for signal conditioners Preamplifiers, Differential
recording system Amplifier, Isolation Amplifier, Vectorcardiograph, Phonocardiograph,
Other biomedical records, Patient isolation, and accident prevention.

Chapter 3 Tissue Engineering: Recombinant DNA technology, Stem cell biology

Tissue Engineering & Tissue Engineering.
Tissue Engineering
• An interdisciplinary field that applies
the principles of engineering and life
sciences toward the development of
biological substitutes that restore,
maintain, or improve tissue functions
or a whole organ.
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 Recombinant
DNA Technology
Recombinant DNA technology is the joining together of
DNA molecules from two different species. The
recombined DNA molecule is inserted into a host
organism to produce new genetic combinations that are of
value to science, medicine, agriculture, and industry.

6
 Recombinant DNA Technology
• Recombinant DNA technology is the joining
together of DNA molecules from two different
species.
• Using Recombinant DNA technology, we can isolate
and clone single copy of a gene or a DNA segment
into an indefinite number of copies, all identical.
• These new combinations of genetic material or
Recombinant DNA (rDNA) molecules are
introduced into the host cells, where they propagate
and multiply.
• The technique or methodology is called
Recombinant DNA technology.
• The rDNA molecule is inserted into a host organism
to produce new genetic combinations that are of
value to science, medicine, agriculture, and industry.
• e.g. Bacterial DNA + human gene for insulin
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Discovery of DNA structure by Watson & Crick in 1953

Isolation of DNA ligase in 1967

Isolation of Restriction Enzymes in 1970

Paul Berg generated rDNA technology in 1972

Cohen & Boyer in 1973 produced the first plasmid vector

capable of being replicated within a bacterial host
8
 Tools used in Recombinant DNA technology
• The vectors help in carrying and integrating the desired
gene.
• These form a very important part of the tools of
Vector: A vehicle
recombinant DNA technology as they are the ultimate
to transfer rDNA
vehicles that carry forward the desired gene into the
into the cells
host organism.
• Plasmids and bacteriophages are the most common
vectors in recombinant DNA technology.
Restriction • Restriction enzymes cut DNA molecules at specific
enzyme: Scissors
DNA sequences called restriction sites
to cut DNA
DNA Ligase: Help • DNA ligase is an enzyme that seals the bonds between
to join DNA restriction fragments
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 Plasmids
• Plasmids are small circular double-stranded DNA
molecules that replicate independently of the
chromosomal DNA.
• e.g. pUC18, pBR322
• They are present in prokaryotes (e.g. E. coli) in
addition to the chromosomal DNA.
• Plasmids contain one or more antibiotic-resistance
genes.
• These genes provide antibiotic-resistance to the
bacteria.
• Plasmids can be transferred from one bacterium to
another.
10
 Restriction Enzymes
• A restriction enzyme is a protein isolated from bacteria that cleaves DNA
sequences at sequence-specific sites, producing DNA fragments with a known
sequence at each end.
• They protect the bacteria against viral infections
• When a virus infects a bacterial cell, restriction enzymes split the viral DNA thus,
the virus is destroyed
• Restriction endonucleases cleave DNA strands in the middle whereas
exonuclease cleaves nucleic acid strands from the ends.
• Hundreds of restriction enzymes have been discovered so far
• They are named after the bacterium in which they are found e.g. Hin I, Hae III,
Eco RI
• These enzymes serve as molecular scissors to cut DNA into defined fragments
• Each restriction enzyme: Recognizes a specific base sequence in double-stranded
DNA
• Splits the DNA at this site called the restriction site yielding restriction fragments.
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 DNA Ligase
• DNA ligases are the enzymes that
are used to join (ligate) DNA
fragments
• The DNA ligase joins two DNA
strands by forming a
phosphodiester bond between them.
• It is known as a molecular glue.
• It is used to join genes of interest
with the cloning vector.
• e.g. T4 DNA ligase is used in
rDNA technology.

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 Enzymes used in rDNA
Technology
• Recombinant DNA is the method of joining two
or more DNA molecules to create a hybrid.
• The technology is made possible by two types
of enzymes, restriction endonucleases, and
ligase.
• A restriction endonuclease recognizes a specific
sequence of DNA and cuts within, or close to,
that sequence. E.g. EcoRI
• DNA fragments generated by digestion with a
restriction endonuclease can be joined together
again by the enzyme ligase (e.g. T4 DNA
ligase).
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Host organism

• The organism in which

the gene of interest
(with or without
vector) is inserted and
multiplied is known as
the host organism.
• Escherichia coli is
commonly used for
multiplication of
various genes.
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rDNA Cloning

• DNA cloning is a molecular biology

technique that is used for the creation of
exact copies or clones of a particular gene
or DNA.
• The DNA molecules produced through
the cloning techniques are used for many
purposes, which include:
• DNA cloning can be used to make
proteins such as insulin with biomedical
techniques.
• It is used to develop recombinant
versions of the non-functional gene to
understand the functioning of the
normal gene. This is applied in gene
therapies also.
• It helps to analyze the effect of
mutation on a particular gene. 15
St • The DNA fragment containing the gene sequence to be cloned (also known as the gene
ep of insert) is isolated.
1
St
• Cutting DNA with the help of restriction enzymes
ep • Insert gene of interest into vector with the help of ligases
2

St • Transformation: Insertion of these DNA fragments into the host cell using a “vector”
ep (carries DNA molecule).
3
St • Selection: Selection of the host cells carrying the rDNA molecule using a marker.
ep
4
St • Cloning of rDNA: The rDNA molecules are generated when the vector self-replicates
ep in the host cell.
5
St • Isolation of rDNA or its product
ep
6
St • Testing of biological properties of gene of interest
ep
7 16
Steps for Recombinant DNA Technology
• Bacterial plasmids with a resistance gene present (usually
ampicillin or kanamycin-resistant) are used as vector DNA
• (1) Isolate the DNA sequence gene of interest.
• (2) Insert your DNA of interest into the vector plasmid:
• (a) cut both vector DNA and gene of interest with the same
restriction enzymes
• (b) mix the DNAs: they join by base pairing,
• (c) add DNA ligase to bond the pieces covalently. The result
is a recombinant plasmid.
• (3) Put recombinant plasmid into E. coli by transformation.
• (4) Clone cells:
• (a) plate cells onto agar containing ampicillin (or
kanamycin)
• (b) each colony contains the recombinant plasmid by its
ability to grow in the presence of ampicillin in the agar.
• (5) Pick colonies and inoculate a large culture to expand the
plasmid carrying the gene of interest.
• (6) Isolate and clean the DNA from the expanded culture.
• (7) Transfer the plasmid DNA to desired cells (e.g., bacteria,
yeast, mammalian cells, etc.) and test the biological properties
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of the gene of interest.
Applications of recombinant
DNA in genetic engineering
are: Production of an Insulin

• For the production of vaccines like the

hepatitis B vaccine.
• Production of transgenic plants with
improved qualities like insect and
drought resistance and nutritional
enrichment.
• Therapeutic protein production like
insulin.
• Gene therapy in diseases like cancer,
SCID etc.
• Production of transgenic animals with
improved quality of milk and egg.
 Agriculture:
Genetically modified crops
• Resistance to diseases, insects and pests, herbicides, drought; metal toxicity tolerance; induction of male
sterility for plant breeding purposes; and improvement of quality can be achieved through this
recombinant DNA technology.
• BT-cotton, resistant to bollworms; Herbicide or pesticide-resistant corn & soybean
• “Golden rice” with beta-carotene which is required to make vitamin A, helps to prevent blindness

Pharmacology: Large-scale production of human hormones and other proteins with therapeutic
uses
• Production of safer vaccines
• A number of therapeutic gene products —insulin, interleukins, interferons, growth hormones,
erythropoietin, and coagulation factor VIII—are now produced commercially from cloned genes

Medicine: Production of antibiotics, hormones, vaccines and interferon

• Human Gene Therapy
• Diagnosis of genetic disorders
• Forensic Evidence
• Production of Antibiotics like Penicillium and Streptomyces 19
• Production of Hormone Insulin
Gene Therapy
• Gene therapy is a technique that involves the replacement of
defective genes with healthy ones in order to treat genetic disorders.
• It is an artificial method that introduces DNA into the cells of the human body.
• In some genetic disorders, patients lack the functional form of a particular gene.
Gene therapy attempts to provide a normal copy of the gene to the cells of a
patient’s body.
• For example, DNA cloning was used to build plasmids containing a normal
version of the gene that's nonfunctional in cystic fibrosis.
• When the plasmids were delivered to the lungs of cystic fibrosis patients,
lung function deteriorated less quickly.
• Application of Gene Therapy
• It is used in the replacement of genes that cause medical ill-health
• The method generally destroys the problem causing genes
• It helps the body to fight against diseases by adding genes to the human body
• This method is employed to treat diseases such as cancer, ADA deficiency,
cystic fibrosis, etc.
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21
Stem Cell Biology

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Stem cells are one of most fascinating areas of biology today

Stem cells are a specific type of cell capable of evolving into

many different types of specialized cells within the body.

• They serve as a repair system for the body.

Due to their ability to repair, regenerate, and develop into

certain specialized cell types, stem cells offer great promise as
therapy for a number of diseases.
• Research on stem cells is going on, and it is believed that stem cell
therapies can cure ailments like paralysis and Alzheimer’s as well. 23
 Stem Cells
• Stem cells which are
the undifferentiated
master cells have the
ability to continuously
divide and develop into
various other kinds of
cells.
24
Stem cells are different from other cells in the
body in three ways:
• Proliferation: They can divide and renew themselves
over a long time
• Differentiation: They are unspecialized, so they
cannot do specific functions in the body
• Plasticity: They have the potential to become
specialized cells, such as muscle cells, blood cells, and
brain cells
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There are two major sources of stem cells:
• Embryonic stem cells and adult stem cells are also called tissue stem
cells.
• Embryonic stem (ES) cells are stem cells that are derived from the
inner cell mass of a mammalian embryo at a very early stage of
development, when it is composed of a hollow sphere of dividing
cells (a blastocyst).
• Adult stem cells: Some tissues in the adult body, such as the
epidermis of the skin, the lining of the small intestine, and bone
marrow, undergo continuous cellular turnover. 26
 Embryonic stem cells
• Embryonic stem cells (often
referred to as ES cells) are
stem cells that are derived
from the inner cell mass of a
mammalian embryo at a
very early stage of
development, when it is
composed of a hollow
sphere of dividing cells (a
blastocyst).
27
Adult stem cells: Depending upon the source of their origin
they are of different types
• Hematopoietic stem cells (HSCs): These are found in bone marrow
and are a continuous source of progenitors of red cells, platelets,
monocytes, granulocytes and lymphocytes.
• Mesenchymal stem cells or Marrow stromal cells (MSCs): These are
also isolated from bone marrow and, in culture can differentiate into
osteoblasts, chondrocytes, adipocytes, and even myoblasts.
• Neural stem cells: These are isolated from the central nervous system
(CNS) and have the potential to differentiate into neurons, astrocytes,
and oligodendrocytes. There are other kinds of stem cells.
• Umbilical cord and Placental stem cell: These are isolated from
umbilical cord and placenta. It is relatively easy to collect, with no risk
to the mother or baby. It contains haematopoietic (blood) stem cells. 28
 Types of Stem Cells
• There are 5 major types:
• Totipotent Stem Cells: These can differentiate into all possible types of cells.
• Each cell can form a complete organism.
• These types of cells are found in plants and in early embryos (1-3 days)
• Pluripotent Stem Cells: These are the cells from an early embryo (blastocyst
5-14 days) and can differentiate into over 200 types of cell type.
• Multipotent Stem Cells: These differentiate into a closely related cell type.
• e.g., the hematopoietic stem cells differentiate into red blood cells and white
blood cells.
• Oligopotent Stem Cells: Adult lymphoid or myeloid cells are oligopotent.
They can differentiate into a few different types of cells.
• Unipotent Stem Cells: They can produce cells only of their own type. Since
they have the ability to renew themselves, they are known as unipotent stem
cells.
• e.g., Muscle stem cells. 30
31
 Application of Stem Cell

• Basic research: Clarification of complex events that occur during

human development and understanding molecular basis of cancer
• Biotechnology (drug discovery and development): Stem cells
can provide specific cell types to test new drugs
• Tissue Regeneration: This is the most important application of
stem cells. The stem cells can be used to grow a specific type of
tissue or organ.
• This can be helpful in kidney and liver transplants.
• The doctors have already used the stem cells from beneath the
epidermis to develop skin tissue that can repair severe burns or
other injuries by tissue grafting.
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 Application of Stem Cell

• Cancer treatment: They are applied especially to treat cancers,

which require high-dose chemotherapy within the scope of medical
care.
• The patient’s own stem cells are extracted from bone marrow or
peripheral blood prior to high-dose chemotherapy, stored
temporarily and transplanted after the treatment in order to
minimize the side effects of the aggressive chemotherapy and to
support the regeneration of destroyed cells.
• Treatment of Brain Diseases: Stem cells can also treat diseases
such as Parkinson’s disease and Alzheimer’s.
• These can help to replenish the damaged brain cells.
• Researchers have tried to differentiate embryonic stem cells into
these types of cells and make it possible to treat diseases. 34
 Areas of active research on stem cell biology
• Treating liver disease with stem cells that have been manipulated to
become specialized liver cells
• Creating stem cell-derived neurons for the study of motor neuron
disease
• Creating insulin-producing pancreatic beta cells for clinical trials in
diabetes
• Stimulating an anti-brain tumor immune response via manipulated
stem cells
• Investigating the use of stem cells to study and treat Gaucher disease
and parkinsonism
• Reprogramming tumor-specific immune cells from stem cells for
cancer immunotherapy
• Manipulating stem cells to become bone and cartilage 35
 Tissue Engineering
An interdisciplinary field that applies the principles of engineering
and life sciences toward the development of biological substitutes
that restore, maintain, or improve tissue functions or a whole organ.

36
 TISSUE ENGINEERING

• Tissue engineering utilizes living cells

as engineering materials.
• Tissue engineering evolved from the
field of biomaterials development and
refers to the practice of
combining scaffolds, cells, and
biologically active molecules into
functional tissues.
• The goal of tissue engineering is to
assemble functional constructs that
restore, maintain, or improve damaged
tissues or whole organs.
• Artificial skin and cartilage are
examples of engineered tissues;
however, currently they have limited
use in human patients. 37
Why do we need Tissue Engineering?

38
GOALS OF TISSUE ENGINEERING

39
 Tools of Tissue Engineering
• Three tools of tissue engineering
• Cell
• Living part of the tissue
• Produce proteins and provide functions of cell
• Scaffold
• Provides structural support and shape to
construct
• Provide place for cell attachment and growth
• Usually biodegradable and biocompatible
• Cell signalling
• Signals that tell the cell what to do
• Protein and mechanical stimulation
• Growth factors
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 Cell
• Cells are often categorized by their source:
• Autologous cells- obtained from same source to which they
will be implanted. Have fewest problem of rejection and
pathogen transmission
• Allogenic cells- comes from body of same donor species
• Xenogenic cells- isolated from individuals of other species, like
use of animal cells for cardiovascular implants
• Isogenic cells- isolated from genetically identical organisms
like twins, clones
• Stem cells- undifferentiated cells with the ability to divide in
culture , give rise to different specialized cells . Can be
multipotent, pluripotent and totipotent
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 Scaffolds
• Scaffolds are structures
manufactured for the sole purpose of
allowing cells to grow
• Key elements of scaffolds and cell
development
• Structures that are enable to support 3-
D cell structures
• Allow for cell attachment, migration,
and growth
• Enable diffusion of cell nutrients
• Allow the manipulation of cells to
form as correctly shaped
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 Signalling Molecules

•Hormones: Secreted by endocrine glands, by dispersed glandular cells.

•Neurohormones: Secreted by Neurons into blood circulation.
•Neurotransmitters: Secreted by neurons at nerve endings.
•Cytokines: Secreted by immune-competent cells.
•Growth Factors: Secreted by various types of cells. 43
 Steps of Tissue Engineering
• Isolated cells are taken to the
culture system and cultivated
to selective cell differentiation
time
• Different biomaterials are
used for constructing scaffolds
• Different techniques are used
for scaffold fabrication
• For in vitro tissue engineering
tissue culture is done either on
petri dishes or bioreactors 44
 APPLICATIONS
OF TISSUE
ENGINEERING
•Replacing the old cell: When there is damage in our body cells or organs
we use tissue engineering techniques to overcome the damage by
replacing the old cell.
•Regenerative Medicines: Regenerative medicines repair damaged tissues
and organs.
• They stimulate the body’s own repair mechanisms to heal previously
irreparable tissues or organs.
• If the body cannot heal itself, the tissues or organs can be grown in the
laboratory and then implanted.
• Regenerative Medicine also involves the use of stem cells or progenitor
cells obtained through directed differentiation

45
 APPLICATIONS OF TISSUE
ENGINEERING
• Drug Discovery- For effective drug
screening, 3D cultures are introduced
to analyze the effect of drug action
• Organ Transplantation: The
transplantation of cells that perform a
specific biochemical function (e.g.
Artificial pancreas or Artificial liver).
• Therapeutic Cloning: Bioartificial
liver device, artificial pancreas,
artificial bladders, and Cartilage.
• There is a wide range of Tissue
Engineered products or materials that
are used to cure diseases in humans
and save life.
• Even if Tissue Engineering is life-
saving, it is expensive. 46
 REFERENCES

• C.B.Powar, 2010.Cell Biology.5th Ed,Himalyan Publishing House.

• Sandhu, Sardul Singh. Recombinant DNA Technology. India: I.K. International
Publishing House Pvt. Limited, 2013. John G. Webster 1998.
• Principles of Tissue Engineering. Switzerland: Elsevier Science, 2000.
• Jeremy M. Berg, John L. Tymoczko and Lubert Stryer. 2006. “Biochemistry,” 6th
Ed. W.H. Freeman and Co. Ltd.
• Robert Weaver. 2012 “Molecular Biology,” 5th Edition, MCGraw-Hill.
• Concepts and Applications of Stem Cell Biology: A Guide for
Students. Germany: Springer International Publishing, 2020.
• Martin Alexander, 1994 “Biodegradation and Bioremediation,” Academic Press.

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THANK YOU

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