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Introduction to biotechnology

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 Dr. Khaled El-Sayed El-Gayar
- Biology Department, Faculty of Science, Jazan University, KSA

- Egyblood, Vacsera, Cairo, Egypt

 What is Biotechnology?

Biotechnology is any technological application

that uses biological systems, living organisms
or derivatives, to make or modify products and
processes for specific use.
The main areas of application of biotechnology
• Bioprocess technology ( e.g. polysaccharides, medically important drugs,
solvents, protein-enhanced foods).
• Enzyme technology
• Waste technology, recycling of resources; foods and fertilizers, biological
fuels.
• Environmental technology(pollution control, removing toxic wastes);
recovery of metals from mining wastes.
• Renewable resources technology (The use of renewable energy sources, in
particular lignocellulose, to generate new sources of chemical raw materials and
energy – ethanol, methane and hydrogen).

• Plant and animal agriculture

• Healthcare
Biotechnology: a three-component central core

Many biotechnological processes may be considered as

having a three component:
1- Central core, in which one part is concerned with
obtaining the best biological catalyst for a specific process.
2- Construction and technical operation (the best possible
environment for the catalyst to perform).
3- Downstream processing to separate and purify of an
essential products from a fermentation process.
 DNA Composition

Campbell et al.,2008
 Each chromosome has one very long DNA molecule, with
hundreds or thousands of genes arranged along its length.
 The DNA of chromosomes replicates as a cell prepares to
divide, and each of the two cellular offspring inherits a
complete set of genes.
 The entire "library" of genetic instructions that an organism
inherits is called its genome.
 DNA controls the development and maintenance of the entire
organism.
Nucleotides
 The monomers units of DNA are nucleotides.
 A nucleotide is a nucleoside with one or more phosphate groups
covalently attached to the 3'- and/or 5'-hydroxyl group(s).

Phosphate group

Nitrogenous base

Deoxyribose sugar
Nitrogenous bases of DNA
There are four different types of nucleotides found in DNA.

 Adenine (A)
 Guanine (G)
 Cytosine (C)
 Thymine (T)
 In RNA, the four bases include Adenine, Uracil, Guanine,

and Cytosine.
 Adenine and Guanine are double-ring molecules known as
Purines; Cytosine, Thymine, and Uracil are single-ring
molecules called Pyrimidines.
 Deoxyribose sugar
 The deoxyribose sugar of the DNA backbone has 5
carbons and 3 oxygens.
 The hydroxyl groups on the 5'- and 3'- carbons link to
the phosphate groups to form the DNA backbone.
 Deoxyribose lacks an hydroxyl group at the 2'-
position.
2-Deoxyribose sugar
 Structure of RNA
 RNA is a single-stranded molecule and has a much shorter chain
of nucleotides.

 RNA contains ribose sugar. These hydroxyl groups make RNA

less stable than DNA.

 The complementary base to adenine is Uracil (pyrimidine base).

 Most active RNAs, including mRNA, tRNA, rRNA, and other

non- coding RNA, contain self-complementary sequences.

 Comparison between DNA and RNA

RNA DNA
Nucleus and
Presence Nucleus
cytoplasm
Function Help DNA Inheritance
)tRNA(, (mRNA( and
Types No types
)rRNA(
Sugar Ribose De-oxyribose
Nitrogenous
bases
One strand from
Shape Double helix
nucleotides
 Gene Expression

1- Transcription
RNA polymerase makes a copy of information in the
gene complementary to one strands of DNA.

2- Translation
Occurs on ribosomes, messenger RNA is decoded or
translated to determine the sequence of amino acid in the
protein being synthesized.
 DNA

Transcription

mRNA

Translation
Gene Expression

Amino acids chains

Folding

Protein
Ribosomes

 Factory for protein synthesis.

 Composed of ribosomal RNA and ribosomal
proteins (known as a Ribonucleoprotein ).
 Translate (mRNA) to build polypeptide chains
using amino acids delivered by (tRNA).
 Proteins

Proteins are chain like polymers of a few or many

thousands of amino acids.

Amino acids: 3-nucleotide RNA sequences (codon).

Methods in Biotechnology
a- The Human Genome
Project
 What is the Human Genome Project?
- The Human Genome Project (HGP) is a global scientific
research program created to understand the hereditary
instructions that make each of us unique.
- The HGP will create a vast resource of detailed scientific
information about the structure, organization and function
of human DNA.
- U.S. govt. project coordinated by the Department of
Energy and the National Institutes of Health, launched in 1986
by Charles DeLisi.
Genome definition: the whole hereditary information
of an organism that is encoded in the DNA.
Aims of the HGP project:
1- To identify the approximate 22,000 genes in the human
DNA.

2-To determine the sequences of the 3 billion bases that

make up human DNA.

3-To store this information in databases.

4- To develop tools for data analysis.

5- To address the ethical, legal, and social issues that arise

from genome research.
 Benefits of Human Genome Project research

- Improvements in medicine (Improved diagnosis of disease).

- Microbial genome research for fuel and environmental cleanup.
- DNA forensics (Identifying potential suspects at a crime scene).
- Improved agriculture and livestock(More nutritious production).
- Understanding of evolution and human migration (Study migration of
different population groups).
- More accurate risk assessment(Reduce the likelihood of heritable
mutations).
 b- Bacterial restrictions and ligase enzymes
Restriction enzymes cut DNA molecules at a limited
number of specific DNA sequences, called restriction sites
yielding a set of restriction fragments.
The most useful restriction enzymes cut DNA in a
staggered way producing fragments with “sticky ends” that
can bond with complementary “sticky ends” of other
fragments
DNA ligase is an enzyme that seals the bonds between
restriction fragments.
 c- Cloning an Eukaryotic Gene in a Bacterial Plasmid

In gene cloning, the original plasmid is called a cloning

vector.
Defined as a DNA molecule that can carry foreign DNA into
a cell using a restriction enzyme and DNA ligase to make
recombinant DNA.
 Protein production steps from gene (Gene cloning)

1- DNA extraction from human cell/ Plasmid extraction from bacterial

cell

2- Restriction digestion for both DNA and plasmid using the same
restriction enzymes to make sticky ends

Desired gene linear plasmid

3- Ligation by DNA ligase forming recombinant plasmid DNA

4- Transformation into E.coli

5- Protein expressed from gene of interest.
6- Bacterial growth in Bioreactor (Mass production)
7- Protein extraction
8- Protein purification
Khaled E. El-Gayar 2018
 d-Bacterial Expression Systems

Protein production can occur after gene expression. Commonly used

protein expression systems include those derived from bacteria, yeast,
baculovirus/insect, and mammalian cells.
Escherichia coli as a Bacterial system
E. coli is one of the most widely used expression hosts, and DNA is
normally introduced in a plasmid expression vector. The techniques for
over expression in E. coli are well developed and work by increasing
the number of copies of the gene.
Why E. coli in Biotechnology?
• It is one of a family of Enterobacteriaceae, most of which are rod-
shaped Gram negative.
• E. coli metabolism can generate ATP either by oxidative degradation of
organic compounds in the presence of air or by fermentation of simple
sugars under anaerobic conditions.
• Because it grows rapidly on well-defined simple media and because of
the wealth of information on its genetics, biochemistry, and physiology
• E. coli has been a favorite choice for the production of foreign proteins
by means of recombinant DNA technology as Human insulin and
growth hormone.
 e-Amplifying DNA in Vitro :
The Polymerase Chain Reaction (PCR)

PCR is an exponentially progressing synthesis of the

defined target DNA sequences in vitro.
 PCR: Polymerase chain reaction
(30-40cycles of 3 steps)

1- Denaturation
1min. at 94⁰C

2- Annealing
45 sec. at 54⁰C

3- Extension
2 mins. at 72 ⁰C
 f-Gel Electrophoresis

 Gel electrophoresis is a method for separation and analysis

of macromolecules as DNA, RNA and proteins and their

fragments, based on their size and charge.

 DNA is separated using agarose gel electrophoresis or SDS-

polyacrylamide gel electrophoresis(SDS-PAGE)

 Proteins are separated using SDS-PAGE

 Agarose gel electrophoresis
 Agarose gel electrophoresis is the most effective way of
separating DNA fragments of varying sizes ranging from 100
bp to 25 kb.
 Agarose is isolated from the seaweed and consists of repeated
agarobiose (L- and D-galactose) subunits.
 The use of agarose gel electrophoresis is the separation of
DNA.
 To separate DNA using agarose gel electrophoresis, the DNA is
loaded into pre-cast wells in the gel and a current applied.
The phosphate backbone of the DNA (and RNA) molecule is
negatively charged, therefore when placed in an electric field,
DNA fragments will migrate to the positively charged anode.
The rate of migration of a DNA molecule through a gel is
determined by the following: 1) Size of DNA molecule; 2)
Agarose concentration; 3) DNA conformation; 4) Voltage
applied, 5) Presence of ethidium bromide, 6) Type of agarose
and 7) Electrophoresis buffer.
 After separation, the DNA molecules can be visualized under
UV light after staining with an appropriate dye.
 SDS-PAGE
It is a method used to separate proteins according to
their size and charges.
SDS, an anionic detergent, is used in SDS-PAGE to
reduce proteins to their primary (linearized) structure
and coat them with uniform negative charges.
 g-Southern blot

A Southern blot is a method used for detection of a

specific DNA sequences in DNA samples.
Southern blotting combines transfer of electrophoresis
separated DNA fragments to a filter membrane and
subsequent fragment detection by probe hybridization.
h. Western blotting

 "blotting" means the transfer of biological samples from a

gel to a membrane and their subsequent detection on the
surface of the membrane.
The specificity of the antibody-antigen interaction enables a
target protein to be identified in a complex protein mixture.
Western blotting can produce qualitative and semi-
quantitative data about the protein of interest.
Microbes as Tools of Biotechnology
The microbial biotechnology covers many scientific
fields, ranging from production of recombinant
hormones to that of microbial insecticides, from
mineral leaching to bioremediation of toxic
wastes……etc.
 Fermentation Technology
Fermentation technology is the process of deriving energy from
the oxidation of organic compounds, such as carbohydrates.
Fermentation has the ability to produce:
(1)Essential primary metabolites as acetic and lactic acids, glycerol,
acetone, butyl alcohol, organic acids, amino acids, vitamins and
polysaccharides.
(2)Secondary metabolites as penicillin, streptomycin, cephalosporin,
etc.
(3)Many forms of industrially useful enzymes, e.g. amylases,
pectinases and proteases.
(4)Monoclonal antibodies, vaccines and novel recombinant products,
e.g. therapeutic proteins.
A bioreactor refer
to any system
supports a
biologically active
environment. This
process can either
be aerobic or
anaerobic.

https://commons.wikimedia.org/wiki/File:Real_life_bioreactor.png
 Closed bioreactor used in ethanol research

http://www.bioreactors.eu/en/bioreactor

Lab bioreactor https://en.wikipedia.org/wiki/Bioreactor#/media/File:Pg166_bioreactor.jpg

 To achieve optimization of the bioreactor system:
(1) The bioreactor should be designed to exclude entrance of
contaminating organisms as well as containing the desired
organisms.
(2) The culture volume should remain constant.
(3) The dissolved oxygen level must be maintained above
critical levels of aeration.
(4) Environmental parameters as temperature, pH, etc., must
be controlled and the culture volume must be well mixed.
Natural raw materials
Natural raw materials originate mostly from agriculture and
forestry. These are mainly carbohydrates of varying
chemical complexity, and include sugar, starch, cellulose,
hemicellulose and lignin. Sugar-bearing raw materials as
sugar beet, sugar cane or millet are the most suitable and
available to serve as feedstock for biotechnological
processing.
Some Applications of Biotechnology
1-Single – Cell Protein (SCP)
SCP is the protein - rich cell mass containing all the essential
amino acids, derived from microorganisms grown on a
large scale for either animal or human consumption.

Microorganisms are excellent source of SCP because of:

1-Their rapid growth rate.
2- Their ability to use very cheap raw materials as carbon
sources.
3-The uniquely high efficiency, expressed as grams of
protein produced per kilogram of raw material, with which
they transform these carbon sources to protein.
2-Antibiotics Production

An antibiotic is a substance, usually produced by a

microorganism which, in very small quantities, inhibits or
kills other microorganisms .

Traditional method of antibiotic production:

The traditional genetic approach to improving the yield of
an antibiotic producing organism depends entirely on
random mutagenesis and screening of high producers.
Genetics of antibiotic production:
The efficiency of production of antibiotics is
determined largely by the genetic makeup of the
producing strains, and much effort has been spent on
improving these strains.
 3- Industrial Enzymes Production
The use of microorganisms as a source material for enzyme
production has developed for several important reasons:
(1) There is a high activity per unit dry weight of product.
(2) Seasonal fluctuations of raw materials and possible shortages
due to climatic change or political upheavals do not occur.
(3) In microbes a wide spectrum of enzyme characteristics, as pH
range and high-temperature resistance, is available for selection.
(4) Industrial genetics has greatly increased the possibilities for
optimizing enzyme yield.
The organism producing the enzymes should have a GRAS-
status, which means that it is Generally Regarded as Safe.
Most of the industrially used microorganisms have been
genetically modified to overproduce the desired activity.
Several thermostable enzymes, like the Taq polymerase have
been identified and widely used in PCR from Thermus
aquaticus.
Cellulase is obtained from E.coli and degrades cellulose.
Xylanase from fungus Trichoderma is used in paper industry.
Bacterial proteases are still the most important detergent
enzymes. Some products have been genetically engineered to be
more stable in the hostile environment of washing machines.
Protease and lipase containing enzyme solutions are used for
lens cleaning.
Some toothpaste contains glucoamylase and glucose oxidase.
Also enzymes are also used for applications in skin and hair care
products.
The use of starch degrading enzymes, amylase, was the first
large-scale application of microbial enzymes in food industry.
4-Food Technology
Microbial fermentation is essential for production of beer,
butter milk, cheeses, kefir, olives and many more.
The metabolic end products produced by the microorganisms
flavor fermented foods. For example, mold-ripened cheeses
owe their distinctive flavors to the mixture of aldehydes,
ketones, and short-chain fatty acids produced by the fungi.
Lactic acid bacteria are widely used to produce fermented
foods.
 5-Microbial Enhanced Oil Recovery (MEOR)

MEOR is a biological based technology consisting

in manipulating function or structure, or both, of
microbial environments existing in oil recovery.
The aim of MEOR is to improve the recovery
of oil entrapped in porous media while increasing
economic profits.
http://eor.ir/facilities/meor/
6- Application in Forensics

Forensic science can be defined as the intersection of

law and science.
DNA profiling (also called DNA testing, DNA typing,
or genetic fingerprinting) is a technique employed by
forensic scientists to assist in the identification of
individuals via their DNA profiles.
DNA profiling has helped to acquit or convict suspects
in many of the most violent crimes, including rape and
murder.
Because DNA evidence is so specific, it is actually
much easier to exclude a suspect than to convict
someone based on a DNA match.
How can DNA evidence be planted?

Blood and seminal fluid

Sneezing or coughing
Person touches the mouth, nose or other part of the face.
Hairs, fibers, or trace material from their clothing.
Wind can carry in contaminants.
 Criminal Investigation Steps

I- DNA Collection & Comparison

Investigators gather samples from the crime scene and

suspects and then analyze them.
DNA samples can be collected from: Saliva, blood, hair
strands, skin, finger or toe nails, and/or a tooth with a
set of specific DNA regions or markers.
II-PCR: Polymerase Chain Reaction

Used to make millions of exact copies of DNA from a

biological sample.
Allows very small samples to be analyzed, as a sample
of a few skin cells.
Must be very careful about contamination in this
process.
Mitochondrial DNA Analysis

Uses DNA extracted from mitochondrion rather than

nuclear DNA.
Especially useful in old cases and old samples.
III- Digestion of DNA using restriction enzymes.
IV- Separation of the DNA fragments by size using gel
electrophoresis.
V- Transfer of fragments to a nitrocellulose or nylon
membrane.
VI- Hybridization of a probe to the fragment or fragments
of interest.
VII- Probe detection (autorad development).
7- Pharmaceutical Products of DNA Technology
(HUMAN THERAPEUTICS)
• Insulin, expressed from human insulin genes on plasmid
inserted into Escherichia coli, was the first genetically
engineered therapeutic agent to be approved for clinical
use in humans.
• Bacterially produced insulin, used widely in the treatment
of diabetes, is typically in its structure and clinical effects
from natural insulin.
Human growth hormone (hGH), a protein made naturally
by the pituitary gland, was the second product.
Inadequate secretion of hGH in children results in
dwarfism.
Before the advent of recombinant DNA technology, hGH
was prepared from pituitaries removed from human
cadavers.
The supply of such preparations was limited and the cost
prohibitive.
Recombinant human insulin and Human growth
hormone offered impressive proof of the clinical

efficacy and safety of human proteins made by

biotechnology.
 DNA VACCINES

DNA vaccines consist of appropriately engineered plasmid

DNA prepared on a large scale in microorganism.

The obvious advantages of DNA plasmid vaccines are that

they are not infectious, do not replicate, and encode only the

protein(s) of interest.
 8- Gene Therapy

Gene therapy is a process where cells are taken from the

patient, alter their chromosomes by adding genes, and
then replace the cells back into the patient using a
carrier such as a virus.
9-BIOREMEDIATION
 Conventional methods of remediation

1- Dig up and remove it to a landfill

• Risk of excavation, handling and transport of hazardous

material

• Very expensive to find another land to finally dispose

these materials
2-High temperature incineration
3- Chemical decomposition as dechlorination, UV
oxidation
The disadvantages of these methods:
 The cost of the application is very expensive
 Lack of public acceptance as in case of incineration
 Incineration generates more toxic compounds
 Materials released from imperfect incineration cause Ozone depletion.
 Fall back on earth and pollute some other environment.
 Dioxin production due to burning of plastics leads to cancer
Bioremediation is defined as :

The treatment of pollutants or waste using of

microorganisms that break down the undesirable
substances .

The advantages of bioremediation:

 Relatively low cost
 Low technology techniques
 Generally has general public acceptance
 Can often be carried out on site, no transport
 Types of Bioremediation

1- Intrinsic Bioremediation
2- Engineered Bioremediation.
Role of Biotechnology in Bioremediation
 It provides natural mechanisms for the removal of
contaminants from the environment, water and soil.
Biotechnology is applied when the contaminants are
composed of industrial wastes.
Produces microorganisms through genetic engineering
techniques which will have higher metabolic activities
to digest chemicals more efficiently.
 Microorganisms

Enzymes produced:
Environmental Less wastes
Alkaline protease.
wastes
Neutral protease.
Amylases. Enzymatic
bioconversion Animal feed
Cellulases.
etc
Soluble proteins
Human need
Peptides &
Amino acids
New industry Growing
Ammonia
algae

New industry Fertilization to

plants

A possible utilization of environmental wastes by microorganisms.

10- Farm Products (Transgenic Plants).
 Methods dependent on biotechnology increases the diversity of
genes that can be incorporated into crop plants and shorten the
time required for the production of new varieties of plants.
 Transgenic plants that are viable and fertile can be regenerated
from these transformed cells, and the genes that have been
introduced into these transgenic plants are as stable as other
genes in the plant nuclei and show a normal pattern of
inheritance.
 Transgenic plants are most commonly generated by
exploiting a plasmid vector carried by Agrobacterium
tumefaciens, a bacterium.

 Foreign DNA carrying from one to 50 genes can be

introduced into plants in this manner, with the donor
DNA originating from different plant species, animal
cells, or microorganisms.
The soil bacterium Agrobacterium tumefaciens causes
crown gall disease in plants by transferring the T-DNA
region of a tumor-inducing (Ti) plasmid into host cells.

Integration of the foreign DNA disrupts tumor formation,

and only those plant cells with the kanR gene will grow in
culture containing antibiotic. Plants are easily regenerated
from cultured cells (calluses): the adult transgenic plant
expresses the foreign gene.
Agrobacterium mediated transformation
https://www.mun.ca/biology/scarr/Transgenic_Plants.html
 Higher yield

Industrial Nutritional
products quality

Transgenic
plants
provide
Pharmaceut
icals and Enhanced
edible shelf life
vaccine
Biotic and a
biotic stress
tolerance
11- Farm Products (Transgenic Animals).
 Creating a Transgenic Animal. To create a transgenic
animal, the gene of choice is manipulated and prepared in the
laboratory. The transgene is then injected into the egg of an
animal, which is implanted into the surrogate.

Nuclear Transfer Technology. One method of cloning animals,

nuclear transfer involves removing the nucleus from a donor
animal and planting it into an egg cell that has had its nucleus
previously removed.
12- Bioethanol from biomass

• The production of alcohol by fermentation of

sugars and starch is an ancient science and is often
considered to be one of the first microbial processes
used by humans.
• C6H12O6 → 2CH3CH2OH + 2CO2
 Main References
-Alcamo E,(1999).DNA technology textbook.
- Campbell, N. A., Raise, J. B., Ury, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V.,
Jackson ,R. B.(2008).Biotechnology. In:Biology.8th edition. Benjamin Cummings.
- David P Clark(2010).Academic cell molecular biology. Elsevier INC.
- F. Sambrook, R.W. Russell (2008) .Molecular Cloning. Laboratory Manual. Cold Spring
Harbour Laboratory Press.
- Glazer A.N., Nikaido H. (2010) .Microbial Biotechnology - Fundamentals of Applied
Microbiology, Cambridge University Press, Cambridge.
- Kendal, Hunt (1996) .Introduction to Biotechnology. Debuque Publishing Co. Iowa

- John E. Smith.(2009). Biotechnology. Fifth edition. CAMBRIDGE UNIVERSITY PRESS

- Singh U.S and Kapoor K. (2010). Microbial biotechnology,Oxford book company,Jaipur,

India
 Thank you

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