Chapter 11 Biotechnolgy: Principles and Processes

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

(b) DNA ligase

(d) DNAvirus

2. Who discovered Recombinant DNA technology?

(a) Hargobind Khorana

(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.

(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.

(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

(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.

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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

(d) Bacillus

(2) In 1963, the restriction enzymes were discovered by:

(a)Linn and Arber

(b) Mendel

(d)None of above

(3) Tools for Recombinant DNA technology are:

(a) Restriction enzyme

(b) Phosphatase

(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

(d) None of above

(6) Ethidium Bromide stain is used to see clearly

(a) DNA fragments

(b) nucleus

(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

(d) BAC

3. Which of these is not a sequence of DNA in expression vectors?

(a) Origin of replication,

(b) Selectable markers

(d) Ti Plasmid

4. The DNA molecule used for integrating foreign gene for cloning:

(a) Carrier

(b) Template

(d)Transformer

5. Find incorrect statement about plasmid:

(a) They are circular.

(b) They are transferrable

(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

(d) Peptidase

2) The mechanism that causes a gene to move from one linkage group to another is called:

(a) Translocation

(b) Crossing over

(d) Duplication

3) The DNA fragments separate on an agarose gel can be visualized after staining with:

(a) Acetocarmine

(b) Aniline blue

(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

(d) Penicillium expansum

5) Which of the following is correctly matched?

(a) Agrobacterium tumefaciens - Tumour

(b) pBR322 - Enzyme

(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

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

(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

(d) None of these

Q3. Which of the following bacteria is not a source of restriction endonuclease?

(a) Haemophilus influenza

(b) Eschenchia coli

(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

(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

(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

(d) All of the above

2. Which enzyme is used to break cell wall of bacterial cells?

(a) Lysozyme

(b) Cellulase

(d) All of the above

3. Name the enzyme used to removed RNA from DNA.

(a) Chitinase

(b) Ribonuclease

(d) Protease

4. Purified DNA can be precipitates out with help of:

(a) Chitinase

(b) Chilled ethanol

(d) All of the above

5. In Agarose gel electrophoresis DNA moves towards:

(a) Anode

(b) Cathode

(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)

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

(d) Kary Mullis

Q.2) Which of the following is basic requirement of PCR?

(a) Two oligonucleotide primers

(b) DNA segment to be amplified

(d) All of above

Q.3) Denaturation takes place at temperature:

(a) 40°-50°C

(b) 60°-70°C

(d) 90°-98°C

Q.4) The PCR is:

(a) DNA sequencing technique

(b) DNA amplification technique

(d) All of above

Q.5) Thermus aquaticus is source of:

(a) Vent polymerase

(b) Primase enzymes

(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

(d) any of the above

2. The small scale bioreactors have volume of-

(a) 5-10 litres

(b) 10-20 litres

(d) 1-20 litsre

3. What is the function of carbon in stainless steel bioreactors?

(a) Improves resistance to corrosion

(b) Improves ductility

(d) Improves halogen resistance

4. The bioreactor is not capable of-

(a) Producing aseptic conditions

(b) Meeting containment regulations

(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

(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.

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

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.

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

Plus Two

Saturday, 30 January 2021

Chapter 11 Biotechnolgy: Principles and Processes

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