Biotechnology power point.pptx

INTRODUCTION TO
BIOTECHNOLOGY (MLS 231)
1

CHAPTER ONE
• Biotechnology: is the use of an organism, or a component of an
organism or other biological system, to make a product or process.
• Many forms of modern biotechnology rely on DNA technology.
• DNA technology is the sequencing, analysis, and cutting-and-pasting
of DNA.
• Common forms of DNA technology include DNA sequencing,
polymerase chain reaction, DNA cloning, and gel electrophoresis.
• Biotechnology inventions can raise new practical concerns and
ethical questions that must be addressed with informed input from all
of society.
• Every single cell in our body has a the entire DNA code.
2

INTRODUCTION
• The term biotechnology was coined by Karl Ereky, a Hungarian scientist,
• in his book entitled Biotechnology of Meat, Fat and Milk Production in
an Agriculture Large-scale Farm) in 1917.
• Bio’ refers to life and ‘technology’ refers to the application of
information for practical use,
• i.e. the application of living organisms to create or improve a product.
• First, biotechnology involves the exploitation of biological entities
• (i.e. micro-organisms, cells of higher organisms—either living or dead),
their components or constituents
• (e.g. enzymes), in such a way that some functional product or service is
generated.
• Second, this product or service should aim to improve human welfare. 3

• In summary, biotechnology is the application of the theory of
engineering
• and biological science to generate new products from raw
materials of biological origin,
• e.g. vaccines or food’, or, in other words, it can also be
defined as ‘the exploitation of living organism/s or their
product/s to change or improve human health and human
surroundings.
• Examples of products of biotechnology include insulin from
genetically modified bacteria and alcohol, bread, yoghurt,
cheese, vitamins, enzymes, pesticide.
4
INTRODUCTION

INTRODUCTION
• Examples of services of biotechnology include; sewage treatment,
detection of pollution using biosensor.
• Some microbes used in biotechnology
• Yeast (fungi)
• Lactobacillus (bacteria)
• Escherichia coli (bacteria)
• Viruses e.g bacteriophage
5

BRANCHES OF BIOTECHNOLOGY
• The definition of biotechnology can be further divided into different
areas known as red, green blue and white.
• Red biotechnology: This area includes medical procedures such as
utilizing organisms for the production of novel drugs or employing
stem cells to replace/regenerate injured tissues and possibly
regenerate whole organs.
• It could simply be called medical biotechnology. •
• Green biotechnology: Green biotechnology applies to agriculture
• and involves such processes as the development of pest-resistant
grains and the accelerated evolution of disease-resistant animals.
6

• Blue biotechnology: Blue biotechnology, rarely mentioned,
encompasses processes in the marine and aquatic
environments, such as controlling the proliferation of noxious
water-borne organisms.
• White biotechnology: White (also called gray) biotechnology
involves industrial processes such as the production of new
chemicals
• or the development of new fuels for vehicles. A distinction is
made between ‘non-gene biotechnology’ and ‘gene
biotechnology’:
7

• Non-gene biotechnology: Non-gene biotechnology works with
whole cells, tissues or even individual organisms.
• Non-gene biotechnology is the more popular practice, involving
plant tissue culture, hybrid seed production, microbial
fermentation, production of hybridoma antibodies and
immunochemistry.
• Gene biotechnology: Gene biotechnology deals with genes, the
transfer of genes from one organism to another and genetic
engineering.
8

• Fields in Biotechnology Biotechnology has benefited human existence
in a variety of ways through innovations that have made life easier for
him.
• Biotechnology benefits several scientific disciplines by providing
products for their progress.
• A. Genetic Engineering
• Genetic engineering, often known as genetic modification, is the use
of biotechnology to directly manipulate an organism’s DNA.
• Genes are the chemical blueprints for an organism’s characteristics.
The characteristics are transferred when genes are moved from one
organism to another.
9

• Organisms can be given specific combinations of new genes,
• and therefore novel combinations of characteristics that do not exist
in nature and, indeed, cannot be created by natural methods through
genetic engineering.
• This is in contrast to traditional plant and animal breeding, which
works by selecting features of interest over many generations.
• B. Tissue culture
• i. Tissue culture is a biological research approach that involves
transferring segments of tissue from an animal or plant to an artificial
environment where they may survive and function.
10

• ii. A single cell, a population of cells, or a full or part of an organ can
all be cultured.
• Cells in culture can proliferate, alter size, shape, or function, perform
specialised tasks (muscle cells, for example, can contract), and
interact with other cells.
• C. Cloning Cloning: is the process of creating a genetically identical
copy of another cell, tissue, or organism.
• A clone is a material that has been duplicated and has the same
genetic composition as the original
11

• Dolly, a Scottish sheep, was the most famous clone.
• Cloning may be divided into three categories:
• a. Gene cloning: Gene cloning is the process of making copies of genes or DNA
segments.
• b. Reproductive cloning: Reproductive cloning is the process of making
duplicates of entire animals.
• Therapeutic cloning: Embryonic stem cells are created by therapeutic cloning.
• Researchers aim to employ these cells to produce healthy tissue in the human
body to replace sick or injured parts.
12

HISTORY OF BIOTECHNOLOGY
• Different developmental stages have taken place in biotechnology to
meet the various needs of humans at the time.
• Its development was principally based on observations, and the
application of these observations to practical scenarios.
• Owing to the evolution of new technologies and a better
understanding of various principles of life science, the complexity of
biotechnology has increased. The development of biotechnology can
be divided into broad stages or categories, including:
• Ancient biotechnology (8000–4000 BC): Early history as related to
food and shelter; includes domestication of animals.
13

• Classical biotechnology (2000 BC; 1800–1900 AD):
• Built on ancient biotechnology; fermentation promotes food production and
medicine.
• 1900–1953: Genetics. • 1953–1976: DNA research, science explodes.
• Modern biotechnology (1977):
• Manipulates genetic information in organisms; genetic engineering; various
technologies enable us to improve crop yield
• and food quality in agriculture and to produce a broader array of products in
industries.
• Over the last 100 hundred years or so, biotechnology emerged with the
following discoveries and advancements:
14

• 1919. Hungarian scientist Karl Ereky coins the term biotechnology.
• 1928. Alexander Fleming discovers penicillin, the first true antibiotic.
• 1943. Oswald Avery proves DNA carries genetic information.
• 1953. James Watson and Francis Crick discover the double helix structure of
DNA.
• 1960s. Insulin is synthesized to fight diabetes, and vaccines for measles,
mumps and rubella are developed.
• 1969. The first synthesis of an enzyme in vitro, or outside the body, is
conducted.
• 1973. Herbert Boyer and Stanley Cohen develop genetic engineering with the
first insertion of DNA from one bacteria into another.
• 1980s. The first biotech drugs to treat cancer are developed. 15

• 1890. The United States Supreme Court rules that a "live human-
made microorganism is patentable subject matter," meaning GMOs
can be intellectual property.
• 1982. A biotech-developed form of insulin becomes the first
genetically engineered product approved by the U.S. Food and Drug
Administration (FDA).
• 1983. The first genetically modified plant is introduced.
• 1993. GMOs are introduced into agriculture with the FDA approval of
growth hormones that produce more milk in cows.
• 1997. The first mammal is cloned.
16

• 1998. The first draft of the Human Genome Project is
created, giving scientists access to over 30,000 human genes
and facilitating research on treatment of diseases such as
cancer and Alzheimer's.
• 2010. The first synthetic cell is created.
• 2013. The first bionic eye is created.
• 2020. MRNA vaccine and monoclonal antibody technology is
used to treat the SARS-CoV-2 virus.
17

SCOPE AND IMPORTANCE OF BIOTECHNOLOGY
• A. Plant Biotechnology:
• Biotechnology boosts agricultural pest resistance, herbicide tolerance, and
the adoption of more ecologically friendly farming techniques.
• Creating crops with improved nutritional profiles to address vitamin and
nutrient deficiency.
• Producing allergen-free and toxin-free meals.
• Increasing crop yields while reducing inputs.
• Reducing the number of agricultural chemicals used by crops and limiting
product run-off into the environment.
• Using biotech crops that require fewer pesticide applications.
• Viruses, bacteria, Amoeba, fungus, and other microbes are used to
manage plant diseases and insect pests
18

• Human insulin, human and bovine growth hormone, human interferon and other
important medicines have been synthesised.
• DNA fingerprinting is used to identify parents and offenders.
• B. Medical Biotechnology :
• Biotechnology is assisting in the healing of the planet by utilising nature’s toolbox
and our own genetic composition.
• Infectious illness rates are being reduced thanks to biotechnology. Providing
different therapies to reduce health risks and negative effects.
• Developing more accurate disease detection techniques Combating severe
diseases and other challenges that the poor world faces on a daily basis.
• Improving the nutritional value of foods and agricultural oils in order to enhance
cardiovascular health.
19

• C. Industrial Biotechnology Improving the efficiency of the
manufacturing process. Reducing petrochemical consumption and
dependence. Biofuels are being used to reduce greenhouse gas
emissions.
• Reduced water use and trash creation. Antibiotics such as Penicillin,
Erythromycin, Streptomycin, Mitomycin, Cycloheximide, and others
are manufactured.
• Single cell proteins (SCP) derived from bacteria, yeast, fungus, or
algae for human and animal use (as supplements).
• Enzyme immobilisation for repetitive industrial use.
20

• The output of transgenic animals for improved milk generation, growth
rate, illness resistance, and the production of important proteins in milk,
urine, and blood.
• Superovulation and/or embryo splitting caused by hormones in farm
animals;
• includes embryo transfer and, in many situations, in vitro fertilization.
• D. Animal Biotechnology
• In vitro fertilization and embryo transfer are used to create test-tube
babies in humans.
• The output of transgenic animals for improved milk generation, growth
rate, illness resistance, and the production of important proteins in milk,
urine, and blood.
21

• Superovulation and/or embryo splitting caused by hormones in farm
animals; includes embryo transfer and, in many situations, in vitro
fertilization.
• E. Environmental Biotechnology
• Deodorization of human excreta and efficient sewage treatment.
• The process of breaking down contaminants in soil, air, or
groundwater using organisms, generally bacteria.
• Petroleum degradation and oil spill control. Waste and industrial
effluent detoxification
22

OVERVIEW OF DNA REPLICATION
• The central dogma of molecular biology is a theory stating that
genetic information flows only in one direction, from DNA, to RNA, to
protein, or RNA directly to protein.
• Key Points
• Deoxyribonucleic acid, commonly known as DNA, is a nucleic acid
that has three main components: a deoxyribose sugar, a phosphate,
and a nitrogenous base.
• Since DNA contains the genetic material for an organism, it is
important that, it should be copied when a cell divides into daughter
cells.
23

OVERVIEW OF DNA REPLICATION
• The process that copies DNA is called replication.
• Replication involves the production of identical helices of DNA from one
double-stranded molecule of DNA.
• Enzymes are vital to DNA replication since they catalyze very important steps in
the process.
• The overall DNA replication process is extremely important for both cell growth
and reproduction in organisms.
• It is also vital in the cell repair process.
24

• DNA or deoxyribonucleic acid is a type of molecule known as a nucleic
acid.
• It consists of a 5carbon deoxyribose sugar, a phosphate, and a
nitrogenous base.
• Double-stranded DNA consists of two spiral nucleic acid chains that
are twisted into a double helix shape.
• This twisting allows DNA to be more compact.
• In order to fit within the nucleus, DNA is packed into tightly coiled
structures called chromatin.
25
DNA Structure

REPLICATION
• Replication is the process by which a double-stranded
DNA molecule is copied to produce two identical DNA
molecules.
• DNA replication is one of the most basic processes
that occurs within a cell.
• Each time a cell divides, the two resulting daughter
cells must contain exactly the same genetic
information, or DNA, as the parent cell.
27

• Step 1: Replication Fork Formation
• Before DNA can be replicated, the double stranded molecule must be
“unzipped” into two single strands.
• DNA has four bases called adenine (A), thymine (T), cytosine (C) and
guanine (G) that form pairs between the two strands.
• Adenine only pairs with thymine and cytosine only binds with
guanine.
• In order to unwind DNA, these interactions between base pairs must
be broken.
28
Preparation for Replication

• This is performed by an enzyme known as DNA helicase.
• DNA helicase disrupts the hydrogen bonding between base pairs to
separate the strands into a Y shape known as the replication fork.
• This area will be the template for replication to begin.
• Replication Begins
• Step 2: Primer Binding
• The leading strand is the simplest to replicate.
• Once the DNA strands have been separated, a short piece of RNA
called a primer binds to the 3' end of the strand.
• The primer always binds as the starting point for replication. Primers
are generated by the enzyme DNA primase.
29

• DNA polymerase III binds to the strand at the site of the primer
and begins adding new base pairs complementary to the strand
during replication.
• In eukaryotic cells, polymerases alpha, delta, and epsilon are
the primary polymerases involved in DNA replication.
• Because replication proceeds in the 5' to 3' direction on the
leading strand, the newly formed strand is continuous.
• The lagging strand begins replication by binding with multiple
primers. Each primer is only several bases apart.
30

• Step 3: Elongation
• DNA polymerase then adds pieces of DNA, called Okazaki fragments, to the
strand between primers.
• This process of replication is discontinuous as the newly created fragments are
disjointed.
• Step 4: Termination
• Once both the continuous and discontinuous strands are formed, an enzyme
called exonuclease removes all RNA primers from the original strands.
• These primers are then replaced with appropriate bases.
• Another exonuclease “proofreads” the newly formed DNA to check, remove and
replace any errors.
• Another enzyme called DNA ligase joins Okazaki fragments together forming a
single unified strand. 31

• The ends of the linear DNA present a problem as DNA polymerase can
only add nucleotides in the 5′ to 3′ direction.
• The ends of the parent strands consist of repeated DNA sequences
called telomeres.
• Telomeres act as protective caps at the end of chromosomes to prevent
nearby chromosomes from fusing.
• A special type of DNA polymerase enzyme called telomerase catalyzes
the synthesis of telomere sequences at the ends of the DNA.
• Once completed, the parent strand and its complementary DNA strand
coils into the familiar double helix shape.
32

• In the end, replication produces two DNA molecules, each with one
strand from the parent molecule and one new strand.
• Replication Enzymes
• The following enzymes are responsible for replication
• DNA helicase: unwinds and separates double stranded DNA as it
moves along the DNA.
• It forms the replication fork by breaking hydrogen bonds between
nucleotide pairs in DNA.
• DNA primase: A type of RNA polymerase that generates RNA primers.
Primers are short RNA molecules that act as templates for the starting
point of DNA replication.
33

34

• Semi-Conservative Replication When DNA replication is
complete,
• there are two identical sets of double stranded DNA, each with
one strand from the original template,
• DNA molecule, and one strand that was newly synthesized
during the DNA replication process.
• Because each new set of DNA contains one old and one new
strand, we describe DNA as being semi-conservative.
35

Transcription
• Transcription is the first step in gene expression, in which
information from a gene is used to construct a functional product
such as a protein.
• The goal of transcription is to make a RNA copy of a gene's DNA
sequence.
• For a protein-coding gene, the RNA copy, or transcript, carries the
information needed to build a polypeptide (protein or protein
subunit).
• Eukaryotic transcripts need to go through some processing steps
before translation into proteins.
38

39
Transcription
• Transcription is the first step in gene expression, in which
information from a gene is used to construct a functional product
such as a protein.
• The goal of transcription is to make a RNA copy of a gene's DNA
sequence.

• RNA polymerase
• The main enzyme involved in transcription is RNA polymerase,
• which uses a single-stranded DNA template to synthesize a complementary
strand of RNA.
• Specifically, RNA polymerase builds an RNA strand in the 5' to 3' direction,
adding each new nucleotide to the 3' end of the strand.
40
Transcription

• Stages of transcription of a gene takes place in three stages:
• initiation, elongation, and termination. Here, we will briefly see how
these steps happen in bacteria.
41
Transcription

42
Transcription
• 1. RNA polymerase binds to a sequence of DNA called the
promoter, found near the beginning of a gene.
• Each gene (or group of co-transcribed genes, in bacteria) has its
own promoter.
• Once bound, RNA polymerase separates the DNA strands, providing
the single-stranded template needed for transcription.
• 2. Elongation: One strand of DNA, the template strand, acts as a
template for RNA polymerase.
• As it "reads" this template one base at a time, the polymerase builds
an RNA molecule out of complementary nucleotides, making a chain
that grows from 5' to 3'.

• The RNA transcript carries the same information as the non-template
(coding) strand of DNA,
• but it contains the base uracil (U) instead of thymine (T).
43
Transcription

• Termination: Sequences called terminators signal that the RNA transcript is
complete.
• Once they are transcribed, they cause the transcript to be released from the
RNA polymerase.
• An example of a termination mechanism involving formation of a hairpin in the
RNA is shown below.
44
Transcription

• Eukaryotic RNA modifications
• In bacteria, RNA transcripts can act as messenger RNAs (mRNAs)
right away.
• In eukaryotes, the transcript of a protein-coding gene is called a
pre-mRNA and
• must go through extra processing before it can direct translation.
• Eukaryotic pre-mRNAs must have their ends modified,
• by addition of a 5' cap (at the beginning) and 3' poly-A tail (at the
end).
45
Transcription

• Many eukaryotic pre-mRNAs undergo splicing.
• In this process, parts of the pre-mRNA (called introns) are chopped
out, and the remaining pieces (called exons) are stuck back together.
46
Transcription

Translation
• • During translation, the nucleotides of the mRNA are read in groups of
three called codons.
• Each codon specifies a particular amino acid or a stop signal.
• This set of relationships is known as the genetic code
The nucleotide sequence of the mRNA is decoded to specify the amino acid
sequence of a polypeptide.
• This process occurs inside a ribosome and requires adapter molecules called
tRNAs.
• Transfer RNAs, or tRNAs, are molecular "bridges" that connect mRNA codons
to the amino acids they encode.
47

• One end of each tRNA has a sequence of three nucleotides called an
anticodon, which can bind to specific mRNA codons.
• The other end of the tRNA carries the amino acid specified by the
codons.
• There are many different types of tRNAs.
• Each type reads one or a few codons and brings the right amino acid
matching those codons
48
Translation

• Ribosomes are the structures where polypeptides (proteins) are built. They are
made up of protein and RNA (ribosomal RNA, or rRNA).
• Each ribosome has two subunits, a large one and a small one, which come
together around an mRNA.
• The ribosome provides a set of handy slots where tRNAs can find their
matching codons on the mRNA template and deliver their amino acids.
• The ribosome has three slots for tRNAs: the A site, P site, and E site.
• tRNAs move through these sites (from A to P to E) as they deliver amino acids
during translation.
• Steps of translation :To see how cells make proteins, let's divide translation
into three stages:
• initiation (starting off), elongation (adding on to the protein chain), and
termination (finishing up). 49
Translation

• Initiation: In initiation, the ribosome assembles around the mRNA to
be read and
• the first tRNA (carrying the amino acid methionine, which matches
the start codon, AUG).
• This setup, called the initiation complex, is needed in order for
translation to get started
• Elongation: Is the stage where the amino acid chain gets longer.
• In elongation, the mRNA is read one codon at a time,
• and the amino acid matching each codon is added to a growing
protein chain.
• Each time a new codon is exposed:
50
Translation

• A matching tRNA binds to the codon
• The existing amino acid chain (polypeptide) is linked onto the amino acid of the
tRNA via a chemical reaction
• The mRNA is shifted one codon over in the ribosome, exposing a new codon for
reading
• During elongation, tRNAs move through the A, P, and E sites of the ribosome, as
shown above.
• This process repeats many times as new codons are read and new amino acids
are added to the chain.
51
Translation

• Finishing up: Termination: is the stage in which the finished polypeptide chain is
released.
• It begins when a stop codon (UAG, UAA, or UGA) enters the ribosome,
• triggering a series of events that separate the chain from its tRNA and allow it to
drift out of the ribosome.
• After termination, the polypeptide may still need to fold into the right 3D shape,
• undergo processing (such as the removal of amino acids), get shipped to the right
place in the cell,
• or combine with other polypeptides before it can do its job as a functional
protein
52
Translation

CHAPTER 2: Natural and artificial mechanisms of DNA transfer (Bacterial
Transformation)
• Transformation is the direct uptake, incorporation and expression of
exogenous genetic material from its surroundings.
• In transformation, a bacterium takes up a piece of DNA floating in its
environment.
• In transduction, DNA is accidentally moved from one bacterium to another
by a virus.
• In conjugation, DNA is transferred between bacteria through a tube
between cells.
• Transposable elements are chunks of DNA that "jump" from one place to
another.
• They can move bacterial genes that give bacteria antibiotic resistance or
make them disease-causing.
55

CHAPTER 2:
• Transformation results in the genetic alteration of the recipient cell.
• Exogenous DNA is taken up into the recipient cell from its surroundings
through the cell membrane (s).
• Transformation occurs naturally in some species of bacteria, but it can
also be affected by artificial means in other cells.
Key Terms • eukaryotic: Having complex cells in which the genetic material
is organized into membrane-bound nuclei.
56

CHAPTER 2
• expression: Gene expression is the process by which
information from a gene is used in the synthesis of a functional
gene product.
• exogenous: Produced or originating outside of an organism.
• translocase: An enzyme that assists in moving another
molecule, usually across a membrane.
transformation: In molecular biology transformation is genetic
alteration of a cell resulting from the direct uptake,
•incorporation and expression of exogenous genetic material
(exogenous DNA) from its surroundings and taken up through
the cell membrane(s). 57

CHAPTER 2
• Genetic Alteration
• In transformation, a bacterium takes in DNA from its environment, often DNA
that's been shed by other bacteria.
• In a laboratory, the DNA may be introduced by scientists
• If the DNA is in the form of a circular DNA called a plasmid, it can be copied in
the receiving cell and passed on to its descendants.
58

CHAPTER 2
• Natural Transformation
• Transformation occurs naturally in some species of bacteria, but it can also
be effected by artificial means in other cells.
• For transformation to happen, bacteria must be in a state of competence,
• which might occur as a time-limited response to environmental conditions
such as starvation and cell density.
• Transformation is one of three processes by which exogenous genetic
material may be introduced into a bacterial cell;
• the other two being conjugation (transfer of genetic material between two
bacterial cells in direct contact),
• and transduction (injection of foreign DNA by a bacteriophage virus into
the host bacterium). 59

CHAPTER 2
• “Transformation” may also be used to describe the insertion of new genetic
material into nonbacterial cells, including animal and plant cells;
• however, because “transformation” has a special meaning in relation to animal
cells,
• indicating progression to a cancerous state, the term should be avoided for
animal cells when describing introduction of exogenous genetic material.
• Introduction of foreign DNA into eukaryotic cells is often called “transfection“.
• Competence refers to the state of being able to take up exogenous DNA from
the environment.
• There are two forms of competence: natural and artificial.
60

CHAPTER 2
• There are numerous bacteria found on planet earth.
• They divide quickly by binary fission producing identical daughter cells.
• Thus, the genetic information is transferred from the mother to the
offspring and is known as vertical transmission.
• The mutations are transferred from one bacteria to another through
horizontal transmission.
• There are three different types of horizontal transmission for the
transfer of genetic information.
• Conjugation • Transduction • Transformation
61

CHAPTER 2
• Bacterial Conjugation Conjugation is the method of transfer of genetic
material from one bacteria to another placed in contact.
• This method was proposed by Lederberg and Tatum.
• They discovered that the F-factor can move between E.coli cells and
proposed the concept of conjugation.
• There are various conjugal plasmids carried by various bacterial
species.
• Conjugation is carried out in several steps:
• Mating pair formation • Conjugal DNA synthesis • DNA transfer •
Maturation
64

• Pilus Formation The donor cells (F+ cells) form a sex pilus and begin contact
with an F- recipient cell.
65

CHAPTER 2
• Physical Contact between Donor and Recipient Cell.
• The pilus forms a conjugation tube and enables direct contact
between the donor and the recipient cells.
• Transfer of F-Plasmid The F-factor opens at the origin of replication.
One strand is cut at the origin of replication,
• and the 5’ end enters the recipient cell.
66

CHAPTER 2
• Synthesis of Complementary Strand Fertility Factor:
• The F (fertility) factor is a circular double stranded DNA molecule
of around 100 x 103 base pairs.
• Bacterial cells with the F factor, denoted as an F+ cell,
• are capable of transferring genes to an F- cell (without F-factor)
by means of conjugation (Fig. 1).
• The donor and the recipient strand both contain a single strand
of the F-plasmid.
• Thus, a complementary strand is synthesized in both the
recipient and the donor.
68

CHAPTER 2
• The recipient cell now contains a copy of F plasmid and becomes
a donor cell.
• Donor cells typically act as donors because they have a chunk of
DNA called the fertility factor (or F factor).
• This chunk of DNA codes for the proteins that make up the sex
pilus.
• It also contains a special site where DNA transfer during
conjugation begins.
• If the F factor is transferred during conjugation, the receiving cell
turns into an F+ donor that can make its own pilus and transfer
DNA to other cells
69

CHAPTER 2
• Bacterial Transduction: Transduction is the process of transfer of
genes from the recipient to the donor through bacteriophage.
• In transduction, viruses that infect bacteria move short pieces of
chromosomal DNA from one bacterium to another "by accident."
• The viruses that infect bacteria are called bacteriophages.
• Bacteriophages, like other viruses, are the pirates of the biological
world;
• they commandeer a cell's resources and use them to make more
bacteriophages.
70

CHAPTER 2
•However, this process can be a little sloppy.
•Sometimes, chunks of host cell DNA get caught inside the new
bacteriophage as they are made.
•When one of these "defective" bacteriophages infects a cell, it
transfers the DNA.
•Some bacteriophages chop the DNA of their host cell into
pieces, making this transfer process more likely.
71

• Step 1: A bacteriophage adsorbs to a susceptible bacterium.
• Step 2: The bacteriophage genome enters the bacterium.
• The genome directs the bacterium's metabolic machinery to manufacture
bacteriophage components and enzymes.
• Bacteriophage-coded enzymes will also breakup the bacterial chromosome.
• Step 3: Occasionally, a bacteriophage capsid mistakenly assembles around either
a fragment of the donor bacterium's chromosome or around a plasmid instead of
around a phage genome.
• Step 4: The bacteriophages are released as the bacterium is lysed.
• Note that one bacteriophage is carrying a fragment of the donor bacterium's DNA
rather than a bacteriophage genome.
72
CHAPTER 2

CHAPTER 2
• Step 5: The bacteriophage carrying the donor bacterium's DNA
adsorbs to a recipient bacterium.
• Step 6: The bacteriophage inserts the donor bacterium's DNA it is
carrying into the recipient bacterium.
• Step 7: Homologous recombination occurs and the donor bacterium's
DNA is exchanged for some of the recipient's DNA.
• Archaea, the other group of prokaryotes besides bacteria,
• are not infected by bacteriophages but have their own viruses that
move genetic material from one individual to another.
73

CHAPTER 2
• Transposable elements Transposable elements are also important
in bacterial genetics.
• These chunks of DNA "jump" from one place to another within a
genome, cutting and pasting themselves
• or inserting copies of themselves in new spots.
• Transposable elements are found in many organisms (including
you and me!), not just in bacteria.
• In bacteria, transposable elements sometimes carry antibiotic
resistance and pathogenicity genes
• (genes that make bacteria disease-causing)
75

• If one of these transposable elements "jumps" from the chromosome into a
plasmid,
• the genes it carries can be easily passed to other bacteria by transformation
or conjugation.
• That means the genes can spread quickly through the population.
76
CHAPTER 2

• Artificial mechanisms of DNA transfer: Bacterial transformation & selection
• Transfer of plasmid DNA into bacteria. How bacteria are selected. Protein
production and purification.
• Key points:
• Bacteria can take up foreign DNA in a process called transformation.
Transformation is a key step in DNA cloning.
• It occurs after restriction digest and ligation(Opens in a new window) and
transfers newly made plasmids to bacteria.
• After transformation, bacteria are selected on antibiotic plates.
• Bacteria with a plasmid are antibiotic-resistant, and each one will form a
colony.
• Colonies with the right plasmid can be grown to make large cultures of
identical bacteria, which are used to produce plasmid or make protein. 77
CHAPTER 2

• The big picture:
• DNA cloning Transformation and selection of bacteria are key steps in DNA
cloning(Opens in a new window).
• DNA cloning is the process of making many copies of a specific piece of DNA,
such as a gene.
• The copies are often made in bacteria.
• In a typical cloning experiment, researchers first insert a piece of DNA, such as a
gene, into a circular piece of DNA called a plasmid.
• This step uses restriction enzymes and DNA ligase and is called a ligation.
• After a ligation, the next step is to transfer the DNA into bacteria in a process
called transformation.
78
CHAPTER 2

CHAPTER 2
• Then, we can use antibiotic selection and DNA analysis methods to
identify bacteria that contain the plasmid we’re looking for.
• Steps of bacterial transformation and selection
• Here is a typical procedure for transforming and selecting bacteria:
79

CHAPTER 2
• 1. Specially prepared bacteria are mixed with DNA (e.g., from a ligation).
• 2. The bacteria are given a heat shock, which causes some of them to take up a
plasmid.
• 3. Plasmids used in cloning contain an antibiotic resistance gene.
• Thus, all of the bacteria are placed on an antibiotic plate to select for ones that
took up a plasmid.
80

CHAPTER 2
• 4. Bacteria without a plasmid die.
• Each bacterium with a plasmid gives rise to a cluster of identical, plasmid-
containing bacteria called a colony.
• 5. Several colonies are checked to identify one with the right plasmid (e.g., by
PCR or restriction digest).
• 6. A colony containing the right plasmid is grown in bulk and used for plasmid or
protein production.
• Why do we need to check colonies?
• The bacteria that make colonies should all contain a plasmid (which provides
antibiotic resistance).
• However, it’s not necessarily the case that all of the plasmid-containing colonies
will have the same plasmid.
81

CHAPTER 2
•When we cut and paste DNA, it's often possible for side
products to form, in addition to the plasmid we intend to
build.
•For instance, when we try to insert a gene into a plasmid
using a particular restriction enzyme,
• we may get some cases where the plasmid closes back up
(without taking in the gene),
•and other cases where the gene goes in backwards.
82

CHAPTER 2
• If the gene were backwards, the wrong strand of DNA would be
transcribed and no protein would be made.
• Because of these possibilities, it's important to collect plasmid DNA
from each colony and check to see if it matches the plasmid we were
trying to build.
• Restriction digests, PCR, and DNA sequencing are commonly used to
analyze plasmid DNA from bacterial colonies.
83

CHAPTER THREE: VECTORS AND MARKERS IN BIOTECHNOLOGY
• A. VECTORS
• A vector, as related to molecular biology, is a DNA molecule (often
plasmid or virus)
• that is used as a vehicle to carry a particular DNA segment into a host
cell as part of a cloning or recombinant DNA technique.
• The vector typically assists in replicating and/or expressing the
inserted DNA sequence inside the host cell.
• Key points
• Vectors act as vehicles to transfer genetic material from one cell to the
other for different purposes like multiplying, expressing, or isolation.
86

CHAPTER THREE
• Vectors are used as a tool in molecular cloning procedures so as to
introduce the desired DNA insert into a host cell.
• The DNA insert that is transmitted by a vector is termed recombinant
DNA,
• and the process is also known as recombinant DNA technology.
• Usually, the vectors are DNA sequences that carry different parts
involved in different functions.
• Vectors usually have an insert, also known as a transgene,
• that carries the recombinant DNA and a larger sequence called the
backbone of the vector responsible for the structure of the vector.
87

CHAPTER THREE
• Vectors can be classified into different types depending on different
characteristics.
• The selection of vectors thus depends on the purpose of the process.
• Vectors are an important component of the genetic engineering
process as these form the basis for the transfer of DNA fragments
from one cell to another.
• Vectors have particular features that carry the gene sequences and
enable them to survive within the host cell.
88

CHAPTER THREE
• The process of gene transfer also differs in different vectors where
some enter the host cell and get incorporated into the host DNA,
• whereas the others just pass the genetic material into the host cell and
recover themselves.
• Even though vectors are usually DNA sequences, viruses and other
particles can also function as vectors in processes like transduction.
• Vectors can be reused for multiple processes as these can be
recovered at the end of the process.
89

CHAPTER THREE
• A cloning vector is a category of vectors that are essential for cloning
procedures.
• These vectors have different sequences that enable them to initiate
replication in host cells as well as propagate within the host.
Types of vectors
• Vectors can be classified into different groups depending on the
purpose of the process
and the type of particles used in the process.
• The following are the commonly studied group of vectors that are used
for different purposes;
90

CHAPTER THREE
• Properties of an ideal vector
• It should be replicate autonomously.
• A vector should be less than 10 KB in size.
• It should be easily isolated and purify.
• It should be easily introduced into the host cell.
• It should have suitable marker genes.
• Vector should consists a unique target sites and recognition sites for
various restriction enzymes.
• It should have the capability to incorporate either itself or the DNA
insert in the Genome of the host cell.
91

CHAPTER THREE: VECTORS AND MARKERS IN BIOTECHNOLOGY
• A. VECTORS
• Introduction A vector, as related to molecular biology, is a DNA molecule
• (often plasmid or virus) that is used as a vehicle to carry a particular
• DNA segment into a host cell as part of a cloning or recombinant DNA technique.
• The vector typically assists in replicating and/or expressing the inserted DNA
sequence inside the host cell.
• Key points
• Vectors act as vehicles to transfer genetic material from one cell to the other for
different purposes like multiplying, expressing, or isolation.
93

• Vectors are used as a tool in molecular cloning procedures so as to introduce the
desired DNA insert into a host cell.
• The DNA insert that is transmitted by a vector is termed recombinant
DNA, and the process is also known as recombinant DNA technology.
• Usually, the vectors are DNA sequences that carry different parts involved in
different functions.
• Vectors usually have an insert, also known as a transgene, that carries the
recombinant DNA and a larger sequence called the backbone of the vector
responsible for the structure of the vector.
94

• Vectors can be classified into different types depending on different
characteristics.
• The selection of vectors thus depends on the purpose of the process.
• Vectors are an important component of the genetic engineering
• process as these form the basis for the transfer of DNA fragments from one cell to
another.
• Vectors have particular features that carry the gene sequences and enable them
to survive within the host cell.
• The process of gene transfer also differs in different vectors where some enter the
host cell and get incorporated into the host DNA,
• whereas the others just pass the genetic material into the host cell and recover
themselves.
95

• Even though vectors are usually DNA sequences, viruses and other particles can
also function as vectors in processes like transduction.
• Vectors can be reused for multiple processes as these can be recovered at the
end of the process.
• A cloning vector is a category of vectors that are essential for cloning
procedures.
• These vectors have different sequences that enable them to initiate replication
in host cells as well as propagate within the host.
96

Properties of an ideal vector
• It should be replicate autonomously.
• A vector should be less than 10 KB in size.
• It should be easily isolated and purify.
• It should be easily introduced into the host cell.
• It should have suitable marker genes.
• Vector should consists a unique target sites and recognition sites for various
restriction enzymes.
• It should have the capability to incorporate either itself or the DNA insert in the
Genome of the host cell.
97

Types of vector
• Vectors are of two types:
• Cloning vector
• Expression vector
• Cloning vectors
• Cloning vectors are small piece of DNA which have the ability and used to
introduce foreign gene of interest into the host cell.
• They can be stably maintained insides the host cell.
• Cloning vector are generally used to obtain multiple copies of desired foreign
gene.
• Example- Plasmid, Cosmid and Phages, BACs, YACs.
• These type of vectors generally contains selectable marker, origin of replication
and a restriction site.
98

• Expression vector
• Expression vector is a type of vector which not only introduces a gene of interest
into the host cell
• but also aids in the analysis of the foreign gene via relevant protein product
expression.
• It is type of vector which is used to obtain or analyses the gene product, which
may be RNA or protein of the inserted desired gene.
• Example- Only plasmid vector.
• Expression vector contains enhancer, promoter region,
• start/stop codon, transcription initiation, selectable marker, ori sites, and
restriction site.
99

Plasmid vector
• A plasmid is a naturally occurring extrachromosomal double stranded DNA,
circular DNA.
• It replicates autonomously within bacterial cell.
• Plasmid carries an origin of replication.
• Plasmid vectors are the simplest cloning vectors.
• It is most widely used for gene cloning.
100

Characteristics of Plasmid vector:
• It contains an ori site or origin of replication.
• It also Contain selective marker such as antibiotic resistance, blue white
screening).
• Small in size (1.0 to 250kb)
• Contains multiple cloning site.
• Easily isolated from the host cell.
Examples of plasmid vectors are:
1. pBR322
2. 2. pUC 18/19
101

pBR322 vector
• It is one of the first vectors to be developed by Boliver and Rodriguez in 1977.
• The name pBR322 denotes the following:
• 1. P- plasmid
• 2. B- Boliver 3.
• R- Rodriguez
• 322- Differentiate it from the other plasmid produced in the same laboratory E.g.
– pBR325, pBR327, etc.
• It is 4363 base pair long
102

• It carries two sets of antibiotic resistance gene:
• Ampicillin
• Tetracycline
Cosmids vector •
It is a type of hybrid plasmid.
• It contains lambda phage cos sequence.
• Cosmids = cos sites + plasmid.
• Genomic size of cosmids is about 30 to 52 kb.
• If they have suitable origin of replication than they can replicate as Plasmid
within the host cells, E.g.- SV40 Ori, ColE1 ori.
• It also contains selectable marker such as Ampicillin resistance gene.
• Collins and Hohn in 1978 was first to described cosmids.
103

Phagemid Vector
• A plasmid vector which contains an origin of replication from a phage, in addition
to that of the plasmid, is termed as Phagemids.
• Phage vector or Bacteriophage vector
• Bacteriophages are viruses that attacks bacteria.
• The Phages are simple in structure.
• It consists of DNA molecules having several gene for replication which is
surrounded by Capsid.
On the basis of structure bacteria phases are of two types:
1. Head and Tail Phages- E.g.: lambda phage.
2.Filamentous phage- E.g.: M13 phage.
104

Lambda phage vectors
• Its genome size is about 48,502 bp.
• It contains origin of replication, genes for head and tail protein and enzymes for
DNA replication
• It has more than one recognition sequence for almost all the restriction
enzymes.
• It should be larger than 38 kb and smaller than 52 kb to packaged into phage
particles.
105

M13 phage vectors
• M13 vectors are used to obtain single- stranded copies of cloned DNA.
• It is 6407 nucleotides long.
• It is circular and 6.4kb in size.
• M13 vector only cause infection in F+ and F’ cells.
• It is used to produce several copies of M13 mp series of vectors.
• Example- M13mp8, M13mp9 etc.
106

Artificial chromosome vectors
• Artificial chromosomes are circular or linear vectors.
• They are stably maintained in, usually 1 to 2 copy per cell.
There are several types of such vectors:
1. Bacterial artificial chromosomes (BAC).
2. Yeast artificial chromosomes(YAC).
3. Mammalian artificial chromosomes(MAC).
4. Human artificial chromosome (HAC).
107

Shuttle vector
• Shuttle vectors are created to replicates in cell of different type of
species.
• They contain two origin of replication, in which one is particular for
each host species, also those genes required for their replication and
not provided by the host cell.
• This type of vectors are developed by recombinant techniques.
108

MARKERS
• INTRODUCTION
• Marker systems are tools for studying the transfer of genes into an experimental
organism.
• In gene transfer studies, a foreign gene, called a transgene, is introduced into an
organism, in a process called transformation.
• A common problem for researchers is to determine quickly and easily if the
target cells of the organism have actually taken up the transgene.
• A marker allows the researcher to determine whether the transgene has been
transferred,
• where it is located, and when it is expressed (used to make protein).
• Marker systems exist in two broad categories: selectable markers and
screenable markers.
109

• Selectable markers are typically genes for antibiotic resistance,
• which give the transformed organism (usually a single cell) the ability to live in
the presence of an antibiotic.
• Screenable markers, also called reporter genes or scorable genes, typically
cause a color change or other visible change in the tissue of the transformed
organism.
• This allows the investigator to quickly screen a large group of cells for the ones
that have been transformed.
110

SELECTABLE MARKERS
• A selectable marker gene encodes a product that allows the transformed
cell to survive and grow under conditions that kill or restrict the growth of
non transformed cells.
• Most such genes used in plants are dominant selectable markers that
confer resistance to antibiotics or herbicides
111

Biotechnology power point.pptx

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Biotechnology power point.pptx

Similar to Biotechnology power point.pptx (20)

More from KingslyNdanga1

More from KingslyNdanga1 (14)

Recently uploaded

Recently uploaded (20)

Biotechnology power point.pptx