11-BIOTECHNOLOGY PRINCIPLES AND PROCESSES
CHAPTER NO.11 BIOTECNOLOGY:PRINCIPLES
AND PROCESSES
A122
INTRODUCTION
BIOTECHNOLOGY:The term biotechnology is derived from
a fusion of two words: biology and technology.It deals with techniques of using
live microorganisms, plants or animals or enzymes to produce entities like
antibodies, vaccines etc. which benefits the
mankind.The term “Biotechnology” was coined by Karl
Ereky in Hungry.The old biotechnology is the use of natural strains of
microorganisms and cell lines.The modern biotechnology is the use of
Genetically Modified Organisms.The European Federation of Biotechnology (EFB)
has given a new definition of biotechnology as “The integration of natural
science and organisms, cells,parts thereof and molecular analogues for products
and services.”
PRINCIPLES OF BIOTECHNOLOGY:Two main techniques of
modern biotechnology are:
1. GENETIC ENGINEERING: It includes techniques to alter the nature of genetic
material, to introduce these into host organisms and thus change the phenotype
of the host organism.
2. CHEMICAL ENGINEERING:
it involves maintenance of sterile microbial contamination free condition for
the manufacture of biotechnological products
such as antibiotics, vaccines, enzymes, medicine
etc. on large scale.
CONCEPTUAL DEVELOPMENT OF THE
PRINCIPLES OF GENETIC ENGINEERING:-
Genetic engineering is based on two important
discoveries:
1. Presence of plasmids in bacteria which can
undergo replication along with alien DNA, independent of chromosomal DNA.
2. Restriction endonucleases which can break DNA at
specific sites. They are called Molecular Scissors or Biological Scissors.
The technique of genetic engineering
includes:
1. Formation of Recombinant DNA
2. Use of gene cloning
3. Gene transfer
A piece of DNA which is introduced from the alien
organism would not be able to multiply itself unless integrated in host genome.
When it gets integrated into the genome of the
recipient,(HOST), it may multiply
and be inherited along with the host DNA.
This is because the alien or foreign piece of DNA
has become part of host chromosome, which has the ability to replicate in the
chromosome.There is a specific DNA sequence called the origin of replication,
(Ori) which is responsible for initiating replication.
An alien DNA is linked with the origin of
replication and multiply itself along with the host DNA, this is called
CLONING.
CONSTRUCTION OF AN ARTIFICIAL
RECOMBINANT DNA MOLECULE: -
It was constructed by Stanley Cohen and Herbert
Boyer in 1972.They cut the piece
of DNA from a plasmid containing antibiotic
resistance gene in the bacterium Salmonella typhimurium. Cutting of piece of
DNA from a plasmid with the help of Restriction Enzymes. The piece of foreign
DNA, was linked with the plasmid DNA, acting as vector.
Linking of the piece of foreign DNA with vector was
done with the help of enzyme
DNA ligase which act on cut DNA molecules and join
their ends .This newly formed DNA is called Recombinant DNA. The vector is used
to transfer Recombinant DNA, to E.coli
When this Recombinant DNA is transferred into E.
coli, it could replicate in the new
host cell in the presence of DNA polymerase enzyme
and make multiple copies of
recombinant DNA. This is known as cloning of
antibiotic resistance gene in E .coli.
Hence, it can be concluded that the three basic
steps in creating genetically
modified organism are;-
1. Identification of DNA with desirable genes.
2. Introduction of the identified DNA into the host.
3. Maintenance of introduced DNA in the host and
transfer of the DNA to its
progeny.
Let us know what we have learnt!
PART: A VERY SHORT ANSWER TYPE
QUESTIONS
(a) MULTIPLE CHOICE QUESTIONS:
1. Which enzyme is used as molecular
scissors in genetic engineering?
(a) restriction endonuclease
(c) DNA polymerase
(b) DNA ligase
(d) DNAvirus
2. Who discovered Recombinant DNA
technology?
(a) Hargobind Khorana
(c) James Watson
(b) Stanley Cohen and Herbert boyar
(d) Sutton and boveri
3. Construction of a Recombinant DNA
involves:
(a) Cleaving and re-joining DNA segments with
endonuclease alone
(b) Cleaving DNA segments with endonuclease and
re-joining them with the ligase.
(c) Cleaving DNA segments with the Ligase and
re-joining them with endonuclease
(d) cleaving and re-joining DNA segments with the
ligase alone
4. Cloning gene is a process where :
(a) gene is cloned in an animal
(b) Fragments of DNA are transferred from one
organism to another
usually carried on a DNA vector.
(c) fragments of DNA cloned in the same organism
using carrier
(d) DNA is cloned in plants
5. In Recombinant DNA techniques the term
vector refers to:
(a) plasmid that can transfer foreign DNA into a
living cell
(b) cosmids that can cut DNA at specific base
sequence
(c) plasmid that can join different DNA fragments
(d) Cosmic Rays that can degrade harmful proteins
(b) FILL IN THE BLANKS:
1. EFB stands for-——---—
2. ---------—Joins the DNA fragments.
3. -----------are used as vector in r DNA
technology.
(c)TRUE/ FALSE:
1. Restriction enzymes help in cutting DNA at
specific site
2. Origin of replication in a chromosome is
responsible for initiating replication
ANSWER KEY: PART-A
a) MULTIPLE CHOICE QUESTIONS:
1. (a) Restriction endonucleases are called
molecular scissors because they can break DNA at specific sites hence, they are
used in genetic engineering
2. (b) The science of Recombinant Technology was discovered
by Stanley Cohen and Herbert boyar
3. (b) Recombinant DNA can be formed by cutting DNA
segments with endonuclease and then Re-joining them with the help of enzyme
ligase
4. (b) Cloning is the process of forming multiple
identical copies of any template DNA
5. (a)
b) FILL IN THE BLANKS:
1. European Federation of Biotechnology
2. Ligase
3. Plasmids
C) TRUE/ FALSE:
1. True
2. True
PART: B SHORT ANSWER TYPE QUESTIONS
1. Who constructed first recombinants DNA?
2. Which two important discoveries lead to concept
of genetic engineering?
3. Define biotechnology?
PART: C LONG ANSWER TYPE QUESTIONS
1. Explain in detail the process of formation of
first Recombinant DNA molecule.
A123
INTRODUCTION
Recombinant DNA technology or genetic engineering is
the field of biotechnology which specialises the development of new
combinations of qenetic material called Recombinant DNA technology (Cohen &
Boyer)
TOOLS FOR RECOMBINANT DNA TECHNOLOGY: Restriction
enzymes ,Alkaline phosphatase, sources of Donor DNA, reverse transcriptase,
polymerase enzyme, T- 4 ligase, vectors and host
organism.Dear students, in this topic we will discuss about various enzymes
needed
in r DNA technology.
RESTRICTION ENZYMES: In 1963, Linn and Arber
isolated two enzymes which restrict the growth of bacteriophage in Escherichia
coli bacterium.These are called restriction enzymes.
(1) Restriction Endonuclease
(2) Methylase.
(1) RESTRICTION ENDONUCLEASE :
It recognises palindromic sequence of DNA and cut the same. It restricts the
propagation of bacteriophage in bacterium.
(2) METHYLASE:
This adds methyl group to the particular palindromic region of bacterial DNA so
that restriction endonuclease is unable to harm the bacterial genome
Restriction enzyme is also called restriction
endonuclease.The first restriction endonuclease Hind II was discovered from
bacterium Haemophilus influenzae by Smith in 1968.It cuts DNA at particular
points having specific sequence of six pairs GTC
GAC/CAG CTG ( Pallindromic Sequence)
This is a sequence of specific base pairs is called
recognition sequence for Hind Il.
NUMBER OF RESTRICTION ENZYMES: There are more
than 900 restriction enzymes isolated from 230 strains of bacteria which
recognise different recognition sequences.
NAMING OF RESTRICTION ENZYMES:--
The first letter of enzyme comes from genus and second name of two letters
comes from species of prokaryotic cell.e.g EcoRI.
Here E stands for genus Eschenchia,’ co’ stands for
species ‘coli ‘ Third name of
letter ‘R’ comes from strain and fourth name of
Roman number ‘I’ following the
name indicate the order from which the enzymes were
isolated.
Arber, Smith and Nathans were awarded Nobel Prize in
1978 for the discovery
of restriction endonuclease.
KINDS OF NUCLEASES:-There
are two kinds of nucleases
(1) Exonucleases
(2) Endonucleases
(1) Exonuclases remove nucleotides from ends of DNA.
(2) Endonuclases remove nucleotides from specific
position within the DNA.
ACTION OF ENDONUCLEASES: Each restriction
endonuclease inspects the length of a DNA sequence. It finds its specific
recognition sequence and bind to the DNA. It cuts each of two strands of DNA at
specific points in their sugar phosphate backbone. Each restriction
endonuclease recognise a specific
palindromic nucleotide sequence in the DNA. It is
4-8 nucleotides long.
PALINDROMIC SEQUENCE :
These are group of letters that form the same word when read both forward and
backward direction. e.g. RAMAR . The two strands of DNA in palindromic region
was the same sequence but, in opposite fashion , 5'—3' and 3'—5'
MOLECULAR SCISSORS OR CHEMICAL SCALPEL: Restriction
endonucleases attach itself between the same two bases on the opposite strand
and cut them litle away from palindromic region. So restriction endonucleases
are called molecular Scissors.
STICKY ENDS:-The
molecular scissors leaves a single stranded portion at the ends and overhanging
stretches called sticky ends are formed on both strands of DNA. These are named
so because they form hydrogen bond with their complementary cut counterparts.
Eco RI (Escherichia coli) and BamHI (Bacillus
amyloliquefaciens) belong to this category.
RECOMBINANT DNA:
When DNA strands are cut by the same restriction endonuclease, the
complementary sticky end of the two DNA strands are joined end to end to create
Recombinant DNA. This is done with the help of enzyme DNA ligase.
BLUNT ENDS: second group of endonuclease cut both strands of DNA so that single stranded pieces are not left on the ends. The ends without single stranded sequences are called blunt ends. Smal (from Serratia marcescens) and Scal (Streptomyces caespitosus) belong to this category of endonucleases which produce blunt ends.
Blunt ends are also made sticky by addition of poly
A and poly T sequence with the help of terminal transferase enzyme.
SEPARATION AND ISOLATION OF DNA
FRAGMENTS: The cutting of DNA by restriction endonuclease
produces a number of DNA fragments of variable length.These can be separated by
a technique known as gel electrophoresis.
DNA fragments are negatively charged and move towards
anode in a matrix of agrose which is obtained from natural polymer of seaweed
named Gelidium.
Agrose has sieving effect that is large fragments move slowly while smaller
fragments move faster and present at farther
locations.
STAINING WITH ETHIDIUM BROMIDE:The
separated DNA fragments can be made visualised only after staining with
ETHIDIUM BROMIDE followed by exposure to UV radiation.
We can see bright orange coloured bands of DNA in
ethidium bromide.The separated bands of DNA are cut from Agrose gel and
extracted from gel piece. This is known as Elution.
The DNA fragments purified in this way are used in
constructing Recombinant DNA by joining with cloning vector.
LET US KNOW WHAT WE HAVE LEARNT!!
PART A: VERY SHORT ANSWER TYPE
QUESTIONS:
(l) MULTIPLE CHOICE QUESTIONS:
(1) The most
common bacterium used in genetic engineering:
(a) Salmonella
(b) Escherichia coli
(c) Clostridium
(d) Bacillus
(2) In 1963, the restriction enzymes were
discovered by:
(a)Linn and Arber
(b) Mendel
(c) Hargobind Khurana
(d)None of above
(3) Tools for Recombinant DNA technology
are:
(a) Restriction enzyme
(b) Phosphatase
(c) DNA polymerase
(d)All the above
(4) Which enzyme is used as molecular
Scissors?
(a) DNA ligase
(b) Helicase
(c)Restriction endonuclease
(d) DNA polymerase
(5) In gel electrophoresis, small DNA
fragments move
(a) Slow
(b) fast
(c) Slow and fast
(d) None of above
(6) Ethidium Bromide stain is used to see
clearly
(a) DNA fragments
(b) nucleus
(c) cell membrane
(d) none of above
(Il) TRUE/FALSE:
1. EcoRI is a restriction enzyme
2. Gel electrophoresis is a technique used to
separate DNA fragments.
3. Ligase enzymes are used to cut DNA strands
(Il) FILLING THE BLANKS:
1. DNA fragments are ............... charged.
2. Agrose gel is isolated from ............ Seaweed.
ANSWER KEY: PART -A
(A) MULTIPLE CHOICE QUESTIONS:
1.(b) Escherichia coli is used by scientists to
store DNA from other organisms.
2. (a) Linn and Arber discovered restriction enzymes
3. (d) Because restriction enzymes, ligase,
phosphatase, reverse transcriptase,
. DNA, polymerase, vectors and host organism are
tools.
4. (c) Because restriction endonuclease cuts the
palindromic sequence of DNA
5. (b) because small fragments squeeze through pores
easily, so move fast.
6. (a) DNA fragments are stained bright orange &
are seen clearly.
(B) TRUE/FALSE:
1. TRUE
2. TRUE
3. FALSE
(C) FILLING THE BLANKS:
1. Negatively
2. Gelidium.
PART: B_ SHORT ANSWER TYPE QUESTIONS:
1. What are molecular scissors?
2. What does EcoRI stand for?
3. What is a palindromic sequence?
PART: C LONG PART ANSWER TYPE QUESTION:
1. Explain in detail the role of restriction
endonuclease enzyme.
2. Explain the process of separation and isolation
of DNA fragments by gel
electrophoresis.
A124
INTRODUCTION
WHAT ARE VECTORS?
A vector is a vehicle, often a virus or a plasmid
that is used to carry a DESIRED
DNA or PASSENGER DNA sequence into a host cell as
part of a molecular cloning
procedure. Depending on the purpose of the cloning
procedure, the vector may
assist in multiplying, isolating, or expressing the
foreign DNA insert.
It can undergo independent replication to increase
the copies of desired genes. The
vector which can replicate in two different hosts is
called, SHUTTLE VECTOR. The
introduced vector will multiply equal to the number
of plasmids or bacteriophages
that normally developed in the particular bacterium.
CLONING VECTORS:
A cloning vector is to obtain numerous copies (clones) of
our gene of interest. These are mostly used in
construction of gene libraries. A
number of organisms can be used as sources for
cloning vectors. Some are created
synthetically, as in the case of Yeast Artificial
Chromosomes and Bacterial Artificial Chromosomes, while others are taken from
bacteria and bacteriophage.
EXPRESSION VECTORS.
An expression vector is to obtain the protein
product of gene of interest.To get that protein we need to allow the expression
of our gene of interest by employing the process of transcription and
translation.
VECTORS IN DNA RECOMBINANT TECHNOLOGY:
1. Plasmids (Plasmid vectors for plants are
different)
2. Bacteriophages or Viruses
3. Cosmids
4. Artificial chromosomes (bacterial, yeast and
human)
1. PLASMID VECTORS:These
are the most common vectors for the PROKARYOTIC host cells.
Bacteria are able to express foreign genes inserted
into plasmids.Plasmids are small, extranuclear, circular, double- stranded DNA
molecules;carrying extrachromosomal genes, but lacking protein coat that naturally
exists in the cytoplasm of many strains of
bacteria.Some of the examples of naturally occurring plasmids are Ti plasmids
in
Agrobacterium, F-factors, R-factors, Co/E1 plasmid,
etc.The best known commercially available plasmids:
pBR-322 (plasmid Boliver & Rodriguez — number
322) & pUC-18, (plasmid University of Califomia- number 18) are modified
from natural plasmids of Escheerichia coli.
Plasmids are independent of the chromosome of
bacterial cell and range in size
from 1000 to 200000 base pairs.
2. BACTERIOPHAGE DERIVED VECTORS:
Bacteriophages, or phages as they are commonly
known, are viruses that
specifically infect bacteria.Like all viruses,
phages are very simple in structure, consisting merely of a DNA (or ribonucleic
acid (RNA)) molecule carrying a number of genes, including several for
replication of the phage, surrounded by protective coat or capsid made up of
protein molecules.
3. ARTIFICIAL CHROMOSOMES AS VECTORS:
Artificial chromosomes are synthetically designed
DNA molecules of known
structure, which are assembled in vitro (in the
laboratory) from specific DNA
sequences that act like a natural chromosome.
Artificial chromosomes are circular
or linear vectors that are stably maintained in,
usually, 1-2 copies per cell. They are
huge in size in comparison to other vectors but can
clone very large segments of
chromosomes.
4. VECTORS FOR CLONING GENES IN PLANTS
AND ANIMALS:Agrobacterium tumifaciens, a pathogen of
several dicot plants is able to deliver a
piece of DNA known as ‘T-DNA’ to transform normal
plant ceils into a tumor and
direct these tumor cells to produce the chemicals
required by the pathogen.
Similarly, retroviruses in animals have the ability
to transform normal cells into
cancerous cells.A better understanding of the art of
delivering genes by pathogens in their
eukaryotic hosts has generated knowledge to
transform these tools of pathogens
into useful vectors for delivering genes of interest
to humans.
FEATURES REQUIRED FACILITATING CLONING
IN THE VECTOR:
(i) ORIGIN OF REPLICATION (ORI): This is a sequence
from wherereplication starts and any piece of DNA when linked to this sequence
can
be made to replicate within the host cells.
(ii) SELECTABLE MARKER: The vector requires a
selectable marker, which helps in identifying and eliminating non transformants
and selectively permitting the growth of the transformants. : 2*s' 0020: is a
procedure
through which a piece of DNA is introduced in a host
bacterium.
(iii) CLONING SITES: In order to link the alien DNA,
the vector needs to have
very few, preferably single, recognition sites for
the commonly used restriction enzymes. Presence of more than one recognition
sites within the vector will generate several fragments, which will complicate
the gene cloning.The ligation of alien DNA is carried out at a restriction site
present in one of the two
antibiotic resistance genes.
LET US KNOW WHAT WE HAVE LEARNT!
PART-A VERY SHORT ANSWER TYPE
QUESTIONS:
(A) MULTIPLE CHOICE QUESTIONS:
1. Which of the following gene helps in
identifying transformed cells?
(a)Plasmid
(b)Selectable marker
(c)Structural gene
(d)Vector
2. A vector that can clone only small DNA
fragment is:
(a) Cosmid
(b) Plasmid
(c) YAC
(d) BAC
3. Which of these is not a sequence of DNA
in expression vectors?
(a) Origin of replication,
(b) Selectable markers
(c) Multiple cloning sites
(d) Ti Plasmid
4. The DNA molecule used for integrating
foreign gene for cloning:
(a) Carrier
(b) Template
(c) Vector
(d)Transformer
5. Find incorrect statement about plasmid:
(a) They are circular.
(b) They are transferrable
(c) They lack protein coat
(d) They are single stranded
(B) FILLIN THE BLANKS:
1.Bacteriophages are commonly known
4S......................
2.There are ..................fypes of artificial
chromosomes.
(C)TRUE/FALSE:
1. Plasmids are most commonly used vectors for
eukaryotes.
2. Bacteriophages are types of cloning vector.
3. YAC and BAC are the examples of artificial
chromosomes.
ANSWER KEY: PART-A
A) MULTIPLE CHOICE QUESTIONS:
1 (b) Selectable marker helps in differentiating
transformants and non
transformants.
2 (b) Plasmid helps in cloning small DNA fragments.
3 (d) Three basic DNA sequences of expression vector
include origin of replication, selectable markers and multiple cloning sites
4 (c) Vectors are vehicles which carry genes for
cloning.
5 (d) Because they are double stranded.
B) FILL IN THE BLANKS:
1. Viruses.
2. Three.
C) TRUE/FALSE:
1. False
2. True
3. True
PART -B SHORT ANSWER TYPE QUESTIONS:
1. What are Vectors?
2. What is Plasmid?
3. What is ori?
PART -C LONG ANSWER TYPE QUESTION:
1. What are cloning vectors? Explain.
A125
INTRODUCTION
BIOTECHNOLOGY is a broad area of biology, involving
the use of living systems and organisms to develop or make products.
The concept of biotechnology encompasses a wide
range of procedures for modifying living organisms according to human purposes,
going back to domestication of animals, cultivation of the plants and
improvements to
these through breeding programs.
In this assignment we will discuss about the Passenger
DNA one of the tools of recombinant DNA technology.
TOOLS OF RECOMBINANT DNA TECHNOLOGY: PASSENGER DNA
Passenger DNA is a piece of DNA that is isolated
from the desired organism
and inserted into a vector for cloning where it
combines with vector DNA. The
passenger DNA contains the gene of desired traits to
be introduced in the host
organism.
The gene ts identified on a genome and pulled out
from it etther before or after cloning.
The cloned foreign DNA fragment expresses normally
as in parental cell.
Thus the foreign DNA fragments can be procured from
a variety of sources
depending on the aim and scope of cloning
experiments.
Identification and characterization of DNA sequences
are rather more difficult
on its genome than using mRNA, if it is in pure
form.
If the gene product translated by mRNA is not well
characterized it can be most difficult procedure for cloning.
In an average cell or tissue 1-2% of total
cytoplasmic RNA population is mRNA which carries transcripts for coding various
proteins.
When mRNA is present in low amount it is rather
difficult to isolate cDNA
clone.
DNA fragments are also isolated from the donor
organism by using restriction endonucleases or can be procured from gene bank.
TYPES OF PASSENGER DNA:Generally,
three types of passenger DNA are used:
COMPLEMENTARY DNA (cDNA):
It is synthesized on RNA template (usually mRNA)
with the help of reverse
transcriptase enzyme discovered by Temin and
Baltimore in 1970 and essential nucleotides.
The DNA is separated from the RNA — DNA complex in
the presence of alkaline phosphatase enzyme.
A cDNA strand is formed on the separated single-
stranded DNA template with the help of polymerase enzyme.
SYNTHETIC DNA (sDNA)
It is synthesized on DNA template or without a template.Artificial
Synthesis of DNA on Template:Kornberg and his coworkers (1959) produced DNA
from deoxyribonucleoside
triphosphates in the presence of DNA polymerase
enzyme, metal ions and a
segment of viral DNA which acts as a primer.
Artifical Synthesis of DNA without a
Template:Hargobind Khorana and his coworkers in
1970, synthesized the gene which
responsible for coding for tyrosine tRNA of E.coli.
RANDOM DNA:Small fragments are formed by breaking a
chromosome of an organism in the
presence of restriction endonucleases.
LET US KNOW WHAT WE HAVE LEARNT!!
1. VERY SHORT ANSWER TYPE QUESTIONS:
(A) MULTIPLE CHOICE QUESTIONS:
1) Molecular scissor is:
(a) Restriction endonuclease
(b) Helicase
(c) Urease
(d) Peptidase
2) The mechanism that causes a gene to move
from one linkage group to another is called:
(a) Translocation
(b) Crossing over
(c) Inversion
(d) Duplication
3) The DNA fragments separate on an agarose
gel can be visualized after staining with:
(a) Acetocarmine
(b) Aniline blue
(c) Ethidium bromide
(d) Bromophenol blue
4) Which one of the following is commonly
used in transfer of foreign DNA in plants?
(a) Trichoderma harzianum
(b) Meloidogyne incognita
(c) Agrobacterium tumefaciens
(d) Penicillium expansum
5) Which of the following is correctly
matched?
(a) Agrobacterium tumefaciens - Tumour
(b) pBR322 - Enzyme
(c) Ligase - Molecular scissors
(d) Hind Il - Plasmid vector
(B) FILL IN THE BLANKS:
1) There are ......... distinct types of restriction
enzymes.
2) Recombinant DNA technology is also popularly
called as genetic .........
3) Since DNA is a ......... molecule, it cannot pass
through cell membranes.
(C) TRUE / FALSE:
1) Exonucleases remove nucleotides at specific
positions within DNA.
2) Bacteriophages are insects that infect animal
cells by injecting their DNA
into these cells.
ANSWER KEY: PART -A
(A) 1. (a) Ristriction endonuclease is also called
molecular scissors.
2. (a) Translocation occurs when chromosomes broke
during meiosis and the resulting fragment joined another chromosome.
3. (c) Ethidium bromide is a molecule commonly used
to visualize DNA in agrose gel electrophoresis experiments.
4. (c) Agro bacterium tumefaciens is used as vector
for gene transfer.
5. (a) Agro bacterium tumefaciens contain tumour
inducing (Ti) plasmid
(B) 1. Three
2. Engineering
3. Hydrophilic
(C) 1. False: Exonucleases remove nucleotides from
the ends of DNA.
2. False: Bacteriophage is a type of virus that
infects bacteria.
B) SHORT ANSWER TYPE QUESTIONS:
1. What does competent represents in component cells
in transformation
experiment?
2. What do you mean by passenger DNA?
C) LONG ANSWER TYPE QUESTIONS:
1. In plants how alien DNA introduced into host
cell?
2. What are the types of passenger DNA? Explain each
of them.
A126
INTRODUCTION
With MICRO-INJECTION, recombinant DNA is directly
injected into the nucleus of an
animal cell.
In another method, suitable for plants, cell are
bombarded with high velocity micro-
particles of gold or tungsten coated with DNA in a
method known as BIOLISTICS or
GENE GUN METHOD.
And the last method “DISARMED PATHOGEN” vectors,
which when allowed to infect the cell, transfer the recombinant DNA into the
host.
MICROINJECTION
DNA can also stably introduce into tissue culture
cells by its direct micro-injection into the nuclei of the cells, using a glass
micro pipette that has been drawn out to on
extremely thin diameter that is from 0.1 to 0.5
micrometer.
PROCEDURE
Micropipette puller for making the needles and a
micromanipulator to position
the needles correctly for injections but given this
equipment & enough practice
one can inject 500 to 1000 cells per hour with DNA
and have up to 50 percent
of the injected cells stably integrated and express
the injected DNA will integrated at random into the nuclear DNA and if injected
gene is attached to a suitable promoter it might be expressed.
ADVANTAGE
In this procedure any piece of DNA can be introduced
into any cell, no selective pressure needs to be applied to maintain the
transferred gene.
1. Itis a very precise method.
2. It is highly efficient method.
Example: This method has been used to transfer the
gene for Rat growth
hormone into mice, in a few of which the gene was
expressed.
Resulting in the production of “giant mice” the same
procedure of introducing
pieces of DNA into plant cells is also followed.
DISADVANTAGE:Microinjection
is the expensive equipment that require, the extensive
practice needed to master this tedious technique and
the relatively small number
of cells that can be treated in one experiment.
after micro injection the egg must be re-implanted
in a surrogate-mother and
after gestation can the progeny be screened for
expression and correct regulation of the foreign DNA.
this is therefore a slow, labour-intensive method of
genetic manipulation.
ELECTROPORATION
This method is based on the use of short electrical
impulses of high field
strength. These impulses increase the permeability
of protoplast membrane and
facilitate entry of DNA molecule into the cell,
direct contact with the membrane
delivery of DNA to protoplasts; electroporation is
one of the several routine
techniques for efficient transformation.
The electroporation impulse is generated by
discharging a capacitor across the electrodes in a specially design electroporation
chamber.
Protoplasts in an ionic solution containing the
vector DNA, are suspended between
the electrons,electroporated and then plated as
usual. Transformed colonies are
selected.
Using electroporation method, successful transfer of
genes was achieved with
the protoplasts of Petunia, maize, rice, wheat and
sorghum.
SHOT-GUN/GENE GUN METHOD OF DNA
INTRODUCTION
In recently years, it has been shown that DNA
delivery to plant cells is also possible,
when heavy metallic pellets (tungsten or gold) coated
with the DNA of interest
are accelerated to a very high initial velocity
(1,400 ft/ sec.) Thes
microprojectiles, normally um in diameter, carried
by a “macroprojectile” or th
‘bullet’ and accelerated into living plant cells
(target cells can be pollen, cultured
cells, cells in differentiated tissues and
meristems) so that they can penetrate cell
walls of intact tissue. The acceleration is achieved
either by an explosive charge
(Cordite explosion) or by using shock waves
initiated by a high-voltage electri
discharge.
ADVANTAGES:
(1) Thousands of particles are accelerated at the
same time, causing multiple hits in transfer of genes into many cells
simultaneously.
(Il) Since intact cells can be used, some of the
difficulties in encountered the use
of protoplasts are automatically circumvented.
(Ill) The method is universal in its application, so
that cell type, size and shape or the presence/ absence of cell walls do not
significantly alter its effectiveness.
In view of this, particles bombardment method using
micro projectiles has a great promise in a variety of plant species,
particularly the cereals.
LET US KNOW WHAT WE HAVE LEARNT!!
PART-A VERY SHORT ANSWER TYPE
QUESTIONS:
(A) MULTIPLE CHOICE TYPE QUESTIONS
Q1. An antibiotic resistance given in a
vector usually helps in the selection of-
(a) Competent cells
(b) Transformed cells
(c) Recombinant cells
(d) None of the above
Q2. The role of DNA ligase in the
construction of a recombinant DNA is:
(a) Formation of phosphodiester bond between two DNA
fragments
(b) Formation of hydrogen bonds between sticly ends
of DNA fragment
(c) Ligation of all purine and pyrimidine base base
(d) None of these
Q3. Which of the following bacteria is not
a source of restriction endonuclease?
(a) Haemophilus influenza
(b) Eschenchia coli
(c) Agrobacterium tumifaciens
(d) Bacillus amyloliquefaciens
Q4. A bacterial cell was transformed with a
recombinant DNA that was generated using a human gene however the transformed
cell did not produce the desired protein reason could be:.
(a) Human gene may have intron which bacteria can
not process
(b) Amino acid codons for humans and bacteria are
different
(c) Human protein is formed but degraded by bacteria
(d) All of the above
Q5. In a genetic engineering experiment
restriction enzyme can be used for:
(a) Bacterial DNA only
(b) Viral DNA only
(c) Any DNA fragments
(d) Eukaryotic DNA only
(B) FILL IN THE BLANKS:
1. DNA oe enzymes join adjacent nucleotides in
double stranded
DNA molecule.
2. The Plasmid vector is isolated from bacterial
cell and cleaved at one side by
restriction 0.0...
3. Recombinant DNA technology is also popularly
called as genetic .....................
(C) TRUE / FALSE:
1. Bacteriophages are insect that infect animal
cells by injecting their DNA into these
cells.
2. E.coli is a gram negative bacterium is easy to
handle and grow.
ANSWER KEY: PART -A
(A) MULTIPLE CHOICE TYPE QUESTIONS:
1. (b) It helps in selection of transformed cell in
the presence of Ampicillin.
2. (a) It helps in ligation (joining) of separate
fragments of DNA.
3. (c) It has now been modified into cloning vector
and it is able to transfer
Gene of interest in variety of plants.
4. (a) Because introns are non-coding sequences.
5. (c) Restriction enzymes cut DNA fragments at
specific sites.
(B) FILL IN THE BLANKS:
(1) Ligase
(2) Endonuclease
(3) Engineering
(C) TRUE / FALSE:
(1) False (These are viruses that attack on
bacteria. )
(2) True
PART-B SHORT ANSWER TYPE QUESTIONS:
Q1. What does “Competent” refer to component cells
in transformation experiments?
Q2. How bacteria take up plasmid during the process
of recombinant DN technology?
Q3. What do you means by Recombinant DNA technology?
PART-C LONG ANSWER TYPE QUESTIONS:
Q1. Describe direct methods for transformation in
Recombinant DNA Technology?
A127
INTRODUCTION
BIOTECHNOLOGY deals with techniques of using live
organisms or enzymes from
organisms to produce products and processes useful
to humans.
RECOMBINANT DNA TECHNOLOGY:It
is a set of techniques,that enable the DNA from different sources to be,
(i) isolated
(2) identified and
(3) recombined
so that the new character can be introduced in an
organism.
PROCESSES OF RECOMBINANT TECHNOLOGY:
1. ISOLATION OF THE GENETIC MATERIALS:
Nucleic acid is the genetic material of all organisms.
In most of the organism genetic material is DNA or (Deoxy Ribonucleic Acid).
Restriction enzymes help to cut DNA from its pure form.
DNA is enclosed within the membrane we have to break
the cell open to release
DNA along with other macromolecules such as RNA,
proteins, polysaccharides and also lipids this can be achieved by using some
enzymes such as: -
Lysozymes for bacterial cel!
Cellulase for plant cell
Chitins for fungus
RNA can be removed by treatment with ribonuclease.
Protein can be removed by treatment with protease.
Purified DNA ultimately precipitates out after the
addition of chilled
ethanol.This can be seen as collection of fine
threads in the suspension.
2. CUTTING OF DNA AT SPECIFIC
LOCATIONS:
RESTRICTION ENZYME digestions are performed by
incubating purified DNA molecules with the restriction enzyme, at the optimal
conditions for that specific enzyme.
Agarose Gel Electrophoresis employed to check
progression of a restriction enzyme digestion.The fragments of DNA can be separated
by a technique known as GEL
ELECTROPHORESIS. Since DNA fragment are Negatively
Charged molecules,they can be separated by forcing them to move towards the
Anode under an electric filed through matrix. Nowadays most commonly used
matrix is
AGAROSE. The DNA fragments separate according to
their size through sieving effect provided by agarose gel. Smaller the fragment
farther it moves.
The separated DNA fragments can be visualised only
after staining DNA with a compound known as ETHIDIUM BROMIDE followed by
exposure to UV radiation. You can see orange coloured bands of DNA.The
separated bands of DNA cut out from the agarose gel and extracted from gel
piece this is called ELUTION.
3. AMPLIFICATION OF GENE OF INTEREST
USING PCR:PCR stands for Polymerase Chain Reaction.It includes
various processes such
as; Denaturation, Primer Annealing and Extension.
This technique was developed by KARRY MULLIS in 1985. In this reaction,
multiple copies of the gene of interest is synthesised in vitro using two sets
of primers and a thermostable enzyme DNA polymerase (Taq polymerase), isolated
from a
bacterium, Thermus aquaticus.The enzyme induces
denaturation of double stranded DNA at high temperature. In this process
replication of DNA is repeated many times.
4. INSERTION OF RECOMBINANT DNA INTO
HOST _ CELL/ORGANISM:There are several methods of
introducing the ligated DNA into recipient cells.Recipient cells after making
them “Competent” to receive, take up DNA present
in its surrounding.So, if a recombinant DNA bearing
gene for resistance to an antibiotic (e.g.ampicillin) is transferred into E
coli cells, the host cells become transformed
into ampicillin resistance cells, only transformants
will grow, untransformed
recipient cells will die since due to ampicillin
resistance gene, one is able to
select a transformed cell in the presence of
ampicillin.The ampicillin resistance gene in this case is called a “Selectable
Marker”.
5. OBTAINING THE FOREIGN GENE PRODUCT:
When you insert a piece of alien DNA into a cloning
vector and transfer it into a
bacterial, plant or animal cell the alien DNA gets
multiplied.The cells can also be multiplied in a Continuous Culture System
where in the used medium is drained out from one side while fresh medium is
added from
the other to maintain the cells in their
physiologically most active logs/
exponential face.This type of culturing method, done
in BIOREACTER, produces a LARGER
BIOMASS leading to Higher Yields of desired gene.
LET US KNOW WHAT WE HAVE LEARNT!!
PART: A_VERY SHORT ANSWER TYPE
QUESTIONS:
(A) MULTIPLE CHOICE QUESTIONS:
1. An enzyme catalysing the removal of
nucleotides form the ends of DNA:
(a) Exonuclease
(b) Endonuclease
(c) DNA Ligase
(d) All of the above
2. Which enzyme is used to break cell wall
of bacterial cells?
(a) Lysozyme
(b) Cellulase
(c) Chitinase
(d) All of the above
3. Name the enzyme used to removed RNA from
DNA.
(a) Chitinase
(b) Ribonuclease
(c) Endonuclease
(d) Protease
4. Purified DNA can be precipitates out
with help of:
(a) Chitinase
(b) Chilled ethanol
(c) Endonuclease
(d) All of the above
5. In Agarose gel electrophoresis DNA moves
towards:
(a) Anode
(b) Cathode
(c) Both a, b
(d) Name of the above
B. TRUE/ FALSE:
1. PCR stands for polymerase count reaction.
2. When we insert alien DNA into cloning vector and
transfer it into bacterial
cells, alien DNA does not multiply.
C. FILL IN THE BLANKS:
1. PCR was discovered by in .
2. are group of letters that formed the same words
when read from both forward and backward.
ANSWER KEY: PART-A
A. MULTIPLE CHOICE QUESTIONS:
1. (a) Exonuclease: -Exonuclease is an enzyme
catalysing the removal of nucleotides
from the ends of DNA
2. (a) Lysozyme: - It helps in breaking cell wall of
bacterial cell
3. (b) Ribonuclease: - RNA can be removed from DNA
with help of Ribonuclease
4. (b) Chilled Ethanol: - Purified DNA precipitates
out after the addition of
Chilled Ethanol.
5. (a) Anode: - DNA is negatively charged molecule,
hence it moves towards positive electrode (Anode).
B. TRUE/ FALSE:
1. False:
Reason: - PCR stands for Polymerase Chain Reaction
2. False:
Reason: When we insert alien DNA into cloning vector
and transfer it into bacterial cell, alien DNA gets multiplied.
C. FILL IN THE BLANKS:
1. Karry mullis in 1985
2. Palindromes
PART: B= SHORT ANSWER TYPE QUESTIONS:
1. Expand the following:
(a) PCR (6) DNA
2. Define r DNA technology?
3. Write various steps involved in recombinant DNA
technology?
PART: C LONG ANSWER TYPE QUESTIONS:
1. A. What does this diagram depict?
B. What is meant by the largest and the smallest in
picture?
C. visualise DNA fragment.
A128
INTRODUCTION
BIOTECHNOLOGY is the branch of biology which deals
with the techniques of using living organisms and enzymes for organisms to
produce products and generic modifications of organisms on a large scale.
POLYMERASE CHAIN REACTION (PCR)
OR
AMPLIFICATION OF GENE OF INTEREST
Discovered by Kary Mullis (1985) it is the method of
gene amplification in which multiple copies of genes can be synthesized with
the help of (chemically synthesized oligonucleotides) primers and enzymes DNA
Polymerase.
1. DENATURATION:
At the temperature 94-98°C denaturation of two strands of DNA takes place. At
this high temperature for 10 minutes and double stranded DNA breaks up into single
stranded DNA.
2. ANNEALING:
When the temperature is lowered, it enables Oligonucleotide
primers to attach to the template DNA. It takes
place at 50°C-60°C.
3. EXTENSION
IN PCR In this step, at final stage of PCR, takes 20 seconds to 1 min at 72°C,
so that DNA polymerase extends the primer sequences from 3’of each primer to
the end of amplicon. DNA polymerase or Taq. polymerase enzyme is isolated from
a bacterium Thermus aquaticus which is a heat tolerant enzyme.
Let Us We Know What Have Learnt!! _
PART: A - VERY SHORT ANSWER TYPE
QUESTIONS:
(A) Multiple Choice Type Questions:
Q.1) The PCR
technique was developed by:
(a) Kohler
(b) Altman
(c) Milstein
(d) Kary Mullis
Q.2) Which of the following is basic
requirement of PCR?
(a) Two oligonucleotide primers
(b) DNA segment to be amplified
(c) A heat stable DNA polymerase
(d) All of above
Q.3) Denaturation takes place at
temperature:
(a) 40°-50°C
(b) 60°-70°C
(c) 70°-80°C
(d) 90°-98°C
Q.4) The PCR is:
(a) DNA sequencing technique
(b) DNA amplification technique
(c) DNA degradation technique
(d) All of above
Q.5) Thermus aquaticus is source of:
(a) Vent polymerase
(b) Primase enzymes
(c) Taq polymerase
(d) Both (a) and (c)
(B) Fill in the blanks:-
1. Annealing of DNA takes place at temperature .
2. The process of binding primer to denatured DNA
strand is called
3. PCR has three steps denaturation, annealing and .
(C) True / False Questions:-
1. The segment of DNA can be amplified billion
times.
2. Primers are chemically synthesized
oligonucleotides those are complimentary to the regions of DNA.
ANSWER KEY: PART - A
(A) Multiple Choice Type Questions:-
1. d (The PCR was discovered by Kary Mullis)
2. d (Basic requirements of PCR are two primers, DNA
segment and DNA Polymerase)
3. d (Denaturation requires temperature 90 — 98°C)
4. b (PCR is amplification technique of DNA)
5. c¢ Thermus aquaticus is source of Taq Polymerase)
(B) Fill in the blanks:-
1. 40°-50°C (Annealing takes place at temperature
40-60°C)
2. Annealing (Binding primer to denatured DNA
strand)
3. Extension (Steps in PCR- Denaturation, Annealing
and Extension)
(C) True/ False Questions:-
1. True (DNA segment can be amplified billion times
by PCR)
2. True (Primers are chemically synthesized
oligonucleotides)
PART: B - SHORT ANSWER TYPE QUESTIONS:
Q.1) What is denaturation?
Q.2) Give two applications of PCR?
PART: C - LONG ANSWER QUESTIONS:
Q.1) Explain three major steps in PCR?
A129
INTRODUCTION
A BIOREACTOR refers to any manufactured device or
system that supports a biologically active environment.In one case, a
bioreactor is a vessel in which a chemical process is
carried out which involves organisms or
biochemicaily active substances derived from such organisms.This process can
either be aerobic or anaerobic.These bioreactors are commonly cylindrical
ranging from 100 liters to 1000 liter and are often made of stainless steel.It
may also refer to device or system designed to grow cells or tissues
in the context of cell culture.
When alien DNA is inserted into cloning vector and
then transferred to the host cell,
DNA gets multiplied. Due to expression of the
foreign gene, proteins can be formed.
Such target proteins (recombination proteins) are to
be produced on large scale.To produce this product on large quantities,
BBOREACTORS are needed.In such bioreactors, 100-1000 liters of culture can be
processed.Most commonly used is STIRRED TYPE BIOREACTOR, whose details are
given below:
The basic design of a STIRRED TANK BIOREACTOR
is shown in the figure.It consists of a large stainless steel vessel with a
capacity of up to 5, 00,000 dm? around which circulation of water is used to
control the temperature within the bioreactor. There is also an agitator,
comprising a series of flat blades,which can be rotated with the
micro-organisms.
The agitator also prevents settling of the cells at
the bottom. Bioreactor also
has adequate arrangement for aeration, temperature
and pH control.
For proper aeration, air can be forced in at the
bottom of the tank through a porous ring, called sparger, by the processcalled
sparging, while there is an outlet to remove air and waste gases at the top of
the tank.
The top of the tank also has a number of inlet tubes
called Ports, through
which materials can be introduced or withdrawn e.g,,
Inoculation port for introducing initial inoculum;
Nutrient port for introducing more nutrients;
Anti foam port for introducing anti foam gadgets;
pH point for introducing acid or alkali to maintain
optimal pH.
At the base of the tank, there is a harvest line to
extract culture medium and
microbial products.
Toregularly detect the pH and temperature changes,
tank is filled with certain
probes.
SIGNIFICANCE:The
STIRRED-TANK BIOREACTOR is a well-tried and tested design for large-
scale production of micro-organisms under aseptic
and a controlled environment for a number of days.SMALL-SCALE BIOREACTOR of 200
liters of capacity is used in research
laboratories. It is also provided with many controls
for the monitoring of physical, chemical and biological parameters that affect
the growth of cells.
LET US KNOW WHAT WE HAVE LEARNT!!!
PART: A VERY SHORT ANSWER TYPE
QUESTIONS:
(A) MULTIPLE CHOICE QUESTIONS:
1. Which of the following should be chosen
for the best yield to produce a Recombinant protein in large amount ?
(a) Laboratory flask of largest capacity
(b) A stirred tank bioreactor without inlets and
outlets
(c) A continuous culture system
(d) any of the above
2. The small scale bioreactors have volume
of-
(a) 5-10 litres
(b) 10-20 litres
(c) 1-10 litres
(d) 1-20 litsre
3. What is the function of carbon in
stainless steel bioreactors?
(a) Improves resistance to corrosion
(b) Improves ductility
(c) Reduces sensitization
(d) Improves halogen resistance
4. The bioreactor is not capable of-
(a) Producing aseptic conditions
(b) Meeting containment regulations
(c) Controlling pH
(d) Produces electricity
5. Which of the following class of
micro-organisms causes less threat toa man?
(a) Low-risk micro-organisms
(b) High-risk micro-organisms
(c) Medium-risk micro-organisms
(d) Environmental risk micro-organisms
(B) FILL IN THE BLANKS:
1. Bioreactor also has adequate arrangement for
temperature and control.
2. In bioreactor, pH port is used for introducing
or. to maintain the optimal pH .
(C) TRUE/FALSE:
1. Bioreactors provides the optimal conditions for
obtaining the desired product.
2. Sparger and stirred tank bioreactor helps in
better sterility.
3. Recombinant protein insulin is used for the
treatment of diabetes mellitus
ANSWER KEY: PART-A
A. MCQs:
1. (c) Because a continuous culture system is
maintained in a bioreactor by continuously and regularly feeding with culture
medium steadily and by providing optinum growth conditions like pH, temperature
and oxygen.
2. (d) The small-scale bioreactors normally have a
volume of 1-20 litres.The bioreactor is used for a number of purposes, scale-up
and scale down studies,clone selection, medium development,process development
etc.
3.(c) Because sensitization is the precipitation of
carbides and grain
boundaries in a stainless steel causing the alloy to
be susceptible to
intergranularcorrosion. So,carbon in the stainless
steel reduces sensitization.
4. (d) because all others are the functions of
bioreactor but only it cannot produce
electricity.
5. (d) Because these microorganisms cause less
threat to man and are very hazardous to the environment. They are also called
as environmental risk microorganisms and are responsible for high economic
losses.
B. Fill in the blanks:
1. aeration,pH
2. acid,alkali
C. True/False:
1. True
2. False because sparger is used to sparge in air
inside a stirred-tank bioreactor.
3. True
PART: B SHORT ANSWER TYPEQUESTIONS:
1. Describe briefly bioreactors.
2. Besides better aeration and mixing properties,
what other advantages do
stirred tank bioreactors have over Shaked flasks?
3. What is the significance of bioreactor?
PART:C _LONG ANSWER TYPE QUESTIONS:
Q.1. Describe the structure of stirred tank
bioreactor with the help of diagram.
A130
DEFINITION:
The various processes used for actual recovery and
purification of biosynthetic
products from fermentation or any other industrial
process together constitute a
down streaming process. It includes two processes:-
1. Separation
2. Purification
DOWNSTREAM PROCESS ANDITS STEPS:
It is an important process for the manufacture of
different products in in
pharmaceutical industry (such as antibiotics,
vaccines, enzymes, hormones like
insulin, human growth hormone), food industries etc.Downstream
processing helps to maximize product recovery and minimizes the
cost.
STEPS OF DOWNSTREAM PROCESSING:
The various steps of downstream processing
involves:-
1. Separation
2. Cell disruption
3. Extraction
4. Isolation
5. Purification
6. Drying
1. SEPARATION OF PARTICLES--It
is the first step of downstream processing and usually involves the separation
of solid substances from the liquid media.It is generally achieved by following
ways:-
a. _ Filtration: - It is used for filamentous fungi
, bacteria and cell debris.
b. Centrifugation: - Used for bacteria , usually
protein precipitates.
c. Flocculation and flotation: - It is used for
small bacterial cells which are
difficult to separate even by centrifugation.
2. CELL DISRUPTION: -
Disruption of microbial cells is done by mechanical disruption, drying lysis of
microbial cells.
5. PURIFICATION: -
It aims at recovery for the product in a highly purified state
and elimination of trace contaminants . Purification
is achieved by following
procedures
a. Crystallization:-This is used for the low
molecular mass compound like antibodies
b. Chromatographic method:- It is generally used for
the purification of low
molecular mass compounds from mixture of similar
molecules for example
antibodies and enzymes.
6. DRYING: -
Drying is the most important step in downstream processing which
makes the product soluble for handling and storage .
Most frequent approaches of
drying are:-
a. Vacuum drying
b. Freeze Dry
c. Spray Drying
DEAR STUDENTS,
LET US KNOW WHAT WE HAVE LEARNT!!
A. MULTIPLE TYPE QUESTIONS:
Q1 ) Flocculation method is associated with
which of these processing methods ?
a) Separation
b) Drying
c) Extraction
d) None of these
Q2) Cell debris and colloids are removed
during downstream processing using which of the following method ?
a) Filtration
b) Chromatography
c) Evaporation
d) Centrifugation
Q3) Filtration method is used to separate
which of the following:-
a) Only bacteria
b) Bacteria and fungi
c) Viruses
d) Allofthese
Q4) Which of these is not a compound of
downstream processing ?
a) Purification
b) Preservation
c) Separation
d) Extraction
Q5) Which of the following is an example of
Membrane filtration?
a) Reverse Osmosis
b) Micro Filtration
c) Bothaandb
d) None of these
B. TRUE AND FALSE:
1. Downstream processing usually involves the
separation of solid substances from liquid media.
2. Purification mainly aims at recovery’ of product
in highly impure state.
3. Disruption of microbial cell is done only by
lysis of microbial cell.
C. FILLIN THE BLANKS:
1. step of downstream processing involves the
elimination of trace contaminants and impurities.
2. Crystallization is used to separate compounds
like
antibodies.
A) MULTIPLE TYPE QUESTIONS:
Ans. Q1) a) Separation
Flocculation method is associated with separation .
Ans. Q2) a) Filtration
Cell debris and colloids are removed by the process
of filtration .
Ans. Q3) 6b) Bacteria and Fungi
Filtration method can separate both bacteria and
fungi
Ans. Q4) 6) Preservation
All other options are part of downstream processing
but preservation not a
component of downstream processing
Ans. Q5) c) Both (a) and (b)
Membrane filtration includes both reverse osmosis
and microfiltration technique .
B) TRUE.AND FALSE:
Q1) True: - It includes the separation of solid
substances from liquid media .
Q2) False: - Purification recovers the products at
highly purified state .
Q3) False: - Disruption of microbial cell is done by
drying and mechanical methods
also .
C) FILL.IN THE BLANKS:
1. Purification
2. Low molecular mass compound
Q1) Define downstream processing.
Q2) Name two processes involved in downstream
processing.
Q3) What are the most frequent approaches of drying.
Q1) Describes the main steps involve in downstream
processing .
A131
RECAPITULATION
BIOTECHNOLOGY:
Biotechnology deals with techniques of using live organisms or enzymes from
organisms to produce products and processes useful to humans.
RECOMBINANT DNA:
These are the molecules of DNA formed by laboratory methods of genetic
recombination by combining at least two
fragments of DNA from two different sources.
ORIGIN OF REPLICATION:
It is a specific DNA sequence responsible for initiating replication.
RESTRICTION ENZYMES:-
These Enzymes are also called “Molecular scissors” as they cut DNA at specific
sites. These are of two types:-
a) Exonucleases: These Enzymes remove nucleotide
from end of the DNA molecule.
b) Endonucleases: These Enzymes cut nucleotide at
specific positions
within the DNA.
PALLINDROMIC NUCLEOTIDE SEQUENCE:It
is the recognition sequence in DNA, recognised by the restriction endonuclease
enzyme. It is 4-8 nucleotides long. In this sequence the
two strands of DNA possess the same sequence of
nitrogen base pairs but in opposite fashion. In one strand it is in 5° — 3’ and
in its complementary strand it is in 3‘ — 5‘ directions.e.g.
5'--------GGATCC--------3'
3’--------C CTAGG--------5'
GEL ELECTROPHORESIS:It
is method for separation and analysis of macromolecules (DNA, RNA and proteins)
and their fragments, based on their size and charge.
VECTORS:A
cloning vector is a DNA molecule which has the ability to replicate ina
host cell and into which the DNA fragment to be
cloned, known as DNA insert, is integrated for cloning.
TRANSFORMATION:Transformation
is a procedure through which a piece of DNA is
introduced in a host bacterium.
BIOREACTORS: Bioreactor is a
vessel that carries out biological reaction on large scale.
DOWNSTREAM PROCESSING:After
completion of Biosynthetic stage, the product has to be subjected through a
series of processes before final product. The processes
include separation and purification, which are
collectively referred to a
downstream processing.
NOW LET US DO NCERT QUESTIONS
Q1. Can you list 10 recombinant proteins
which are used in medical practice? Find out where they are used as
therapeutics?
Ans. Various recombinant proteins obtained from
recombinant DNA technology used in Medical practices are following:
Q2. Make a chart showing a restriction
enzyme, the substrate DNA on which it acts, the site at which it cuts DNA and
the product it produces?
Ans. Name of the restriction enzyme is EcoRI.
Q3. From what you have learnt, can you tell
whether enzymes are bigger or DNA is bigger in molecular size? How do you know?
Ans. Both DNA and enzymes are macromolecules but DNA
is bigger in size
as molecular weight of DNA in prokaryotes is
2.6x10°%(E.coli) and is
1.8x10'*(Humans) in eukaryotes. On the other hand
molecular weight of
protein varies from 10000 to 1 million.
Q4. What would be the molar concentration
of human DNA in a human cell?
Ans. Molar concentration of DNA in human cell is
2.77x10°? moles.
Q5. Do eukaryotic cells have restriction
endonucleases? Justify.
Ans. No, eukaryotic cells do not have restriction
endonucleases. These are
present in prokaryotes and bacteria. In eukaryotes,
DNA molecules are heavily methylated by methylase enzymes, but in prokaryotes,
these endonucleases protect the cells from viral attack by disintegrating viral
DNA without harming self-genome because this genome bear methylation of
sensitive places.
Q6. Besides better aeration and mixing
properties, what other advantages do stirred tank bioreactors have over shake
flasks?
Ans. 1. Capacity of fermenter is more.
2. Small volumes of culture can be taken out from
the reactor for sampling or testing.
3. Bioreactors bear foam control system, pH control
system and temperature control system.
4. Bioreactors are also cost effective.
Q7. Collect 5 examples of palindromic DNA
sequences. Better try to create a palindromic sequence by following base pair
rules.
Ans. Palindrome nucleotide sequences in the DNA
molecule are groups of bases that form the same sequence when read both forward
and
backward in two different strands of DNA. Five
examples of palindromic DNA sequences are as follows:
Q8. Can you recall meiosis and _ indicate
at what = stage a recombinant DNA is made?
Ans. Crossing over results in a recombinant DNA and
this crossing over takes place between non-sister chromatids of homologous
chromosomes during pachytene stage of prophase of meiosis-I.
Q9. Can you think and answer how a reporter
enzyme can be used to monitor transformation of host cells by foreign DNA in
addition to a
selectable marker?
Ans. Reporter enzymes have unique enzymatic activity
and can be used to differentiate recombinants from non-recombinants, due to
their capacity to form colour in presence of chromogenic substrate. e.g. If in
coding sequence of B- galactosidase, recombined DNA is inserted, the
transformed cell shows no colour but if the recombinant DNA is not added, the
blue colour appear due to presence
of chromogenic substrate.
Q10. Describe briefly the following:
(a) Origin of replication.
(b) Bioreactor.
(c) Downstream processing.
Ans: (a) ORIGIN OF REPLICATION: This is a sequence
from where replication starts and any piece of DNA when linked to this sequence
can be made to replicate within the host cells. This sequence is also
responsible for controlling the copy number of the linked DNA. So, if one wants
to recover many copies of the target DNA it should be cloned in a vector whose
origin support high copy number.
(b) BIOREACTOR: Bioreactor is a kind of vessel in
which raw materials are biologically converted into specific products by
microbes, plant and animal cell
and/or their enzymes. The bioreactor provides
optimum growth conditions and facilitates achieving the desired products. The
most commonly used bioreactor is of stirring type. A stirred tank bioreactor is
usually a cylindrical vessel or vessel with a curved base to facilitate mixing
of the contents. In the sparged stirred tank bioreactor, sterile air bubbles
are sparged. The stirrer facilitates the mixing and oxygen availability
throughout the bioreactor. A bioreactor has an agitator has an agitator system,
an oxygen delivery system, a foam control system, a temperature control system,
pH control system and sampling ports.
(C) DOWNSTREAM PROCESSING: The product obtained is
subjected to a series, of processes collectively called downstream processing
before it is made into a finished product ready for marketing. The two main
processes are separation and purification. The product is then formulated with
suitable
preservatives. Such formulations have to undergo
clinical trials, in case of drugs.
Q11. Explain briefly
(a) PCR
(b) Restriction enzymes and DNA
(c) Chitinase
Ans. (a)PCR: Polymerase Chain Reaction is a
molecular biological technique for
enzymatically replicating DNA without using a living
organism, such as E. Coli or
yeast.
THREE STEPS IN PCR ARE:
(i) Denaturation of desired double strand DNA to
ssDNA.
(ii) Annealing of primer to ssDNA (single strand)
(iil) Extension of primer by Taq DNA polymerase
isolated form Thermus aquaticus.
USES: Amplification of desired gene/gene cloning.
ADVANTAGE: More output, greater efficiency, less
error prone, less human interference and cyclic and automated.
(b) RESTRICTION ENZYMES AND DNA: Restriction enzymes
is a group of enzymes used to cleave or cut DNA strands each having a
characteristics base sequence at which it cleaves.
(i) It restricts foreign DNA from entering normal
cell by digesting it at various
recognition sites. Recognition site is palindromic.
(ii) They are endonuclease and exonuclease both
types.
(iii) They produce sticky ends. Cleavage site and
recognition site are different
from each other. Restriction enzymes therefore are
believed to be a mechanism
evolved by bacteria to resist viral attack and to
help in the removal of viral
sequences.
(c) CHITINASE: Chitinase is an enzyme that digests
or breakdown glycosidic bonds in chitin cell wall of fungal cell to facilitate
its transformation.
Q12. Discuss with your teacher and find out
how to distinguish between:
a) Plasmid DNA and Chromosomal DNA
b) RNA and DNA
c) Exonucleases and Endonucleases
A132
INTRODUCTION
BIOTECHNOLOGY deals with techniques of using live
organisms or enzymes from organisms to produce products and the processes
useful to humans.It has various applications in different fields such as_
therapeutics,
diagnostics, processed food, waste management,
energy production,genetically modified crops etc.The European Federation of
Biotechnology defines biotechnology as “The
integration of natural science and organisms, cells,
parts thereof, and molecular analogues for products and services.”
PRINCIPLES OF BIOTECHNOLOGY:The
two core techniques that enabled birth of modern biotechnology are:
1. Genetic engineering: Techniques to alter the
chemistry of genetic material[DNA
and RNAj,to introduce these into host organisms and
thus change the phenotype of the host organism.
2. Maintenance of sterile[microbial contamination
free] ambience in chemical engineering processes to enable growth of only the
desired microbe/eukaryotic cell in large quantities for the manufacture of
biotechnological products like antibiotics,
vaccines, enzymes, etc.
BASIC PRINCIPLES OF BIOTECHNOLOGY:Genetic
engineering allows the isolation and introduction of only the desired genes
into the organism without introducing the undesirable genes.The steps involved
in genetic engineering are:
1) Development of recombinant DNA [rDNA].
2) Cloning of desired gene.
3) Transfer of the cloned gene into suitable host
organisms.
Origin of replication [ori]: A specific DNA sequence
in the chromosome that can initiate DNA replication. The foreign DNA introduce
into the host genome has to be linked to the origin of replication in the host
chromosome for the gene to be able to multiply. If
the foreign gene or DNA
is not linked to the ori sequence it may not be able
to multiply.
Cloning: The process of making multiple identical
copies of template DNA.
Plasmid: A circular extra-chromosomal material that
is capable of autonomous replication. Plasmids are used as vectors for cloning
expression. Foreign gene is introduced into a plasmid and the plasmid is
allowed to multiply. This causes the multiplication
of the desired gene.
Restriction Enzymes: They are enzymes that can cut
DNA at specific points to make fragments. They are also called as “MOLECULAR
SCISSORS”.
Vectors: These are plasmids that are used to
multiply and transfer the desired gene from one organism to the next.
Ligase: Enzymes that is responsible for the joining
of the desired gene fragment with the host DNA. Ligases function by getting DNA
fragments to stick together. It is also known as MOLECULAR GLUE.
THE BASIC STEPS IN GENETIC MODIFICATION
OF AN ORGANISM:Identification of desired DNA fragments
Introduction of desired DNA fragment into suitable host Maintaining foreign DNA
in the host and its transfer to the progeny
TOOLS OF RECOMBINANT DNA TECHNOLOGY:
RESTRICTION ENZYMES OR MOLECULAR SCISSORS: They are
used to cut DNA to be inserted into the vector. These enzymes add methyl group
to the DNA, which help in restricting the digestion of their own DNA. They are
used
to cut DNA fragments with specific recognition
sequences.
RESTRICTION SEQUENCES: The sequence of
DNA bases that can be recognized by the restriction enzyme as the site for
restriction or cutting. They exist as PALINDROMIC SEQUENCES. Palindrome in DNA
is a sequence of base pairs that are present in the
same order on the two strands when orientation of reading is kept same.
RECOGNITION SITE:
Restriction
enzymes belong to larger class of Nucleases. These are -
1. Endonucleases
2. Exonucleases
Endonucleases cut the DNA in the middle whereas
Exonucleases cut the DNA
at the ends. e.g. ECoR1, Hind Ill, etc. are examples
of restriction endonucleases. Restriction enzymes cut at a specific site on DNA
known as restriction site. Each restriction endonuclease identifies a specific
palindromic nucleotide sequences in DNA.
LIGASE: Ligases are the enzyme that joins the two
DNA fragments.Presence of sticky ends helps in ligation.
SEPERATION AND ISOLATION OF DNA
FRAGMENTS:
The DNA fragments obtained through restriction are separated
by a technique
called as gel electrophoresis.
GEL ELECTROPHORESIS:
It involves the migration of negatively charged
DNA to the positive electrode through a porous
polymer gel matrix under the
influence of an electric field. The DNA fragments
separate or resolve depending on their size as well as the pore size of gel.
Hence, the smaller the fragment size, the farther it moves. The most commonly
used matrix is agarose which is a natural polymer extracted from sea weeds.
VISUALIZATION:
The separated DNA fragments can be visualised only after staining the DNA with
a compound known as ethidium bromide followed byexposure to UV radiation.
ELUTION:
The
separated bands of DNA are cut out from the agarose gel and extracted from the
gel piece.
CLONING VECTORS:
A vector is a DNA molecule which has the ability to replicate in an host cell
and into which the DNA fragment to be cloned, known as DNA insert is integrated
for cloning.
PLASMID:
Plasmids are the most widely used cloning vectors in the technique of gene-manipulation
in bacteria. They are circular, double-stranded DNA molecules occurring in
extra chromosomal state and self-replicating.
TO ACT AS VECTOR, DNA MOLECULE SHOULD
BEAR FOLLOWING CHARACTERISTICS:
ORIGIN OF REPLICATION [ORI]: This is a sequence
from where replication starts and any piece of DNA when linked to this sequence
can be made to replicate within the host cells.
SELECTABLE MARKER:
Transformation is a procedure through which a piece of DNA is introduced in a
host bacterium. Normally,
the genes encoding resistance to antibiotics such as
Ampicillin,Chloramphenicol, Tetracycline, Kanamycin, etc., are considered
useful selectable markers for E.coli. The normal
E.coli. cells do not carry resistance against any of these antibiotics.
CLONING SITES: In order to link
the alien DNA, the vector needs to have very few preferably single, recognition
sites for the commonly used restriction enzymes. Presence of more than one
recognition sites within the vector will generate several fragments, which will
complicate the gene cloning. The ligation of alien DNA is carried out
at a restriction site present in one of the two
antibiotic resistance genes. A recombinant DNA is inserted within the coding
sequence of an enzyme called 8 - galactosidase. This results into inactivation
of the enzyme, which is referred to as insertional inactivation.
VECTORS FOR CLONING GENES IN PLANTS AND
ANIMALS:Agrobacterium tumifaciens, a pathogen of several dicot plants is able to deliver a piece DNA known as “T-DNA” to transform normal plant cells into a tumor and direct these tumor cells to produce the chemicals required by the pathogen. Similarly, retroviruses in animals have the ability to transform normal cells into cancerous cells.
COMPETENT HOST
In order to allow bacterial cells to take up the
DNA, bacterial cell should be
made competent. This can be done by treating the
cells with specific concentration of divalent ions such as calcium ions, which
creates pores in the cell wall of the bacteria. Such bacteria are subjected to
heat shock. In this method the calcium treated competent cells are kept in ice.
They are then briefly incubated at 42°C for 1-2 minutes and then immediately
placed in ice.
This forces the rDNA into the competent cell.
Apart from this, DNA can be inserted into host cells
using biolistics,
microinjection, gene gun etc. Using microinjection,
DNA can be directly
inserted into the nucleus of the host cell.
A high velocity microparticles of gold or tungsten
coated with DNA is methodology used in biolistic.PROCESS OF RECOMBINANT DNA
TECHNOLOGY There are several steps involved in the process of recombinant DNA
technology:
1. ISOLATION OF THE GENETIC MATERIAL:
To isolate the DNA, membranes need to be broken
down. Cells can be treated
with lysozyme (in case of bacteria), cellulase (in
case of plant cells) and
chitinase (in case of fungus). Ribonucleases are
used to remove the RNA whereas Proteases are used to remove the proteins. After
this, the pure DNA can be obtained through precipitation via Chilled Ethanol.
DNA is then obtained as fine threads in suspension.
3. RESTRICTION DIGESTION OF THE
ISOLATED DNA:
Agarose gel electrophoresis is used to check the progression
of restriction digestion of the DNA. The gene of interest is now inserted into
specific vector and joined via enzyme known as ligase. This forms a recombinant
DNA
molecule.
4. AMPLIFICATION OF GENE OF INTEREST
USING PCR:Polymerase Chain Reaction (PCR) is used to amplify
the target gene of interest. For these two sets of primers polymerase forward
primer and reverse primer is used. DNA polymerase enzyme is used to amplify the
DNA. The most common polymerase used during PCR is
Taq polymerase.
4. INSERTION OF RECOMBINANT DNAIS INTO
HOST CELL OR ORGANISM:Recipient cell is made competent to
take up the recombiant DNA.
5. EXPRESSION OF DESIRED PROTEIN:The
ultimate goal of recombinant DNA technology is to obtain desired protein of
interest. The protein obtained is known as recombinant protein.To produce large
quantities of recombinant protein, large vessels known as
bioreactors are used. A bioreactor provides the
optimal conditions for achieving the desired product by providing optimum
growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).
BASIC PARTS OF A BIOREACTOR:
1. Agitator
2. Oxygen Control system
3. Foam control system
4. Temperature control
5. Sampling port
6. pH control
7. Iniet
8. Outlet
BIOREACTORS ARE MAINLY OF TWO TYPES:
STIRRING TYPE BIOREACTOR: A stirrer is fixed to a
bioreactor having a curved base to facilitate better mixing of the contents. It
also improves aeration of the medium.
SPARGER TYPE BIOREACTOR: In this air is bubbled into
the bioreactor from the base of the bioreactor. This bubbling of air results in
mixing as well as aeration of the contents.
DOWNSTREAM PROCESSING:The processes and methods
involved in the separation and purification of the desired product are called
as downstream processing.
In case of drugs, the product needs to be suitably
formulated with preservatives and drug tested before being made available
commercially.
Let Us Know What We Have Learnt!!!
PART: A VERY SHORT ANSWER TYPE
QUESTIONS:
A. MULTIPLE CHOICE QUESTIONS:
1. An enzyme catalysing the removal of
nucleotides from the ends of DNA is:
(a)Endonuclease
(b)Exonuclease
(c)DNA ligase
(d)Hind-I|
2. A recombinant DNA molecule can be
produced in the absence of the following:
(a)Restriction endonuclease
(b) DNA ligase
(c)DNA fragments
(d)E.coli
3. In agarose gel electrophoresis,DNA
molecules are separated on the basis
of their:
(a)Charge only
(b)Size only
(c)Charge to size ratio
(d)All of the above
4. Polymerase chain reaction is most useful
in:
(a)DNA synthesis
(b)DNA amplification
(c)Protein synthesis
(d)Amino acid synthesis
5. Molecular scissor is:
(a)Restriction endonuclease
(b)Helicase
(c)Urease
(d)Peptidase
B. TRUE/FALSE:
1. Hind Il always join DNA molecules at specific
site by recognising a
particular sequence of six base pairs.
2. Plasmids are the most widely used cloning vectors
in the technique of gene
manipulation in bacteria.
C. FILL IN THE BLANKS:
1..........48 groups of letters that formed the same
words when read from both
forward and backward.
2. Plasmids and Bacteriophages are
the............... which are used for cloning
purposes in prokaryotes.
3. The plasmid vector is isolated from bacterial
cell and cleaved at one side by
restriction..............
ANSWER KEY: PART-A
A. MULTIPLE CHOICE QUESTIONS:
1. (b) Exonuclease
2. (d) E. coli
3. (b) Size only
4. (b) DNA amplification
5. (a) Restriction endonuclease
B. TRUE/FALSE:
1. True
2. True
C. FILL IN THE BLANKS:
1. Palindrome
2. Vectors
3. Endonuclease
PART -B SHORT ANSWER TYPE QUESTIONS:
1. Distinguish between Plasmid DNA and Chromosomal
DNA.
2. Give differences between Exonuclease and
Endonuclease.
PART -C LONG ANSWER TYPE QUESTIONS:
1. Explain (a) PCR (b) Restriction enzymes and DNA.
A211
RECALL:
Biotechnology is the technique of using live organisms
or their enzymes for products and processes useful to humans.
Biotechnology deals with:
Basic steps in genetically modifying an organism:
1 MARK QUESTIONS (MCQs)
1. The DNA fragments have sticky ends due
to:
a) Endonuclease
b) b) Unpaired bases
c) Calcium ions
d) Free methylation
2. Plasmids are used as cloning vectors for
which of the following reasons?
a. Can be multiplied in culture
b) Self-replication in bacterial cells
c) Can be multiplied in laboratories with the help
of enzymes
d) Replicate freely outside bacterial cells
3. The first transgenic plant to be
produced is
a) brinjal
b) Tobacco
c) rice
d) cotton
4. Which bacterium is used in the
production of insulin by genetic
engineering?
a) Saccharomyces
b) Rhizobium
c) Escherichia
d) Mycobacterium
5. ———— is used as a vector for cloning
into higher organisms
a) Retrovirus
b) Baculovirus
c) Salmonella typhimurium
d) Rhizopus nigricans
6. Plasmid is a:
a) Fungus
b) Plasmid
c) Part of Plasma membrane
d) Extra chromosomal DNA in bacterial cell
7. In genetic engineering, antibiotics are
used
a) as selectable markers
b) to select healthy vectors
c) to keep the cultures free of infection
d) as sequences from where replication starts
8. Which of the following enzyme is used in
the case of fungus to cause a release of DNA along with other macromolecules?
a) Lysozyme
b) Cellulase
c) Chitinase
d) amylase
9. Recombinant DNA is obtained by cleaving
the pro-DNA by
a) primase
b) exonucleases
c) ligase
d) restriction endonuclease
10. DNA ligase is helpful in
a) translation
b) removal of genes
c) insertion of genes in DNA
d) transversion
11. Maximum application of animal cell
culture technology today is the production of
a) insulin
b) interferons
c) vaccines
d) edible proteins
12. The most important feature in a plasmid
to be used as a vector is
a) origin of replication
b) presence of a selectable marker
c) presence of sites for restricting endo-nuclease
d) Its size
13. Genetic engineering is not possible
without biological scissors
a) helicases
b) polymerases
c) ligases
d) restriction endo nuclease
14. Restriction endonucleases are capable
of
a) creating sticky ends
b) breaking DNA at any site
c) causing crossing over
d) breaking DNA at specific site
15. PCR is most useful in
a) DNA synthesis
b) DNA amplification
c) protein synthesis
d) Amino acids synthesis
16. In agarose gel electrophoresis ,DNA
molecules are separated on the basis of their
a) charge only
b) size only
c) charge to size ratio
d) all of the above
17. The tumor inducing capacity of Agro
bacterium tumefaciens is located in large extra chromosomal plasmids called
a) Riplasmid
b) lambda phage
c) PBR 322
d) T1 plasmid
18.Transposons are
a) housekeeping genes
b) jumping genes
c) transporting genes
d) stationary genes
19.For transformation, micro particles are
coated with DNA to be bombarded with gene gun are made up of
a) silver or platinum
b) platinum or zinc
Cc) silicon or platinum
d) gold or tungsten
20.The transfer of genetic material from
one bacterium to another through the mediation of a vector like virus is termed
as
a) transduction
b) conjugation
c) transformation
d) translation
ANSWER KEY
ANS 1. b) Unpaired bases
ANS 2. b) Self-replication in bacterial cells
ANS 3. b) tobacco
ANS4._ c) Escherichia
ANS 5. a) Retrovirus
ANS 6. d) Extra chromosomal DNA in bacterial cell
ANS7. a) as selectable markers
ANS8. c) Chitinase
ANS 9. d) restriction endonuclease
ANS 10. c) insertion of genes in DNA
ANS 11. b) interferons
ANS 12. a) origin of replication
ANS 13. d) restriction endo nuclease
ANS 14. d) breaking DNA at specific site
ANS 15. b) DNA amplification
ANS 16. b) size only
ANS 17. d) T1 plasmid
ANS 18. b) jumping genes
ANS 19. d) gold or tungsten
ANS 20. a) transduction
A212
RECAPITULATION
Biotechnology deals with large scale production and
marketing of products and processes using live organisms, cells or enzymes.
Modern biotechnology using genetically modified
organisms was made possible only when man learnt to alter the chemistry of DNA
and construct recombinant DNA this key
process is called recombinant DNA technology or
genetic engineering.
Genetic engineering process involves the use of
restriction endonucleases, DNA ligase, appropriate plasmid or viral vectors to
isolate and ferry the foreign DNA into host organisms,
expression of the foreign gene, purification of the gene product, i.e., the
functional protein and finally making a suitable formulation for marketing.
Large scale production involves use of bioreactors.
Q:1 Define Biotechnology.
Ans: Biotechnology deals with large scale production
and marketing of products
and processes by the use of natural strains of
microorganisms and cell lines.
Q:2 What are the key tools of recombinant
technology?
Ans: The key tools of rDNA technology are
restriction endonucleases and cloning
vectors.
Q:3 What are Moleculer scissors?
Ans: Molecular scissors are the restriction enzymes
which cut the DNA at specific
location usually they are obtained from Bacteria.
Q:4 What is vector in biotechnology?
Ans: A vector is any vehicle, often a virus or a
plasmid that is use to ferry a desired DNA sequence into a host cell as part of
a molecular cloning procedure.
Q:5 Write full form of PCR.
Ans: Polymerase Chain Reaction.
Q:6 What is probe?
Ans: A probe is a radio-labelled single stranded
sequence of DNA or RNA
(prepared in lab) used to search for its
complementary sequence in a simple
genome.
Q:7 Recalling meosis, indicate at what
stage a recombinant DNA is made.
Ans: At Pachytene stage of prophase-| of Meosis-l.
Q:8 What are exonucleases and
endonucleases?
Ans: Exonucleases.Enzymes which remove nuceleotides
from the end DNA Endonucleases. This enzymes cut or break the DNA at a specific
position within
the DNA.
Q:9 What is microinjection?
Ans: It is a technique of vectorless gene transfer,
wnere recombinant DNA is
directly injected into the nucleases of animal cell
by using micro needles or
micropipettes, like in oocytes, eggs and enbryos
etc.
Q:10 While doing a PCR denaturation step is
missed. What will be the effect of this process ?
Ans: Primers will not anneal the template, thus
extension will not occur. There will be no amplification.
Q:11 Write one use of PCR technique.
Ans: It is use in forming abundant amount of DNA for
analysis in DNA fingerprinting technique, used in forensic science.
Q:12 What are bioreactors?
Ans: Bioreactors are large vessel in which raw
materials in large volume (100-1000
liters) are processed and biologically converted
into large quantities of specific
products using microbial, animal, plant, or human
cell or enzymes.
Q:13 Define pallindromes.
Ans: Pallindromes are groups of letters that form
the same word when read both
forward and backward.In DNA these are the sequence
of base pairs reads the same on both the strands of DNA when the orientation of
reading is kept same in both strands.
e.g.,5'-GAATTC-3'3-CTT AAG-S5'
Q:14 What is downstream processing?
Ans: Downstream processing is a series of processes
involving separation and purification to which the product has to be subjected
before it is ready for
marketing as a finished product.
Q:15 State the role of DNA ligase in
biotechnology.
Ans: DNA ligase is used to join two fragment of DNA
to seal the gaps between
them. Only the matching DNA fragment with
complementary ends can be joined with DNA ligase.
Q:16 How is Agrobacterium tumifaciens able
to transform a normal plant cell into a tumor?
Ans: Plasmid of agrobacterium tumifaciens carries
the genes for tumour, ‘Ti’ this
segment also termed as T-DNA. Agrobacterium
transfers these cancer causing genes in plants thus causing tumour.
Q:17 Why is it essential to have a
selectable marker in a cloning vector?
Ans: A selectable marker helps us to distinguish
between transformed and non-
transfomed cells.
Q:18 Why do DNA fragments move towards the
anode during gel-electrophoresis?
Ans: DNA is negatively charged so it is moves
forward positively charged anode during gel electrophoresis.
Q:19 Would you like to choose an
exo-nuclease enzyme while producing a recombinant DNA molecule?
Ans: No, exonuclease will shorten or degrade the DNA
fragments containing required gene, because it acts at free ends of linear DNA
fragments with spicky ends.circular plasmid (not having free end) will not get
a cut.
Q:20 What does H, in, d and Ill refer in enzyme
hind-lll?
Ans: H : Haemophilus; in : influenza; d: strain, Ill
: Number of endonuvlease.
A213
REVISION
POINTS TO REMEMBER:
Genetic engineering and bioprocess engineering are
the two main techniques that have gives rise to modern biotechnology.
Tools of Recombinant DNA Technology:
The key tools of rDNA technology are:
Restriction Endo nucleases and Cloning
Vectors.Restriction Endonucleases:
(i) Restriction endonucleases cut the desired DNA at
specific sites to produce sticky ends.
(ii) These endonucleases are also called scalpels or
molecular scissors.
(iii) Restriction endonucleases are of three types
such as Typel, Type II and
Type Ill.
(iv) Type Il is used in gene cloning and is most
stable.
Cloning Vectors:
(i).Cloning vectors are also called vehicle DNA or
gene carrier.Plasmids are the most commonly used vectors.
(ii) Other types of the vector include
bacteriophages, cosmids, phagemids,
Yeast Artificial Chromosome (YAC), Bacterial
Artificial Chromosome (BAC)and also some plant and animal viruses.
(iii). The first artificial cloning vector is pBR
322322 from E. coli.
Processes of Recombinant DNA
Technology:
(i) Electrophoresis is a technique used in rDNA
technology for separation of
DNA fragments by the use of the electric field.
(ii) Passenger DNA can also be obtained through the
genomic library, cDNA
library or through Polymerase Chain Reaction (PCR).
(iii) The other antibiotic resistance gene of a
plasmid remains active and acts
as a selective marker in selecting the transformant.
(iv) Alternatively, such selectable markers have
been developed which differentiate recombinants from non-recombinants on the
basis of their ability to produce colour in the form of a chromogenic
substrate.
2- MARKS QUESTION/ANSWERS (SHORT TYPE
QUESTIONS)
Q1:-Name the regions A, B, and C.
Ans. (A) BamHl
(B) Pstl
(C ) Ampicillin resistance gene (ampR)
Q2:- What do “Eco”, “R” and “I” refer to in
the enzyme EcoRI?
Ans: “E co” refers to the species from which it is
taken, “R” refers to the strain, and “I” states that it was the first enzyme
isolated from this strain.
Q3-:How is Ti plasmid of Agrobacterium
tumefaciens modified to convert it into a cloning vector?
Ans. The Ti plasmid is a tumour-inducing plasmid.
The genes responsible for
its pathogenic nature are either removed or altered
so that it does not harm the plants and only delivers the gene of interest.
Q4:- What is the role of Agrobacterium
tumefaciens in plant transformation?
Ans .Agrobacterium tumefaciens is a plant pathogen
that infects crops such as tomato, sunflower, cotton, soybean, etc. It causes
crown gall disease in plants which are induced by Ti plasmid . The Ti plasmid
incorporates a DNA segment called the T-DNA into the DNA of the host plant
cell. This T-DNA causes tumours
Q5:- Identify the steps A, B, C in the
following diagram
Ans: (A) Denaturation:- The DNA strands are treated
with a temperature 94°C and the strands are separated.
(B) Annealing:-The primers anneal to the
complementary strands.
(C) Extension:-The DNA polymerase facilitates the
extension of the strands.
Q6:-What is a bioreactor?
Ans: The bioreactor is a large vessel used to carry
out a biological reaction and to culture aerobic cells for conducting cellular
or enzymatic immobilizations.
Q7:-What would happen if a plasmid without
a selectable marker was chosen as a cloning vector?
Ans. A selectable marker helps to distinguish
transformed cells from the nontransformed ones. If the cloning vector does not
have a selectable marker It will be difficult to distinguish between transformants
and non transformants
Q8:-What is the difference between cloning
and expression vector?
Ans .All vectors that are used for propagation of
DNA inserts in a suitable
host are called cloning vectors. When a vector is
designed for the expression
of, i.e. production of the protein specified by, the
DNA insert, it is termed as
an expression vector.
Q9:-What are molecular scissor?
Ans. Restriction endonuclease enzymes are called
molecular scissors which
can cut double-stranded DNA at specific sites.
Q10:- What is the role of restriction
endonuclease?
Ans. Restriction endonuclease inspects the length of
DNA sequence.
It finds specific recognition sequence, i.e.
palindromic nucleotide sequence in DNA.
These enzymes cut the strand of DNA a little away
from the centre of palindromic sites.
Thus restriction endonucleases leave overhanging
stretches called sticky ends on each strand.
Q11:-List three important features
necessary for preparing a genetically modifying organism.
Ans. Conditions necessary for preparing:
Identification of DNA with desirable genes.
Introduction of the identified DNA into the host.
Maintenance of introduced DNA in the host and
transfer of the DNA to its progeny.
Q12:-Expand PCR. List the three main steps.
Ans .PCR stands for Polymerase Chain Reaction.Three
steps of PCR are:
Denaturation
Annealing
Extension of primers.
Q13:- What is EcoRI ? How EcoRI differ from
an exonuclease?
Ans: EcoRI is an endonuclease restriction enzyme
which cut both the stands of
palindromic DNA at a specific position of nitrogen
base 5’ (GAATTC) 3’ while
exonuclease removes nucleotides from terminals of
DNA strands.
Q14:-Do eukaryotic cells have restriction
endonucleases?
Ans. No, eukaryotic cells do not have restriction
endonucleases. This is
because the DNA of eukaryotes is highly methylated
by a modification enzyme, called methylase. These enzymes are present in
prokaryotic cells where they help prevent the invasion of DNA by virus.
Q 15:-What is the significance of ori in a
cloning vector?
Ans.The origin of replication (ori) is a sequence
from where replication starts.
Any piece of DNA, when linked to this sequence, can
be made to replicate
within the host cells. This sequence is also
responsible for controlling the
copy number of the linked DNA.
Q 16:- What do you understand by gene
cloning?
Ans. Gene cloning or DNA cloning is the process in
which the gene of
interest is copied out of the DNA extracted from an
organism. The process of
gene cloning is carried out in the following steps:
Isolation of DNA fragment or gene
Selection of the appropriate vector
The isolated DNA fragment is incorporated into the
vector.
The recombinant vector is transformed in the host
cell
The recombinant host cell is isolated.
Q 17:-What is the role of enzyme “Ligase” in
genetic Engineering?
Ans.Enzyme “Ligase” acts as molecular Suture which
helps in joining two pieces of DNA. The Joining process requires ATP as it
derive energy to construct bond phosphodiester between cohesive ends.
Q 18:- Name two main steps which are
collectively referred to as down streaming process. Why is this process
significant?
Ans .Separation and Purification .This process is
essential because before
reaching into market, the product has to be
subjected for clinical trial and
quality control.
Q19:- Mention the important properties
which a good vector possess?
Ans. The important properties which a good vector
must possess
i) Size
ii) Origin of Replication
ili) Selectable Marker
iv) Cloning Sites
Q 20:- Expain briefly Chitinase .
Ans. It is an enzyme used to break the cell wall of
fungi to release its cellular
parts. Certain plants and fungus consists of a cell
wall composed of chitin,
which provides strength to the cell. Chitinase is
used when there is a need to
break this wall and allow entry of foreign DNA in
plant cells.
A214
RECAPITULATION
Biotechnology is defined as the broad area of
biology which uses both the technology and the application of living organisms
and their components to develop, modify and produce useful products for human
welfare. The term
‘Biotechnology’ was coined in the year 1919 by an
agricultural engineer Karoly Ereky, hence he is called as the father of
Biotechnology.Let us have an overview of the principles and processes of
Biotechnology.
PRINCIPLES OF BIOTECHNOLOGY:According
to modern Biotechnology, the main principles of Biotechnology are:
1. GENETIC ENGINEERING, which is used to modify the
DNA of the target organism, thereby changing the phenotype of the organism.
2. BIOPROCESS ENGINEERING, which is the maintenance
of sterile conditions to support the growth of large quantities of desired
microbes and other eukaryotic cells which are used for the production of new or
modified biotechnological products such as
antibiotics, enzymes,vaccines, etc.
The techniques of genetic engineering
mainly include:
1. DNA fragment is isolated from the donor organism.
2. It is inserted into the vector DNA.
3. It is transferred into an appropriate host.
4. Cloning of the recombinant DNA in the host
organism.
RECOMBINANT DNA TECHNOLOGY:Recombinant
DNA technology is also known as Genetic Engineering. It is the process of
joining together two DNA molecules from two different organisms. This is known
as the recombinant DNA.
The steps involved in the processes of
Recombinant DNA technology are:
1. Isolation of DNA
2. DNA fragmentation using restriction endonucleases
3. Ligation of the desired DNA fragment into the
vector
4. Transfer of the recombinant DNA into the host
5. Culture of the transformed cells in a nutrient
medium.
6. Extraction of the desired product.
DNA CLONING:DNA
cloning is the process of making multiple, identical copies of a piece of DNA.
This process requires cloning vectors which possess the following properties:
It should be smaller in size but should be able to
carry a large DNA insert.
The cloning vector should have the origin of
replication so that it can autonomously replicate in the host organism.
It should have a restriction site.
It should have a selectable marker to screen
recombinant organism.
It should possess multiple cloning sites.
Bioprocess Engineering Bioprocess engineering is the
multiplication of cells in the bioreactors. A large amount of culture is
obtained in the process which produces a higher yield of the required protein.
The products that are obtained are subjected to
a series of processes. The products are purified by
downstream processing
and subjected to quality check before undergoing
further trials. This process
is used to manufacture antibiotics, vaccines and
other therapeutic drugs.
REVISION QUESTION-ANSWERS (2 MARKS)
1) What are essential features must be present in a
cloning vehicle?
i) Ori site where replication starts.
ii) Selectable markers to distinguish between
recombinant and non-recombinant forms.
iii) | Ori site must support high copy numbers.
2) Write down the names for tools required in
recombinant DNA technology.
i) Restriction enzymes
ii) Competent Host
iii) Ligases, Polymerase enzymes, and Alkaline
Phosphatase.
3) What are restriction Enzymes? What are its types?
Restriction Enzymes belongs to a class of enzymes
called nucleases.They are of two types Exonucleases (cut DNA at terminals) and
Endonucleases (cut DNA at a particular site).
4) Write two uses of gene cloning.
i) In identification of genes responsible for human
diseases.
ii) In production of recombinant human insulin.
5) Write any two uses of PCR.
i) PCR is Polymerase Chain Reaction.
ii) It can quickly amplify a piece of DNA without
cell.
iii) It can help to detect HIV DNA in blood or tissue
samples.
6) Why E. Coli in used as competent host in rDNA
technology?
i) It can grow both in aerobic and anaerobic
conditions.
ii) Simple genome with only 4400 genes.
iii) Complete gene sequence and easy to handle.
iv) Faster rate of growth.
7) What are the basic requirements of PCR technique?
i) DNA template-Desired DNA segment required to be
amplified.
ii) 2small nucleotide (RNA primers) for annealing to
DNA.
8) What are selectable markers? What is their use in
genetic engineering?
Selectable marker is the sequence on DNA, which
helps in identifying and eliminating non-transformants and selectively
permitting the growth of transformants. The vector requires a selectable marker
for this purpose.
12) What is Bioprocess engineering? What are its uses?
Bioprocess engineering is the multiplication of
cells in the bioreactors. A
large amount of culture is obtained in the process
which produces a higher yield of the required protein.This process is used to
manufacture antibiotics, vaccines and other
therapeutic drugs.
A215
RECAPITULATION
LET US RECALL:
Biotechnology is the technique of using live
organisms or their enzymes for products and processes useful to humans.
Biotechnology deals with:
1. microbe- mediated processes (making curd bread
and wine etc.)
2. in-vitro fertilization. (“Test- tube baby
program”).
3. Synthesis and using of gene.
4. Preparation of DNA vaccine.
5. Correcting a defective gene.
Basic steps in genetically modifying an
organism:
Identification of DNA with desirable genes.
Introduction of identified DNA into the host.
Maintainance of introduced DNA in the host and
transfer of DNA to its progeny.
3- MARK QUESTIONS :
1. How is the copy number of plasmid vector
and yield of the recombinant protein related to each other?
A.1. The copy number of plasmid vector is directly
related to the yield of recombinant protein. Higher the copy number of vector
plasmid, greater is the copy number of gene and consequently, the yield of the
recombinant protein is in higher amounts.
Q.2. Can exonuclease is used while
producing a recombinant DNA molecule?
A.2. No, an exonuclease cannot be used while
producing a recombinant DNA. This is because an exonuclease degrades the DNA.
It cannot produce DNA fragments with sticky ends.
Q.3. What are the features of a plasmid
being used as a cloning vector?
A.3. The characteristics of plasmids being used as a
cloning vector are:
It should have an origin of replication
It should have a selectable marker
It should have restriction sites
Q.4. What are competent cells? What does
the word “competent” refer to?
A.4. Competent cells are those that allow the
foreign DNA to incorporate into the
host by a slight alteration in the cell walls.
“Competent” means the ability of a cell to intake foreign DNA.
Q.5. What do “Eco”, “R” and “I” refer to in
the enzyme EcoRI?
A.5. “Eco” refers to the species from which it is
taken, “R” refers to the particular
strain, and “I” states that it was the first enzyme
isolated from this strain.
Q.6. Why are proteases added while isolating
the DNA?
A.6. Proteases are added to degrade the proteins so
that they do not interfere with
the downstream DNA treatment.
Q.7. If the “denaturation” step is missed
during PCR, what would be its effect on the entire process?
A.7. lf the denaturation process is missed, there
will no separation of DNA strands,
the primers will not anneal to the template strands
and eventually, amplification of
DNA will not occur.
Q.8. Name a recombinant vaccine.
A.8. Hepatitis B vaccine is a recombinant vaccine.
Q.9. How is Ti plasmid of Agrobacterium
tumefaciens modified to convert it into a cloning vector?
A.9. The Ti plasmid is a tumour-inducing plasmid.
The genes responsible for its
pathogenic nature are either removed or altered so
that it does not harm the plants and only delivers the gene of interest.
Q.10. Do biomolecules such as DNA, proteins
exhibit biological activity in anhydrous conditions?
A.10. No, biomolecules do not exhibit biological
activity in anhydrous conditions.
That is why life does not sustain without water.
Q.11. What is Biotechnology?
A.11. Biotechnology is defined as the broad area of
biology which uses both the
technology and the application of living organisms
and their components to
develop, modify and to produce a useful product for
human welfare. The term
‘Biotechnology’ was coined in the year 1919 by an
agricultural engineer Karoly
Ereky, hence he is called as the father of
Biotechnology.
Q.12. What is Genetic engineering?
A.12. Genetic engineering is the technique mainly
used to change or to modify the genetic material (DNA/RNA), and to introduce
them into other organisms.
Q.13.What is the Principle of
Biotechnology?
A.13.The modern biotechnology started with two
crucial technologies:
Genetic Engineering
Chemical Engineering.
Q.14.How is Biotechnology useful in
developing food crops and in agriculture process?
A.14.Biotechnology extends its applications over a
broad spectrum in the fields of
agriculture and development of food crops.
In the agriculture field, it helps in improving food
quality, quantity, and processing.Bio-fertilizers and Bio-pesticides are
eco-friendly sources for agriculture, which contains the living microorganisms
that help in promoting growth by increasing the
supply or availability of primary nutrients. Farmers
choose biotech crops to
increase the yield and in lower production costs.
Q.15.What is the different types of
biotechnology?
A15.The scope of Biotechnology has driven a need to
classify Biotech based on
some common features or their final purpose.
Below are some of the main areas of Biotechnology:
Medical- Biotechnology.
Agricultural-Biotechnology.
Industrial -Biotechnology.
Plant -Biotechnology.
Animals- Biotechnology.
Environmental- Biotechnology.
Marine- Biotechnology.
Bio-process engineers.
Biopharma.
Food -Biotechnology.
Q.16. What do you understand by gene
cloning?
A.16. Gene cloning or DNA cloning is the process in
which the gene of interest is
copied out of the DNA extracted from an organism.
The process of gene cloning is
carried out in the following steps:
Isolation of DNA fragment or gene
Selection of the appropriate vector
The isolated DNA fragment is incorporated into the
vector
The recombinant vector is transformed in the host
cell
The recombinant host cell is isolated
Q.17. What is the role of Agrobacterium
tumefaciens in plant transformation?
A.17. Agrobacterium tumefaciens is a plant pathogen
that infects crops such as
tomato, sunflower, cotton, soybean, etc. It causes
crown gall disease in plants
which are induced by Ti plasmid or the
tumour-inducing plasmid. The Ti plasmid
incorporates a DNA segment called the T-DNA into the
DNA of the host plant cell.This T-DNA causes tumours.
Q.18. What is a bioreactor? Explain
different types of bioreactors.
A.18. The bioreactor is a large vessel used to carry
out a biological reaction and to
culture aerobic cells for conducting cellular or
enzymatic immobilizations. The
different types of bioreactors are:
Stirred Tank Bioreactors
Bubble Column Bioreactors
Airlift Bioreactors
Fluidized Bed Bioreactors
Packed Bed Bioreactors
Q.19. What is a polymerase chain reaction?
What are the steps involved?Mention its applications.
A.19. The polymerase chain reaction is the process
used in molecular biology to
obtain several copies of a specific segment of
DNA.The following steps are involved in the process:
Denaturation
Annealing
Extension
Applications:PCR has its applications in the
following fields:
Forensic Science
Research and genetics.
Q.20. What are the properties of a good
vector?
A.20. A good vector must possess the following
properties:
The vector must be small in size so that it is easy
to isolate and purify.
It should have an origin of replication, a base pair
sequence where replication
starts.It should have a selectable marker that helps
in selecting the transformed host
cells.The vector should have at least one unique
recognition site to bind the foreign
DNA.
Chapter 11 Biotechnolgy: Principles and Processes